Enhancement of Fungal Pectinolytic Enzymes Production Using Gamma Radiation Under Solid State Fermentation

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Enhancement of fungal pectinolytic enzymes production using gamma radiation under solid state fermentation. Thesis Submitted in Partial Fulfillment for M.Sc. Degree in Microbiology To Botany and Microbiology Dept., Faculty of Science, Helwan University

Presented BY Shaima Abdel Mohsen Ibrahim B.Sc. Microbiology&Biochemistry (2005)

Under Supervision of Prof. Dr/ Mohamed E. Osman

Prof. of Microbiology, Faculty of Science, Helwan University Prof.Dr/Ahmed Ibrahim ELSayed El- Batal.Prof .of Applied Microbiology& Biotechnology.NCRRT

Faculty of Science Enhancement of fungal pectinolytic enzymes production using gamma radiation under solid state fermentation. Thesis Submitted in Partial Fulfillment for M.Sc. Degree in Microbiology

Presented BY Shaima Abdel Mohsen Ibrahim B.Sc. Microbiology&Biochemistry (2005) Ain Shams University Faculty of Science Botany and Microbiology Department

2013

جحضيي االًحاج الفطري لالًزيوات الوحللة للبكحيي باصحخذام اشعة جاها جحث ظروف الحخور شبه الجافة. رسالة هقدهة هي شيواء عبذ الوحضي ابراهين بكالوريوس العلوم – هيكروبيولوجى&كيوياء حيوي جاهعةعيي شوش)0225( كوحطلب جزئى للحصول على درجة الواجيضحير فى الويكروبيولوجى جحث إشراف

أ.د:-

أستاذ الويكروبيولوجى- قسن النبات والويكروبيولوجى كلية العلوم- جاهعة حلواى

أ.د:-

استاذ الويكروبيولوجيا التطبيقية والتكنولوجيا الحيوية الوركز القوهي لبحوث وتكنولوجيا االشعاع كلية العلوم – جاهعة حلواى قسم النبات والميكروبيولوجى

0223

جاهعة حلواى

كلية العلوم جحضيي االًحاج الفطري لالًزيوات الوحللة للبكحيي باصحخذام اشعة جاها جحث ظروف الحخور شبه الجافة. رسالة هقدهة هي شيواء عبذ الوحضي ابراهين بكالوريوس العلوم – هيكروبيولوجى&كيوياء حيوي جاهعةعيي شوش )0225( كوحطلب جزئى للحصول على درجة الواجيضحير فى الويكروبيولوجى

كلية العلوم – جاهعة حلواى قسم النبات والميكروبيولوجى 0223 Approval Sheet

Title of master thesis

Enahncement of fungal pectinolytic enzymes production using gamma radiation under solid state fermentation.

Submitted to Department of Botany and Microbiology Faculty of Science- Helwan University By Shaima Abdel Mohsen Ibrahim B.Sc. Microbiology&Biochemistry (2005) Supervision Committee: Prof. Dr/ Mohamed E. Osman

Prof. of Microbiology, Faculty of Science, Helwan University Prof.Dr/Ahmed Ibrahim El Sayed El Batal

Prof .of Applied Microbiology& Biotechnology,NCRRT. ACKNOWLEDGMENT

First and foremost my unlimited thanks are to our God who guides and sustains.

My deepest gratitude and appreciation to Prof.Dr.Mohamed .E.Osman, Prof of Microbiology , Botany and Microbiology Department, Helwan University for his closely supervision and kind help.

I am deeply thankful to Prof.Dr.Ahmed Ibrahim El Sayed El-Batal, Prof of Applied Microbiology&Biotechnology Drug Radiation Research Dep. National Center for Radiation Research &Technology (NCRRT) for suggesting the research topic, valuable supervision as this thesis is a part of the Project.“Nutraceuticals and Functional Foods Production by Using Nano/Biotechnological and Irradiation Processes.” that is financially supported by NCRRT.

My sincere thanks extended to all the staff and members of the Microbiology lab in NCRRT.

Gratitude is extended to all the staff and members of the Microbiology lab at the Department of Botany and Microbiology, Faculty of Science, Helwan University.

Lastly my thanks go to my family for their understanding and willingness to assist. Enhancement of Fungal Pectinolytic Enzymes Production Using Gamma Radiation Under Solid State Fermentation

(Shaima Abdel Mohsen Ibrahim)

(Botany and Microbiology Dep.,Faculty of Science,Helwan University)

Summary

14 fungal species were screened for their ability to produce pectinases on sugar-beet pulp medium. The highest producer strain was identified as Penicilium citrinum. The optimum conditions for polygalacturonases production were achieved by growing the fungus on sugar beet pulp mineral salts medium and incubation for 7 days at 250C, pH 5.5and 0.04g N/g dry SBP by using the conventional method and 1.2 % of nitrogen source by using the factorial design method and surfactant of 0.1% Tween 40. The use of gamma irradiation at a dose of 0.7% kGy yields the highest increase of production of PGase. Polygalacturonases were precipitated from culture supernatant using ammonium sulphate then purified by gel filtration chromatography on sephadex G-100. The optimum pH and temperature of the enzyme activity production were found to be 6.0 and 40°C respectively. The enzyme was found to be stable at pH rang 4 – 8 and showed high stability at temperature rang 20°C -60°C. Mg+2 and Zn+2 stimulated PGase activity. Contents

No Title Page 1 Introduction 1 2 Review of literature 4 1-Classification of pectic substance 5 1.5.Pharmaceutical uses of pectin 8 2-Classification of pectic enzymes 10 2.1. Pectic estrases 10 2.2. Depolarizing pectinases 11 2.3. Cleaving pectinases 12 3. Production of Pectinases 14 3.1. Submerged fermentation (SmF) 15 3.2. Solid substrate fermentation (SSF): 15 4. Uses of Pectinases 23 4.1.Fruit juice industry 23 4.2. Wine industry 25 4.3 Textile industry 26 5. Factors controlling the microbial pectinase production 26 5.1. PH and thermal stability of pectinases 26 5.2. Carbon Sources 28 5.3.-Nitrogen sources 29 5.4–Temperature 30 5.5- Incubation period 31 5.6- Inoculum size: 31 5.7- Surfactants: 32 6. Factorial Design: 33 7. Gamma Rays 35 7.1. Ionizing radiation 37 7.2. Responses of pectinases to gamma radiation 37 8. Purification of microbial pectinases 38 9. Applications of pectinases 39

3- Materials and Methods 40 3.1.Microorganisms 40 3.2.Culture media 40 3.3. Fermentation substrates 41 4. Culture condition 41 5. Screening for pectinolytic enzymes using Sugar beet 42 pulp medium 6. Analytical methods 43 6.1. Pectinases assay 43 6.2. Assay for pectin lyase 45 6.3. Protein determination 45 6.4. Statistical analysis: 45 7. Optimization of parameters controlling pectinases 46 production by P.citrinum. 7.1. Effect of different natural products 46 7.2. Effect of different nitrogen sources 47 7.3. Effect of different inoculum sizes 47 7.4. Effect of different incubation periods 48 7.5. Effect of different pH values 48 7.6. Effect of different temperatures 49 7.7. Effect of different surfactants 49 7.8. Application of factorial design for optimization of 50 pectinase production by P.citrinum under Solid state fermentation 7.9. Effect of different gamma irradiation doses 50 8. Purification of pectinases 51 8.1. Production of pectinases and preparation of cell-free 51 filtrate 8.2. Ammonium sulphate precipitation 51 8.2.1. Steps for precipitation by ammonium sulphate 52 8.3. Dialysis 52 8.4. Gel filtration chromatography 53 9. Characterization of the purified polygalacturonase 56 enzyme 9.1. Effect of different pH values 56

9.3. Effect of different temperatures on the enzyme 57 9.4. Effect of different metal ions on the activity of the purified exo-polygalacturonase enzyme produced by 56 P.citrinum 10. Bioextraction of pectin from different agro-residues for 57 different pharmaceutical applications 4- Results 58 4.1.Screening of the most potent fungal pectinase producer 58 4.1.1. polygalacturonase activity 58 4.1.2. Pectin lyase activity 60 4.2. Optimization of the fermentation parameters affecting 61 enzyme production 4.2.1. Effect of some agroindustrial by-products as carbon source on polygalacturonase production by P.citrinum 61 under Solid state fermentation. 4.2.2. Effect of different nitrogen sources on 63 polygalacturonase production using Penicillium citrinum under Solid state fermentation 4.2.3. Effect of inoculum size on polygalacturonases 66 production by P.citrinum under solid state fermentation 4.2.4. Effect of different incubation periods on extracellular polygalacturonase enzyme production by Penicillium 68 citrinum

4.2.5. Effect of different pH values on polygalacturonases 70 production by P.citrinum under solid state fermentation 4.2.6 Effect of different incubation temperatures on 72 polygalacturonase production by P.citrinum under solid state fermentation 4.2.7. Effect of some surfactants on polygalacturonase 74 production by P.citrinum 4.2.8. Application of factorial design for optimization of 76 polygalacturonase production by P.citrinum under Solid state fermentation 4.2.9. Effect of Gamma irradiation doses on polygalacturonase production by P. citrinum under Solid 82 state fermentation using optimized conditions of factorial design 4.3. Purification and characterization of the enzyme 84

4.3.1. Purification steps 84 4.3.2. Characterization of the purified enzyme 86 4.3.2.1. Effect of different pH values 86 4.3.2.2.Effect of different temperatures 90 4.3.2.3. Effect of different metal ions on the activity of the 94 purified polygalacturonase enzyme produced by P.citrinum 4.4. Extraction and determination of pectic substances 96 5- Discussion 98 6- Concluding remarks 126 7- References 127 7

List of tables

No. Title page 1 Composition of pectin in different fruits and vegetables 7 2 Comparison of solid and submerged fermentation for 18 pectinase production 3 Polygalacturonase activity of the tested fungal species under 59 solid state fermentation Effect of some agroindustrial by-products as carbon source 4 on polygalacturonase production by P.citrinum under Solid 62 state fermentation Effect of different nitrogen sources on polygalacturonase 5 production using Penicillium citrinum under Solid state 65 fermentation 6 Effect of inoculum size on polygalacturonase production by 67 P.citrinum under solid state fermentation. 7 Effect of different incubation periods on production of the 69 polygalacturonase enzyme by Penicillium citrinum 8 Effect of different pH values on polygalacturonases 71 production by P.citrinum under solid state fermentation 9 Effect of different incubation temperatures on 73 polygalacturonase production by Penicillium citrinum 10 Effect of some surfactants on polygalacturonase production 75 by P. citrinum under solid state fermentation. Effect of the variables and their interactions in the 25 11 factorial designs for optimization of polygalacturonase 78 production by P.citrinum under Solid state fermentation ANOVA table for the enzyme activity: effect of inoculums 12 size, yeast extract and temperature on the activity of PGase 80 13 Effect of Radiation Dose on polygalacturonase production 83 using Penicillium citrinum. 14 Purification of PGase secreted by P.citrinum 85 Effect of different pH values on the activity of the purified 87 15 polygalacturonase enzyme produced by P.citrinum. Effect of different pH values on the stability of the purified 89 16 polygalacturonase enzyme produced by P.citrinum. Effect of the temperature on the activity of the purified 91 17 polygalacturonase enzyme produced by P.citrinum.

Effect of different temperatures on the stability of the 18 purified exo-polygalacturonase enzyme produced by 93 P.citrinum. 19 Effect of different metal ions on the activity of the purified 95 polygalacturonase enzyme produced by P.citrinum 20 The different weights of pectin extracted from different 97 agroindustrial by products inoculated with P.citrinum

List of Figures

No. Title page 1 Structure of pectin 8 2 Mode of action of pectinases 14 3 polygalacturonases activity of the tested fungal species grown under solid state conditions 60 Effect of different agroindustrial by products as carbon 4 sources on polygalacturonase production by P.citrinum. 63 Effect of different nitrogen sources on polygalacturonases 5 production using P.citrinum under solid state fermentation. 66 6 Effect of inoculum size on polygalacturonase production by P.citrinum under solid state fermentation 68 Effect of different incubation periods on polygalacturonase 7 production by P.citrinum 70 Effect of different pH values on polygalacturonases 8 production by P.citrinum 72 Effect of different incubation temperatures on 9 polygalacturonase production by Penicillium citrinum 74 Effect of some surfactants on polygalacturonase production 10 by P.citrinum. 76 Factorial design for the effects of yeast extract and 11 inoculums size at pH5.5 on the polygalacturonase activity of 80 Penicillium citrinum Plot of predicted versus actual polygalacturonase 12 production 81 Effect of different doses of gamma Radiation on 13 polygalacturonase production using Penicillium citrinum. 84 14 Gel filtration profile of polygalacturonase 86

Effect of different pH values on the activity of the purified 15 polygalacturonase enzyme produced by P.citrinum. 88

16 Effect of different pH values on the stability of the purified exo- 90 polygalacturonase enzyme produced by P.citrinum. Effect of the temperature on the activity of the purified exo 92 17 polygalacturonase enzyme produced by P.citrinum Effect of different temperatures on the stability of the 94 18 purified polygalacturonase enzyme produced by P.citrinu 19 Effect of different metal ions on the activity of the 96 purified polygalacturonase enzyme produced by P.citrinum.

Abbreviations and symbols

Conc. Concentration g gram

µg microgram hr hour

L Liter

M Molar mg milligram min minute ml milliliter mM millimolar

µM Micromolar pH negative logarithm of numerical value

` (hydrogen ion exponent) rpm round per minute

SMF submerged fermentation sp species

SSF Solid state fermentation

3,5 DNS 3,5 Dinitrosalycylic acid.

Aim of the study

Aim of the study:

The present study aimed to investigate some aspects in relation to enhancement of fungal production of pectinolytic enzymes using Gamma radiation under Solid state fermentation.

1. Screening of the most potent fungal isolates for the biosynthesis of extracellular pectinases. 2. Optimization of solid state fermentation parameters for the highest enzyme producion (different carbon sources, nitrogen sources, pH, temperature, duration time, and surfactants). 3. Role of gamma irradiation on pectinase production 4. Characterization of partially purified enzyme. 5. Possible applications of microbial pectinases with extraction of some natural pectin from agrowastes

sources.

Introduction

Application of biotechnology in industrial production, holds many promises for sustainable development, but many products still have to pass the test of economic viability .White biotechnology is biotechnology used for industrial purposes. Industries incorporating white biotechnology use living organisms, organic materials, or chemical components of living organisms such as enzymes in the production process. Applications of white biotechnology currently being used or researched include manufacturing processes, the creation of biomaterials, and alternate energy sources.

In addition to purely commercial benefits, white biotechnology is also being researched as a way to make industry more environmentally friendly by providing less polluting sources of energy, lessening dependence on fossil fuels, and creating industrial processes with fewer polluting by-products.

Biological processes are based on chemical processes, and so white biotechnology is being incorporated into many production processes and

Introduction

Products that involve chemical reactions. Some chemicals used in industry, such as some polymers and acids, can be produced biologically rather than through conventional means. Industrial enzymes can be used in chemical-intensive processes such as the production of paper and the treatment of textiles and leather for clothing. Cleaning products made with this kind of biotechnology, such as laundry and dishwashing detergents, use enzymes in the place of conventional inorganic chemicals.

Pectinases are the first enzymes to be used in homes.Their commercial application was first reported in 1930 for the preparation of wines and fruit juices. Only in 1960, the chemical nature of plant tissues became apparent and with this knowledge, scientists began to use enzymes more efficiently. As a result, pectinases are today one of the upcoming enzymes of the commercial sector. Primarily, these enzymes are responsible for the degradation of the long and complex molecules called pectin that occur as structural polysaccharides in the middle lamella and the primary call walls of young plant cells. Pectinases are now

Introduction

an integral part of fruit juice and textile industries as well as having various biotechnological applications. Microbial sources have occupied an important place in the pectinases production .Among microbes, fungi as enzyme producers have many advantages , since they are normally GRAS (generally regarded as safe) strains and the produced enzymes are extracellular which makes it easy recuperation from fermentation broth (Pushpa and Madhava 2010). The pectinase class of hydrolytic enzymes is one of several enzymes that Penicillium sp can produce to utilize a wide variety of naturally substrates. Accordingly a local isolate of Penicillium sp was chosen to investigate the production and characterstics of its pectinase yield.

Review of literatures

REVIEW OF LITERATURE

Pectinase comprises a heterogeneous group of enzymes that catalyze the breakdown of pectin-containing substrates. They are widely used in the food industry to improve the cloud stability of fruit and vegetable nectars,for production and clarification of fruit juices, and for haze removal from wines (Cavalitto et al., 1996). Furthermore, phytopathologic studies have reported that fungal endo-polygalacturonase (endoPGase) which is a major kind of pectinase has been shown to activate plant defense responses, including phytoalexin accumulation, lignification, synthesis of proteinase inhibitors, and necrosis (Cervone et al., 1989). Further research has confirmed that endoPGase can degrade the plant cell wall releasing pectic oligomers which can stimulate a wide array of plant defence responses (Boudart et al., 1998). With the increasing application of pectinases, decreasing its production cost has become one of the most important targets. For this purpose, selection of carbon source and nitrogen source with low value is a practical consideration. Previous studies reported that many waste products from

Review of literatures the agricultural industry containing pectin, such as sugar beet pulp (SBP), citrus pulp pellets, apple pomace, pulp, lemon pulp and other related materials have been used as carbon source for induction of pectinase by many microorganisms (Said et al., 1991).

1. Pectic substances in plant cell walls

Chemically, pectic substances are complex colloidal acid polysaccharides, with a backbone of galacturonic acid residues linked by a (1, 4) linkages. The side chains of the pectin molecule consist of L-rhamnose, arabinose,galactose and xylose. The carboxyl groups of galacturonic acid are partially esterified by methyl groups and partially or completely neutralized by sodium, potassium or ammonium ions.

Classification of pectic substances

Based on the type of modifications of the backbone chain pectic substances are classified into protopectin, pectic acid, Pectinic acid and pectin (Miller, 1986).

1.1Protopectin:

This is a parent pectic substance and upon restricted hydrolysis yields pectin or Pectinic acid. Protopectin is occasionally a term used to describe the water-insoluble

Review of literatures pectic substances found in plant tissues and from which soluble pectic substances are produced (Kilara, 1982).

1.2Pectic acids:

These are the galacturonans that contain negligible amounts of methoxyl groups. Normal or acid salts of pectic acid are called pectates.

1.3Pectinic acids:

These are the galacturonans with various amounts of methoxyl groups. Pectinates are normal or acid salts of pectinic acids (Kilara, 1982). Pectinic acid alone has the unique property of forming a gel with sugar and acid or, if suitably low in methyl content, with certain other compounds such as calcium salts.

Review of literatures

Table.1.Amount of pectin in different fruits and vegetables: (Kashyap et al., 2001).

Pectic

Fruit /vegetable Tissue Substance (%)

Apple peel Fresh 0.5–1.6

Banana peel Fresh 0.7–1.2

Peaches pulp Fresh 0.1–0.9

Strawberries pulp Fresh 0.6–0.7

Cherries pulp Fresh 0.2–0.5

Peas pulp Fresh 0.9–1.4

Carrots peel Dry matter 6.9–18.6

Orange pulp Dry matter 12.4–28.0

Review of literatures

Fig.1. Structure of pectin (Harholt et al., 2010)

2. Pharmaceutical Uses of Pectin:

1. In the pharmaceutical industry, pectin favorably influences cholesterol levels in blood. It has been reported to help reduce blood cholesterol in a wide variety of subjects and experimental conditions as comprehensively reviewed (Sriamornask, 2001).Consumption of at least 6 g/day of pectin is necessary to have a significant effect in cholesterol reduction. Amounts less than 6 g/day of pectin are not effective (Ginter., 1979).

2. Pectin acts as a natural prophylactic substance against poisoning with toxic cations. It has been shown to be effective in removing lead and mercury from the gastrointestinal tract and respiratory organs (Kohn,

Review of literatures

1982) . When injected intravenously, pectin shortens the coagulation time of drawn blood, thus being useful in controlling hemorrhage or local bleeding (Joseph, 1956).

3. Pectin reduces rate of digestion by immobilizing food components in the intestine. This results in less absorption of food. The thickness of the pectin layer influences the absorption by prohibiting contact between the intestinal enzyme and the food, thus reducing the latter’s availability (Wilson&Dietschy, 1974; Dunaif& Schneeman, 1981; Flourie et al., 1984).

4. Pectin has a promising pharmaceutical uses and is presently considered as a carrier material in colon- specific drug delivery systems (for systemic action or a topical treatment of diseases such as ulcerative colitis, Crohn’s disease, colon carcinomas) The potential of pectin or its salt as a carrier for colonic drug delivery was first demonstrated by studies, of Ashford et al., (1993) and Rubinstein et al ., (1993). The rationale for this is that pectin and calcium pectinate will be degraded by colonic pectinolytic enzymes(Englyst et al.,1987) but will retard drug

Review of literatures

release in the upper gastrointestinal tract due to its insolubility and because it is not degraded by gastric or intestinal enzymes(Sandberg et al.,1983). 3. Classification of pectic enzymes

Pectinases are classified under three headings according to the following criteria: whether pectin, pectic acid or oligo-D-galacturonate is the preferred substrate, whether pectinases act by trans-elimination or hydrolysis and whether the cleavage is random (endo-, liquefying of depolymerizing enzymes) or endwise (exo- or saccharifying enzymes). The three major types of pectinases are as follows.

3.1. Pectinesterases (PE) (Ec 3.1.1.11):

Pectinesterases also known as pectinmethyl hydrolase, catalyzes deesterification of the methyl group of pectin forming pectic acid. The enzyme acts preferentially on a methyl ester group of galacturonate unit next to a non- esterified galacturonate one.

3.2. Depolymerizing pectinases

These are the enzymes:

3.2.1-Hydrolyzing glycosidic linkages

They include:

Review of literatures

3.2.1.1- Polymethylgalacturonases (PMG). Catalyze the hydrolytic cleavage of a-1,4-glycosidic bonds. They may be:

3.2.1.11. Endo-PMG: causes random cleavage of α-1,4- glycosidic linkages of pectin, preferentially highly esterified pectin.

3.2.1.1.2. Exo-PMG: causes sequential cleavage of α -1, 4- glycosidic linkage of pectin from the non-reducing end of the pectin chain.

3.2.1.1.2- Polygalacturonases (PG) (Ec 3.2.1.15).

Catalyze hydrolysis of α -1, 4-glycosidic linkage in pectic acid (polygalacturonic acid). They are also of two types:

3.2.1.1.2.1. Endo-PG: also known as poly (1,4- α -D- galacturonide) glycanohydrolase, catalyzes random hydrolysis of α - 1,4-glycosidic linkages in pectic acid.

3.2.1.1.2.2. Exo-PG (Ec 3.2.1.67): also known as poly (1,4- α -D-galacturonide) galacturonohydrolase, catalyzes hydrolysis in a sequential fashion of a-1,4-glycosidic linkages on pectic acid.

3.3. Cleaving pectinases

Review of literatures

Cleaving α -1,4-glycosidic linkages by trans- elimination, which results in galacturonide with an unsaturated bond between C4 and C5 at the non-reducing end of the galacturonic acid formed. These include:

3.3.1. Polymethylegalacturonate lyases (PMGL).

Catalyze breakdown of pectin by trans-eliminative cleavage. They are:

3.3.1.1. Endo-PMGL (Ec 4.2.2.10): also known as poly (methoxygalacturonide) lyase, catalyzes random cleavage of a-1,4-glycosidic linkages in pectin.

3.3.1.2. Exo-PMGL: catalyzes stepwise breakdown of pectin by trans-eliminative cleavage.

3.3.2.2. Polygalacturonate lyases (PGL) (Ec 4.2.99.3):

Catalyze cleavage of α -1,4-glycosidic linkage in pectic acid by trans-elimination. They are also of two types:

3.3.2.2.1. Endo-PGL (Ec 4.2.2.2):

Also known as poly (1,4- α D-galacturonide) lyase, catalyzes random cleavage of α -1,4-glycosidic linkages in pectic acid.

Review of literatures

3.3.2.2.2. Exo-PGL (Ec 4.2.2.9): also known as poly (1, 4- α -D-galacturonide) exolyase, catalyzes sequential cleavage of a-1, 4-glycosidic linkages in pectic acid.

3.3. Protopectinase

This enzyme solubilizes protopectin forming highly polymerized soluble pectin.On the bases of their applications, pectinases are mainly of two types: acidic pectinases and alkaline pectinases.

Review of literatures

Figure 2. Mode of action of pectinases: (a) R = H for PG and CH3 for PMG; (b) PE; and (c) R = H for PGL and CH3 for PL. the arrow indicates the place where the pectinase reacts with the pectic substances. PMG, polymethylgalacturonases; PG, polygalacturonases; PE, pectinesterase; PL, pectin lyase (Jayani et al., 2005).

4. Production of Pectinases

Microbial enzymes are commercially produced either through submerged fermentation (SmF) or solid substrate fermentation (SSF) techniques.

Review of literatures

4.1. Submerged fermentation (SmF)

SmF techniques for enzyme production are generally conducted in stirred tank reactors under aerobic conditions using batch or fed batch systems. High capital investment and energy costs and the infrastructural requirements for large-scale production make the application of Smf techniques in enzyme production, not practical in a majority of developing countries environments. Submerged fermentation is cultivation of microorganisms on liquid broth it requires high volumes of water, continuous agitation and generates lot of effluents.

4.2. Solid substrate fermentation (SSF):

SSF incorporates microbial growth and product formation on or with in particles of a solid substrate under aerobic conditions, in the absence or near absence of free water and does not generally require aseptic conditions for enzyme production (Mudgett, 1986 and Sanzo et al., 2001).

4.3.Microorganisms commonly used in submerged and solid state fermentation for Pectinases production

Microorganisms are currently the primary source of industrial enzymes: 50 % originate from fungi and yeast; 35 % from bacteria, while the remaining 15 % are either of

Review of literatures plant or animal origin. Filamentous microorganisms are most widely used in submerged and solid-state fermentation for pectinases production. Ability of such microbes to colonize the substrate by apical growth and penetration gives them a considerable ecological advantage over non-motile bacteria and yeast, which are less able to multiply and colonize on low moisture substrate (Smith et al, 1988). Among filamentous fungi three classes have gained the most practical importance in SSF; the phycomycetes such as the geneus Mucor; the ascomycetes genera Aspergillus and basidiomycetes especially the white and rot fungi (Young et al., 1983). Bacteria and yeasts usually grow on solid substrates at the 40%to70% moisture levels (Young et al., 1983). Common bacteria in use are (Bacillus licheniformis, Aeromonas cavi, Lactobacillus etc and common yeasts in use are Saccharomyces and Candida Pectinase production by Aspergillus strains has been observed to be higher in solid-state fermentation than in submerged process (Solis-Pereyra et al., 1996).

4.4. Substrate for fermentation:

Medium require presence of bioavailable nutrients with the absence of toxic or inhibitory constituents medium. Carbon, nitrogen, inorganic ions, and growth

Review of literatures factors are also required. For submerged fermentation besides carbon source, nitrogen growth factors, media requires plenty of water. The most widely used substrate for solid state fermentation for pectinase production are materials of mainly plant origin, which include starchy materials such as grains, roots, tubers , legumes, cellulosic lignin, proteins, and lipid materials (Smith and Aidoo; 1988). Agricultural and food processing wastes such as wheat bran, cassava, sugar beet pulp, Citrus waste,corn cob, banana waste, saw dust and fruit pomace (apple pomace) are the most commonly used substrates for SSF for pectinase production (Pandey et al., 2002).

Review of literatures

3.3 Table.2.Comparison of solid and submerged fermentation for pectinase production: (Raimbault, 1998)

Liquid Substrate Solid Substrate

Factor fermentation Fermentation

Polymer Insoluble Substrates: Starch Substrates Soluble Substrates(sugars) Cellulose Pectins Lignin

Aseptic conditions Heat sterilization and Vapor treatment, non aseptic control sterile conditions

High volumes of water Limited Consumption consumed and effluents of water; low Aw. No Water discarded effluent

Easy control of Low heat transfer capacity Metabolic Heating temperature

4.5. Pectinases production in solid state fermentation 4.5.1. Protopectinases

PPases are classified into two types, on the basis of their reaction mechanism. A-type PPases react with the inner site, i.e. the polygalacturonic acid region of protopectin, whereas B-type PPases react on the outer site, i.e. on the polysaccharide chains that may connect the 08

Review of literatures polygalacturonic acid chain and cell wall constituents.A- type PPase are found in the culture filtrates of yeast and yeast-like fungi. They have been isolated from Kluyveromyces fragilis, Galactomyces reesei and Trichosporon penicillatum and are referred to as PPase-F, - L and -S, respectively. B-type PPases have been reported in Bacillus subtilis and Trametes sp. , and are referred to as PPase- B, -C and -T,respectively. B-type PPases have also been found in the culture filtrate of a wide range of Bacillus sp. All three A-type PPases are similar in biological properties and have similar molecular weight of 30 kDa.PPase-F is an acidic protein, and PPase-L and -S are basic proteins. The enzymes have pectin-releasing effects on protopectin from various origins. The enzymes catalyze the hydrolysis of polygalacturonic acid; they decrease the viscosity, slightly increasing the reducing value of the reaction medium containing polygalacturonic acid .PPase- B, -C and -T have molecular weights of 45, 30, and 55 kDa, respectively.

4.5.2. Polygalacturonases

Endo-PGases are widely distributed among fungi, bacteria and many yeasts. They are also found in higher plants and some plant parasitic nematodes. They have been

Review of literatures reported in many microorganisms, including Aureobasidium pullulans, Rhizoctonia solani, Fusarium moniliforme, Neurospora crassa, Rhizopus stolonifer, Aspergillus sp., Thermomyces lanuginosus, Peacilomyces clavisporus . Endo- PGases have also been cloned and genetically studied in a large number of microbial species.

In contrast, exo-PGases occur less frequently. They have been reported in Erwinia carotovora, Agrobacterium tumefaciens, Bacteroides thetaiotamicron, E.chrysanthemi, Alternaria mali, Fusarium oxysporum, Ralstonia solanacearum, Bacillus sp.Exo-PGases can be distinguished into two types:fungal exo-PGases, which produce monogalacturonic acid as the main end product; and the bacterial exo-PGases,which produce digalacturonic acid as the main end product. Occurrence of PGases in plants has also been reported. Polygalacturonate lyases (Pectate lyases or PGLs) are produced by many bacteria and some pathogenic fungi with endo-PGLs being more abundant than exo-PGLs. PGLs have been isolated from bacteria and fungi associated with food spoilage and soft rot. They have been reported in Erwinia carotovora , Amucala sp., Pseudomonas syringae, Colletotrichum magna , E. chrysanthemi , Bacillus sp. ,Bacillus sp. Very few reports on the production of polymethylgalacturonate

Review of literatures lyases (pectin lyases or PMGLs) have been reported in literature. They have been reported to be produced by Aspergillus japonicus , Penicillium paxilli , Penicillium sp., Pythium splendens Pichia pinus , Aspergillus sp. Thermoascus auratniacus .

4.5.3. Pectinesterase

PE activity is implicated in cell wall metabolism including cell growth, fruit ripening, abscission, senescence and pathogenesis. Commercially PE can be used for protecting and improving the texture and firmness of several processed fruits and vegetables as well as in the extraction and clarification of fruit juices .PE is found in plants, plant pathogenic bacteria and fungi. It has been reported in Rhodotorula sp. , Phytophthora infestans , Erwinia chrysanthemi B341, Saccharomyces cerevisiae , Lachnospira pectinoschiza , Pseudomonas solanacearum, Aspergillus niger, Lactobacillus lactis subsp. Cremoris , Penicillium frequentans , E. chrysanthemi 3604 , Penicillium occitanis , A. japonicus and others.There are many reports of occurrence of PE in plants, viz., Carica papaya , Lycopersicum esculentum , Prunus malus , Vitis vinifera , Citrus sp., Pouteria sapota, and Malpighia glabra

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4.6. Advantages of Solid-State Fermentation

For several products Solid-State Fermentation offer advantages over fermentation in liquid broths/submerged fermentation :( Cook, 1994)

· Higher product yield

· Better product quality

· Cheaper product recovers

· Cheaper technology ·

· Higher substrate concentration

· Less probability of contamination

· Lower capital investment

4.7.Disadvantages:

Despite solid-state fermentation being both economically and environmentally attractive, their biotechnological exploitation has been rather limited (Pandey, 1992; Aidoo et al., 1982)

· Limitation on microorganism

· Medium heterogeneity

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· Heat and mass transfer control growth measurement and monitoring

· Scale up problems

5. Uses of Pectinases

5.1Fruit juice industry

5.1.1 Fruit juice clarification

Addition of pectinase lowers the viscosity and causes cloud particles to aggregate to larger units (break), so easily sedimented and removed by centrifugation. Indeed pectinase preparation was known as filtration enzymes. Careful experiments with purified enzyme have shown that this effect is reached either by a combination of PE and Polygalacturonase or by PL alone in the case of apple juice, which contains highly, esterified pectin (>80%) (Ishii and Yokotsuka; 1972).

5.1.2 Enzymes treatment of pulp for juice extraction

In early periods of pectinase uses for clarification, it was found first for black currents that enzyme treatment of the pulp before pressing improved juice and color yield (Charley, 1969). Enzymatic pectin degradation yields thin free run juice and a pulp with good pressing characteristics

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(Beltman and Plinik; 1971). In case of apples it has been shown that any combination of enzymes that depolymerize highly esterified pectin (DE>90) can be successfully used (Pilnik and Voragen, 1993).

5.1.3 Liquefaction

It is process in which pulp is liquefied enzymatically so pressing is not necessary. Viscosity of stirred apple pulp decreases during treatment with pectinases, cellulase and a mixture of the two-enzyme preparation. Cellulase alone had little effect on pectin and solubilized only 22% of cellulose. Combined cellulase and pectinase activities released 80% of the polysaccharide. A similar effect has been found for grapefruit .(Pilnik and Voragen, 1993).

5.1.4. Maceration

It is the process by which the organized tissue is transformed into a suspension of intact cells resulting in pulpy products used as a base material for pulpy juices and nectars as baby foods The aim of enzyme treatment is transformation of tissue into suspension of intact cells. This process is called enzymatic maceration (The so called macerases are enzyme preparation with only Polygalacturonase or PL activity). A very interesting use of

Review of literatures enzymatic maceration is for the production of dried instant potato mash. Inactivation of endogenous PE is important for the maceration of many products (Pilnik and Voragen, 1993).

5.2 Wine industry

Pectolytic enzymes are added before fermentation of white wine musts, which are made from pressed juice without any skin contact in order to hasten clarification. Another application of Pectolytic enzymes during wine making is associated with the technology of thermovinification. During heating the grape mash to 50°C for few hours large amounts of pectin are released from the grape, this does not occur in traditional processing. It is therefore necessary to add a Pectolytic preparation to the heated mash, so that the juice viscosity is reduced. An additional benefit from the process is that the extraction of anthocyanins is enhanced, probably due to a breakdown in cell structure by the enzyme, which allows the pigments to escape more readily and thus helps in color enhancement (Tucker and Woods, 1991).

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5.3 Textile industry

In the textile industry pectinases are sometimes used in the treatment of natural fibers such as linen and ramie fibers (Baracet et al., 1991).

6. Factors controlling microbial pectinases production

6.1. PH and thermal stability of pectinases

Enzyme deactivation and stability are considered to be the major constraints in the rapid development of biotechnological processes. Stability studies also provide valuable information about structure and function of enzymes. Enhancing the stability and maintaining the desired level of activity over a long period are two important points considered for the selection and design of pectinases. The stability of pectinases is affected by both physical parameters (pH and temperature) and chemical parameters (inhibitors or activators). PH is also one of the important factors that determine the growth and morphology of microorganisms as they are sensitive to the concentration of hydrogen ions present in the medium. The optimal pH for Rhizopus arrhizus endo-PG has been found to be in the acidic range of 3.8-/6.5. Rhizopus stolonifer endo-PG was stable in the pH range 3.0 upto/5.0 and this

Review of literatures enzyme is highly specific to non-methoxylated PGA. The two PGs were stable at pH 5.0 and 7.5 and at a temperature of 50 ºC whereas two PLs exhibited maximum stability at 5.0 and 7.5 and at a temperature of 400C. It has also been reported that PL from Aspergillus fonsecaeus was stable at 5.2. This PL does not react with PGA but it does with PGA pretreated with yeast PG. The optimal pH for A. niger PMG was around 4.0. Most of the reports studied the pH and thermal stability by conventional optimization methods (i.e. the effect of temperature on pectinase stability was studied at constant pH and vice versa). The interaction effect between pH and temperature is another interesting aspect, which alters the stability differently. The combined effect of pH and temperature on stability of three pectinases, viz., PMG, PG and PL from A. niger was studied in this laboratory using response surface methodology. For this purpose, a central composite design was used and a quadratic model proposed to determine the optimal pH and temperature conditions at which pectinases exhibit maximum stability. The optimum pH and temperature were 2.2 and 23 ºC, respectively, for PMG, 4.8 and 280C, respectively, for PG and 3.9 and 29 ºC, respectively, for PL. PL was more stable than PMG and PG.

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6.2. Carbon Sources

The production of food enzymes related to the degradation of different substrates. These enzymes degrade pectin and reduce the viscosity of the solution so that it can be handled easily. Optimization of physical parameters such as pH, temperature, aeration and agitation in fermenters should be done. The different carbon sources on base as apple pectin and the pressed apple pulp stimulated the production of pectinolytic enzymes and the growth of the microorganism (dry biomass). The different carbon sources showed maximum dry biomass (d.b.) with glucose and fructose. The best carbon source on base for better production of pectinolytic enzymes was the pressed apple pulp. Biosynthesis of endo-PG and growth of the culture Aspergillus niger in relation to the carbon sources. Biosynthesis of endo-PG is induced by pectic substances and inhibited in the presence of easy metabolized monosaccharides (glucose, fructose, etc.) and some other compounds. Many results were obtained by many authors who described the use on different inexpensive carbon sources for better production of pectinolytic enzymes (Aguilar and Huitron, 1987; Maldonado et al, 1986; Hours et al., 1988; Larious et al., 1989; Leuchtenberger et al., 1989; Pericin et al., 1992; Shevchik et al., 1992;

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Hang and Woodams, 1994; Berovic and Ostroversnik, 1997; Alkorta et al., 1998; Zheng et al., 2000; Kaur and Satyanarayana, 2004; Joshi et al., 2006; Zhong-Tao et al., 2009; Tsereteli et al., 2009).

6.3.-Nitrogen sources

In most microorganisms, both inorganic and organic forms of nitrogen are metabolized to produce amino acids, nucleic acids, proteins, and cell wall components (Kumar&Takagi, 1999). Different organic and inorganic nitrogen sources: yeast extract, peptone, tryptone, glycine, urea, ammonium chloride, ammonium nitrate, ammonium sulphate, and ammonium citrate were supplemented separately. The purified enzyme retains its full activity after exposure for 1h at 60 and 700C in the presence of 0.6 and 1.8 M ammonium sulphate respectively. However in absence of ammonium sulphate enzyme looses its 60% activity at 60 ºC while 88% activity is lost at 70 ºC. At higher temperature (80–100 ºC) ammonium sulphate is not able to stabilize the activity of pectin lyase. Of the various nitrogen compounds tested for pectinase production, high pectinase activities were obtained with (NH4)2SO4, yeast extract powder, soya peptone, soybean pulp powder and the MGW.

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6.4–Temperature Incubation temperature has been found to be a significant controlling factor for enzyme production(Kitpreechavanich et al., 1984).Various optimum temperature values were reported for maximum pectinase production, maximum enzyme activity was found at 40ºC and lower activity was showed at 30 ºC by Aspergillus Niger. The optimal temperature of PL was detected at 450C. Obi and Moneke, 1985 stated that the maximum activity of their enzyme was observed at this degree. No activity was recorded after heating the enzyme over 55 ºC. A significant amount of biomass was produced by P.clavisporus at temperatures between 20 ºC and 500 C. The highest growth rates were observed at 300C. Endopolygalacturnase production was detected in cultures incubated at 20 ºC, 30 ºC, 40 ºC, 50 ºC with The highest value was attained at 30 ºC,whereas no enzyme production was observed at 10 and 60 ºC.

6.5- Incubation period

With the respect to the role of incubation period on pectinase production by microorganisms , different incubation periods were reported for maximum

Review of literatures pectinase production .The maximum pectinase activity was found at 7th day of incubation by Aspergillus niger.It means that pectinase production activity is correlated with the incubation time, which was also found from other investigations (Venugopal et al., 2007and Pereira et al., 1992).It can be noticed that the optimum time of fermentation was found to be 72 h after which there is decrease in the production of the enzyme by Aspergillus niger. Polygalacturanase production by Moniliella sp peaked between 3rd and 4th day of cultivation .when Penicillium sp was used maximal Pg activity was detected at the 8th day.

6.6- Inoculum size:

It is known that inoculum size has a marked effect on enzyme yields in fungal cultures in solid state conditions (Meyrath &Suchnex, 1972). The inoculum size of 1×10 7 ml-1 resulted in the maximum production of endo-and exo-pectinases in solid state fermentation. (Solis-Pereyra et al., 1996), with the highest level of spores (10 6 spores g-1 about a 10% decrease in the maximum activity was observed. The fact that lower inoculum sizes do not affect enzyme production is very important because large production of spores becomes

Review of literatures unnecessary. Optimum inoculum density is important consideration for SSF process since over crowding of spores can inhibit growth and development (Ghanem et al., 2000).Higher inoculum levels besides increasing spore density increase water content of the medium as well.

6.7- Surfactants:

Previous experiments on fungal cell permeability demonstrated that non-ionic surfactants (NIS, surface active agents) can stimulate the release of enzymes (Reese and Macguire, 1969). The effects of surfactants have been attributed to at least three causes: i) Action on the cell membrane causing increased permeability (Reese and Macguire, 1969). ii) promotion of the release of bound enzymes (Reese and Macguire, 1969). iii) Decrease in growth rate due to reduced oxygen supply (Hulme and Stranks, 1970). Tween 80 (a surfactant) was used to enhance the SSF rate. Addition of tween-80 into the growth medium of citrus peel enhanced pectin lyase production and maximum enzyme yield was noted in SSF medium receiving 0.2% of this surfactant. Growth media

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containing less and more than 0.2% tween-80 showed lower activities of the enzyme. Higher levels of Tween- 80 increased the penetration of water into the solid substrate matrix and increase the surface area more than the requirement of the microbe (Fujian et. aI. 2001). Tween-80 has also been shown to increase enzyme production in fungal species such as T-reesei (Mandel and Weber, 1969). The non-ionic surfactant increases extracellular protein accumulation in culture filtrates by enhancing the export of proteins or enzymes through the cell membrane.

7. Factorial Design:

A factorial design is often used by scientists wishing to understand the effect of two or more independent variables upon a single dependent variable. Factorial experiments permit researchers to study behavior under conditions in which independent variables, called in this context factors, are varied simultaneously. Thus, researchers can investigate the joint effect of two or more factors on a dependent variable. The factorial design also facilitates the study of interactions, illuminating the effects of different conditions of the experiment on the identifiable subgroups of subjects participating in the experiment (Freedman, 2005).

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Factorial ANOVA is used when we want to consider the effect of more than one factor on differences in the dependent variable. A factorial design is an experimental design in which each level of each factor is paired up or crossed with each level of every other factor. In other words each combination of the levels of the factors is included in the design (Rosenbaum, 2002).

This type of design is often depicted in a table.  Intervention studies with 2 or more categorical explanatory variables leading to a numerical outcome variable are called Factorial Designs.  A factor is simply a categorical variable with two or more values, referred to as levels.  A study in which there are 3 factors with 2 levels is called a 2³ factorial Design.  If blocking has been used it is counted as one of the factors.  Blocking helps to improve precision by raising homogeneity of response among the subjects comprising the block. Advantages of factorial Designs are:

 A greater precision can be obtained in estimating the overall main factor effects.

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 Interaction between different factors can be explored.  Additional factors can help to extend validity of conclusions derived.  Procedure used is General Linear Modelling. To determine the effects of different factors (yeast extract, incubation period, inoculum size, pH, temperature) on the production of pectinase enzymes by Penicillium citrinum. Thus we have a study with 5 factors and 2 levels – a 2 Factorial Design

8. Gamma Rays

Radiation is energy in the form of waves (beams) or particles. Radiation waves are generally invisible, have no weight or odor, and have no positive or negative charge. Radioactive particles are also invisible, but they have weight (which is why they are called a particle) and may have a positive or negative charge. Some radiation waves can be seen and felt (such as light or heat), while others (such as x rays) can only be detected with special instrumentation. Gamma rays, alpha particles, and beta particles are ionizing radiation. Ionizing radiation has a lot of energy that gives it the ability to cause changes in atoms—a process called ionization. Radio and TV signals, microwaves, and laser light are non-ionizing types of

Review of literatures radiation. Non-ionizing radiation has less energy than ionizing radiation. When non-ionizing radiation interacts with atoms, it does not cause ionization (hence non- ionizing or not ionizing) (Taflove and Hagness, 2005).

Gamma and X rays (also called photons) are waves of energy that travel at the speed of light. These waves can have considerable range in air and have greater penetrating power (can travel farther) than either alpha or beta particles. X rays and gamma rays differ from one another because they come from different locations in an atom. Gamma rays come from the nucleus of an atom while Xrays come from the electron shells. Even though X rays are emitted by some radioactive materials, they are more commonly generated by machines used in medicine and industry. Gamma and x rays are both generally blocked by various thicknesses of lead or other heavy materials. Examples of common radionuclides that emit gamma rays are technetium-99m (pronounced tech-neesh-e-um, the most commonly used radioactive material in nuclear medicine), iodine-125, iodine-131, cobalt-57, and cesium- 137 (Tipler and Paul, 2004).

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8.1. Ionizing radiation

Ionizing radiation is energy transmitted via X-rays, γ-rays, beta particles (high speed electrons), alpha particles, neutrons, protons, and other heavy ions such as the nuclei of argon, nitrogen, carbon, and other elements. This energy of ionizing radiation can knock electrons out of molecules with which they interact, thus creating ions. X rays and gamma rays are electromagnetic waves like light, but their energy is much higher than that of light (their wavelengths are much shorter). The other forms of radiation particles are either negatively charged (electrons), positively charged (protons, alpha rays, and other heavy ions) or electrically neutral (neutrons).

8.2. Responses of pectinases to gamma radiation

It has been found that at low doses of gamma radiation the pectinase enzyme was slightly increased as this is owed to the induction of gene transcriptions or proteins has been found after low dose effects until it reached to high doses the enzyme activity was obviously decreased and further inhibited, this may be due to the absorbed dose caused rupturing in the cell membrane .This major injury to the cell allows the extracellular fluids to

Review of literatures enter into the cell. Inversely, it also allows leakage out of ions and nutrients which the cell brought inside. Membrane rupture may result in the death of a cell.

9. Purification of microbial pectinases

Purification of microbial pectinases received a great attention particularly in recent years. In general, the purification procedures included several steps, the major steps include: precipitation of the enzyme, application on different chromatographic columns using ion exchange or gel filtration chromatography, and in many cases performing polyacrylamide gel electrophoresis technique (PAGE), high performance liquid chromatographic technique (HPLC) and the electrofocusing technique. Ammonium sulphate widely used for enzyme precipitation, since (i) it has a high solubility in water, (ii) characterized by the absence of any harmful effect on most enzymes, (iii) has stabilizing action on most enzymes and (iv) it is usually not necessary to carry out the fractionation at low temperature (Dixon & Webb, 1964). Many chromatographs were applied in the purification of the enzyme. For example, Penicillium sp pectinase was partially purified with sephadex G-100 column (Patil and Chaudhari, 2010). Furthermore, the endo-

Review of literatures polygalacturonases isolated from Penicillum oxalicum was purified using Sephadex G-100 Gel Filtration (Chun-hui et al., 2009).

10. Applications of pectinases

Over the years, pectinases have been used in several conventional industrial processes, such as textile, plant fiber processing, tea, coffee, oil extraction, treatment of industrial wastewater, containing pectinacious material, etc. They have also been reported to work in making of paper. They are yet to be commercialized.

Materials and Methods

3-Materials and Methods

3.1-Microorganisms Fungal strains were provided from Pharmaceutical Microbiology Lab, Drug Radiation Research Department (NCRRT) Nasr City-Cairo-Egypt. Fungal colonies were maintained on potato-dextrose agar medium, stored at 4ºC and freshly subcultured every four weeks.The strains included (Alternaria alternata, Aspergillus niger 1, Aspergillus niger 2, Aspergillus niger 3, Aspergillus niger 4, Aspergillus oryzae, Gliocladium vierns, Penicillium brevi- compactum, Penicillium chrysogenum, Penicillium citrinum, Pleurotus ostreatus, Rhizoctonia solani ).

3.2.Culture media:

3.2.1.Potato-dextrose agar médium

According to Ricker and Ricker (1936) this medium was used for isolation and maintenance of the fungal strains and it has the following composition (g/ l): Potato (peeled and sliced) 200 g

Dextrose 20 g

Agar 17 -20 g

Materials and Methods

Distilled water 1000ml pH 7.0

3.3. Fermentation substrates

The sugar beet pulp (SBP) used as a carbon source has the following composition (% on dry basis): pectin, 28.7; cellulose, 20.0; hemicellulose, 17.5; protein, 9.0; lignin, 4.4; fat, 1.2; ash, 5.1 (Xue et al., 1992). The high pectin content could be very helpful for pectinase production.

4. Culture condition The used fermentation has the following contents: Ten grams of sugar beet pulp (SBP) were placed in flasks and moistened with 20ml of distilled water containing (0.4g Na2HPO4+ 0.08g KH2PO4+ 0.4g yeast extract) and autoclaved for 30 min, pH has been adjusted to 5.9 using HCl and NaOH.

4.1. pH adjustment (Sodium acetate-acetic acid buffer solution, pH 5.9)

 Sodium acetate trihydrate powder (24.7 gram) was solubilized in 910 ml distilled water.

Materials and Methods

 Glacial acetic acid (1.2ml) has been mixed in 100ml of distilled water.  Ninety ml were taken from the previous step and mixed with the first step.

5. Screening for pectinolytic enzymes using Sugar beet pulp medium:

The tested fungi have been maintained on potato glucose agar slants and kept in the refrigerator, and subcultured monthly. The solid state fermentation medium was mixed and inoculated with 1.8 × 105 spores per gram of wet substrate. The flasks were placed in a humid cultivation chamber with a gentle circulation of air at 30 °C, under static conditions for 7 days. Triplicate flasks were used for each fungal species, and the end of incubation period, the crude pectinase was extracted using the following procedure:

Five grams of the fermented materials were mixed with 50 ml of sodium acetate buffer and shacked for 1 hour, then squeezed , filtered through a cloth filter,and stored at 40C till measuring its pectinolytic activity. The polygalacturonase and pectin lyase activities were taken as a measure to the pectinolytic enzymes.

Materials and Methods

The activity of the polygalacturonase (PGase) was assayed by measuring the reducing groups released from polygalacturonic acid using the 3, 5-dinitrosalicylic acid method with glucose as the standard. One unit of PGase activity was defined as that amount of enzyme which would yield 1 µmol reducing units per minute.

6. Analytical methods:

6.1. Pectinases assay:

6.1.1. Assay for pectinases (polygalacturonase) activity in the cell –free filtrate:

6.1.1.1Reagents:

1) 3,5-Dinitrosalicylic acid (DNS)

One g DNS dissolved by warming in 20 ml (2 N NaOH). Thirty g Pot. Sod. tartarate dissolved by warming in 50 ml distilled water. After cooling , the two solutions combined together and make up to 100 ml with distilled water.

2) 1 % pectin solution

1- One hundred of sodium acetate buffer solution were taken and then warmed in a water bath.

Materials and Methods

2- One gram of pectin powder was added slowly to the buffer solution on the stirrer until it was homogenous.

3) 1g / 10ml of standard glucose

1- One gm of glucose powder was dissolved in 10 ml distilled water. 6.1.1.2. Procedure The assay was carried out using 0.25 ml of 1% pectin; 0.25 ml of culture filtrate .The resulting mixture was incubated at 50 º C for 10 minutes. Polygalacturonase activity was measured by quantifying the amount of reducing sugar groups which had been liberated after incubation with pectin solution, using the method of Miller, (1959). 0.5 ml 3, 5 –Dinitrosalisyclic acid DNS and 0.5 ml of reaction mixture were placed in a test tube and boiled for 5 min, used glucose as a standard. The enzyme activity (U/gdfs) was calculated as the amount of enzyme required to release one micromole (1μmol) equivalent of galactouronic acid per minute.

The absorbance has been measured at 540 nm; determinations were carried out in triplicates.

Materials and Methods

6.2. Assay for pectin lyase

PL activity was determined by measuring the increase in absorbance at 235 nm of the substrate solution (2 ml of 0.5% citric pectin in 0.1 M citrate-phosphate buffer, pH 5.6) hydrolysed by 0.1ml of the crude enzymatic extract, at 25°C for 2 minutes. One enzymatic unit (U) was defined as the amount of enzyme which liberates 1 μmol of unsaturated uronide per minute, based on the molar -1 -1 extinction coefficient (ε235 = 5550 M cm ) of the unsaturated products (Albershein, 1966; Uenojo and Pastore, 2006). The enzymatic activity was expressed in U/g.

6.3. Protein determination The protein content of the crude enzyme was determined by the method of Lowry et al., (1951) using Bovine Serum Albumin (BSA) as the standard.

6.4. Statistical analysis: Statistical analysis of data was carried out by using one way analysis of variance (ANOVA) Followed by homogenous subsets (Duncun) at confidence levels of 5% using the Statistical Package for the Social Science (SPSS) version 11.

Materials and Methods

7. Optimization of parameters controlling polygalacturonases production by P.citrinum. Penicillium citrinum has been chosen for further studies. Factors such as temperature, pH, incubation period, and others may affect polygalacturonases production. So, the effect of such factors was investigated to determine the optimum conditions for the enzyme production.

7.1. Effect of different natural products: Conical flasks (250 ml) each contain 20 ml of the production medium ,autoclaved for 20 minutes, after cooling ,the flasks were inoculated with 1ml of spore suspension (1.8 ×105 ) and incubated at 25 ºC with different raw materials ( 10g Sugar beet pulp ,5g sugar beet pulp +5g wheat bran , 10g wheat bran , 5g sugar beet pulp +5g banana ,10g banana, 5g sugar beet pulp + 5g vicia faba , 10g vicia faba ) for 7days. At the end of incubation period, samples were collected, extracted and centrifugated respectively.The filtrates, used as the crude enzyme extract, were analyzed for enzyme activity to determine the optimum natural nutrient.

Materials and Methods

7.2. Effect of different nitrogen sources: The effect of different nitrogen sources on polygalacturonases production was carried out by supplementing the production media with equimolecular amount of nitrogen at concentration of (0.04 g/ g dry SBP) for each nitrogen source. Inorganic nitrogen sources such as (NH4)2 HPO4, NH4NO3, and NaNO3 were investigated. Organic nitrogen sources such as urea, yeast extract, peptone, tryptone, and malt extract were also tested. All culture conditions which obtained in the previous experiments were adjusted. Samples were collected and analyzed as mentioned.

7.3. Effect of different inoculum sizes: Different concentrations of spore suspension of the highest producer fungus were used. The following concentrations were applied viz., 1.8, 3.6, 5.4 ×105 spores/ ml and 9×104 spores/ml per each flask (250 ml). At the end of incubation period, polygalacturonase activity was determined for each concentration after incubation period as previously mentioned.

7.4. Effect of different incubation periods: Conical flasks (250 ml) each contain 20 ml of the production medium, autoclaved for 20 minutes, after

Materials and Methods cooling, the flasks were inoculated with 1 ml of spore suspension (1.8×105) and incubated at 25 ºC at different incubation periods (2, 3, 4, 5, 6, 7, 8, 9, and 10 days) at the end of incubation periods, samples were collected, extracted, and centrifuged respectively .The filtrates were used as the crude enzyme extract and analyzed for enzyme activity and protein content to determine the optimum incubation period.

7.5. Effect of different pH values:

This experiment was carried out by dissolving the component of the production medium in different pH buffer solutions. pH values from 3 to 7.5 were examined using

Citric acid-Na2HPO4 buffer solutions. Previous optimized conditions were adjusted; samples were collected and analyzed as mentioned.

7.6. Effect of different temperatures:

Flasks containing 20 ml of sterilized production medium were inoculated with 1 ml spore suspension .The flasks were then incubated at different temperatures (20, 25, 30, 35, and 400C). At the end of the incubation period, the cell free filtrates were used to investigate the enzyme activity.

Materials and Methods

7.7. Effect of different surfactants:

This experiment carried out to investigate the production of polygalacturonases in the presence of some surfactants. Production media was supplemented with different surfactants ( Tween 40 , olive oil , Tween 60 , Tween 80 , soybean oil , sunflower oil , Tween 20 , maize oil , and triton x 100 ( 0.1%) .All surfactants were tested for their induction or inhibitory effect on polygalacturonases production compared to the control which carried out without surfactant addition. Production process with all the above mentioned conditions was carried out to detect the best conditions for yield improvement. Samples were collected and analyzed as usual.

7.8. Application of factorial design for optimization of polygalacturonase production by P.citrinum under Solid state fermentation.

A full factorial two-level design(25) was performed to confirm the optimization of independent factors level by taking incubation period (7 and 8 days), pH (5.0 and 5.5), inoculum size (1.8×105and 3.6×105 spores/ml) ,temperature (25 and 30ºC) and nitrogen content(0.5% and 1.2%) in this study. The level of independent factors were optimized by studying each factor in the design at two different levels(-1, and +1),Table 12).The minimum[coded as(-1)] and

Materials and Methods maximum [coded as(+1)] range of experimental values of each factor used. A set of 32 experiments was performed. The quality of fitting the first-order model was expressed by the coefficient of determination R2 and its statistical significance was determined by F-test. The sugar beet pulp had been used as the sole carbon source.

7.9. Effect of different gamma irradiation doses: All irradiation processes were carried out at the National Center for Radiation Research and Technology (NCRRT) Nasr City-Cairo-Egypt. Irradiation facility was Co-60 Gamma chamber 4000-A India. The source gave average dose rate 3.696 kGy/hr during the period of samples radiation. The fungal strain was grown on PDA for 8days and subjected to gamma radiation at doses (0.1, 0.2, 0.5, 0.7, 1, 1.5 and 2 kGy). The tested cultures have been investigated for its enzyme activity.

8. Purification of polygalacturonases:

8.1. Production of polygalacturonase and preparation of cell-free filtrate:

Fungal cultures were grown in conical flasks of 250ml capacity on the optimized medium and incubated at the optimum temperature. At the end of incubation period, the supernatant (500 ml) was harvested by extraction,

Materials and Methods followed by centrifugation at 5,000rpm for 15 minutes at 40C and the supernatant was used as crude enzyme extract.

8.2. Ammonium sulphate precipitation: The cell free filtrate was brought to 75% saturation by mixing with ammonium sulphate slowly with gentle agitation and allowed to stand for 24 hrs at 4ºC. After the equilibration, the precipitate was removed by centrifugation (5000 rpm at 4°C for 15 min).The obtained precipitate has been dissolved in 50ml of 0.2M sodium acetate buffer, pH (5.9) to be dialyzed.

8.2.1. Steps for precipitation by ammonium sulphate: 1- Crude extract was poured in to a beaker with a magnetic bar in it. Beaker volume was chosen 2.5-3 times larger than the volume of the sample. 2- The beaker was placed on the stirrer to mix solution with a speed which allowed a vortex to form in the middle of the sample. 3- The amount of ammonium sulphate powder that needed to precipitate the protein was determined and weighed then added to the sample (with stirring) in small portions. 4- Stirrer was turned off, when all salts had dissolved and sample was left for 24 hrs at 4°C.

Materials and Methods

5- Pellets were collected by centrifugation for 20 minutes at 5000 rpm at 4°C then dissolved in the appropriate buffer. 8.3. Dialysis:

According to Karthik et al., (2011), the precipitate was desalted by dialysis by the following protocol: 10cm dialysis bag was taken and activated by rinsing in distilled water. One end of the dialysis bag is tightly tied and the obtained precipitate is placed into the bag. Then the other end of the dialysis bag is tightly tied to prevent any leakage. After that, dialysis bag has been suspended in a beaker containing 0.2M sodium- acetate buffer (pH 5.5) to remove low molecular weight substances and other ions that interfere with the enzyme activity.

8.4. Gel filtration chromatography (Wilson and Walker, 1995):-

8.4.1:- Packing of the column:- (a)- 10 grams of sephadex G-75 (sigma) was weighed and added into 500 ml acetate buffer (0.5 M, pH6) and allowed to swell for at least 3 days in the fridge.

(b)- Degassing process was carried out by placing the beaker containing the matrix ( Sephadex G-75 ) into

Materials and Methods boiling water bath for several hours with occasional gentle knock on the beaker wall (to get rid of air bubbles).

(c) The gel was allowed to cool to the room temperature then packed in the column by pouring carefully down the walls of the column (2.2 cm × 65 cm).

-The column tap must be kept open during the bed settling to allow the formation of one continuous bed also the bed must not to be allowed to precipitate so that when more gel is poured it will not lead to the formation of 2 beds over each others.

-The bed which was formed was 2.2 × 45 cm.

(d) The sorbent was allowed to reach the equilibrium by passing 2 column volume of the used buffer before the application of the sample.

The column was connected to the buffer reservoir and the flow rate of the buffer was maintained at a constant rate of approximately 5 ml per 7.5 min.

8-4-2:-loading of the sample:- 3-7 ml of the enzyme sample was applied carefully to the top of the gel.

Materials and Methods

8-4-3:-Fractionation:- The protein band was allowed to pass through the gel by running the column. Forty fractions each of 5 ml were collected and separately tested for both the protein content (at 280 nm) and for the pectinase activity. The active fractions that have the highest pectinase activity were collected together and concentrated by dialysis against sucrose, then tested for pectinase activity and protein content. This concentrated partially purified enzyme solution was stored in the refrigerator and used for the further characterization and application study.

8.4.4. Calculation of specific activity, purification fold and yield of the enzyme;

Specific activity (U/mg): Activity of the enzyme (U) / Amount of protein (mg)

Yield of enzyme (%): Activity of fraction / activity of crude enzyme ×100

Purification fold: Specific activity of the fraction / specific activity of the crude enzyme.

Materials and Methods

9. Characterization of the partially purified polygalacturonase enzyme: Several factors have been studied to investigate their effects on the partially purified enzyme activity.

9.1. Effect of different pH values

9.1.1. On the enzyme activity

The activity of PGase was determined in the presence of different buffers using sodium acetate buffer (pH 4.0, 5.0), sodium citrate buffer (pH 6.0, 7.0), and sodium phosphate buffer (pH 8.0).The relative activities were based on the ratio of the activity obtained at certain pH to the maximum activity obtained at that range and expressed as percentage.

9.1.2. On the enzyme stability

The pH stability of the enzyme was determined by exposing the purified enzyme first to various pH values (4 to 8) using the different pH buffer solutions mentioned above for a period of 2 hours. Afterwards, aliquots of the mixtures were taken to measure the residual polygalacturonase activity (%) with respect to the control, under standard assay conditions.

Materials and Methods

9.3. Effect of different temperatures on the enzyme

9.3.1. On the enzyme activity

The optimum temperature was determined by incubating each reaction mixture at variable temperatures (20-70ºC). The relative activities (as percentages) were expressed as the ratio of the purified polygalacturonase obtained activity at certain temperature to the maximum activity obtained at the given temperature range.

9.3.2. On the enzyme stability

Thermal stability of the enzyme was investigated by measuring the residual activity after incubating the enzyme at various temperatures ranging from 20 to 70°C for 30 min.

9.4. Effect of different metal ions on the activity of the purified polygalacturonase enzyme produced by P.citrinum.

+2 For determination the influence of Ca , EDTA, +2 +2 +2 +2 +2 Cu , Zn , Mg , Ba and Co on PGase activity. The

Materials and Methods

listed ions were added to the reaction mixture at concentration (1mM). Activity without added metal ions was taken as 100% activity.

10. Bioextraction of pectin from different agro-residues for different pharmaceutical applications

P.citrinum was cultivated in 50ml aliquots/250ml Erlenmeyer flasks of the following media containing any of the different wastes :Sugar beet pulp 10% ,Orange peel waste 10%,and Banana peel waste 10%, yeast extract 1% , pH 6, and inoculated with 1ml of spore suspension (about 1.8×105 spores/ml) incubated at 30°C for 8 days under static conditions. These favored maximum pectin bioextraction. At the end of fermentation time, the filtrate was separated by centrifugation at 4000 rpm for 20 min and poured in 3 volumes of ethanol. The precipitated pectin was collected by centrifugation , washed with ethanol, dried under vaccum at 37°C and then weighed accurately(Kabil and Al-Garni, 2006) .

Results

4-Results

4.1.Screening of the most potent fungal pectinase producer:

The results showed that Penicillia were the most potent among the tested genera for enzyme production (124.6) among the tested genera, followed by Sclerotium rolfsii (115.7), then Aspergillus niger and Pleurotus ostreatus (102.4). The least enzyme production was detected in case of Trichoderma viride (62.1). Among Penicillia ;Penicillium citrinum was the most potent in the production of pectinase (129U/gdfs, so it has been chosen for further studies.

4.1.1. Polygalacturonase activity

It has been found that polygalacturonase enzyme is the most potent type in the cell free filtrate by using 3,5- Dinitrosalisyclic acid DNS (Miller ,1959).

Results

Table (3): Polygalacturonase production by the tested fungal species under solid state fermentation.

Fungal strains Polygalacturonase Pectin lyase activity(U/gdfs*) activity(U/gdfs)

Alternaria alternata 86.2±2

Aspergillus niger 1 86.2±2.2

Aspergillus niger 2 115.3±1.9

Aspergillus niger 3 92.3±1.1

Aspergillus niger 4 96.3±1.05

Aspergillus oryzae 96.8±1.9 Not detected for all tested fungal Gliocladium vierns 95.7±2.1 species. Penicillium brevi-compactum 123.2±2.2

Penicillium chrysogenum 121.4±1.14

Penicillium citrinum 129.2±2

Pleurotus ostreatus 102.4±2.1

Rhizoctonia solani 83.1±2

Scleortium rolfsii 115.7±1.9

Trichoderma viride 62.1±2.1

*- gdfs: Units of pectinase per gram dry fermented substrate

Results

Fig (3): polygalacturonases production by the tested fungal species grown under solid state conditions.

4.1.2. Pectin lyase assay

Pectin lyase enzyme was not detected in the filtrates of the investigated fungal species.

Results

4.2- Optimization of the fermentation parameters affecting enzyme production.

4.2.1. Effect of some agroindustrial by-products as carbon source on polygalacturonase production by P.citrinum under Solid state fermentation.

The production medium was inoculated with 1 ml of spore suspension (1.8×105 spores/ml) which prepared in Tween 80, 0.1% v/v. The growth medium was supplemented with different carbon sources at concentration of ten gram for each treatment (sugar beet pulp,sugar beet pulp+wheat bran, wheatbran, sugarbeetpulp + banana , sugar beet pulp + broad beans, broad beans) . All culture conditions which obtained in the previous experiments were applied during the present investigation. The results in table (4) showed that the maximum enzyme production was achieved when the medium was supplemented with sugar beet pulp giving activity of (126.2 U/gds), while the addition of other agro by-products gave lower enzyme production except for sugar beet pulp +wheat bran (112.2 U/gds) .There was a significant difference

Results

between all tested by-products .Wheat bran exhibited enzyme activity of 107.02 U/gds. Beans gave the activity of 83.06 U/gds.

Table (4): Effect of some agroindustrial by- products as carbon source on polygalacturonase production by P.citrinum under solid state fermentation. Carbon source Enzyme activity(U/gdfs)

Sugar beet pulp 126.2± 2 a

Sugar beet pulp +wheat 112.2± 1.9 b bran

Wheat bran 107.02± 2.2 c

Sugar beet pulp +banana 100.2± 2 d

Sugar beet pulp + beans 95.1± 1.9 e

Beans 83.06± 1.9 f

Banana 73.02±1.2g

*- gdfs: Units of pectinase per gram dry fermented substrate

* Groups with different letters have significant difference between each other .

Results

Fig (4): Effect of different agroindustrial by products as carbon sources on polygalacturonase production by P.citrinum.

4.2.2. Effect of different nitrogen sources on polygalacturonase production using Penicillium citrinum under Solid state fermentation.

Nitrogen sources were supplemented in the production medium with equimolecular amount of nitrogen from different nitrogen sources (Yeast extract, Malt extract, Urea, Peptone, Ammonium sulfate, Tryptone, Ammonium nitrate, Sodium nitrate). All culture conditions were

Results

adjusted according to the optimum condition determined in the previous experiments. The results showed that the yeast extract was the best nitrogen source in inducing enzyme production (129.2 U/gdfs). Ammonium sulphate as inorganic nitrogen source was also effective in the induction of pectinases production (120.1U/gdfs) but less than the activity produced in the presence of yeast extract as a complex nitrogen source .All other nitrogen sources including organic and inorganic sources produced lower levels of polygalacturonases compared to the medium containing the yeast extract.

Results

Table (5): Effect of different nitrogen sources on polygalacturonase production using Penicillium citrinum under Solid state fermentation.

Nitrogen sources Enzyme activity(U/gdfs)

Yeast extract 129.2± 1.9 a

Malt extract 93.2± 1.7 b

Urea 83.1± 1.8 c

Peptone 89.1± 2.2 d

Ammonium sulfate 120.1± 2e

Tryptone 114.2± 1.8 f

Ammonium nitrate 99.1± 2.2 b

Sodium nitrate 95.2± 1.8 b

*-gdfs: Units pectinase per gram dry fermented substrate

* Groups with different letters have significant between each other.

Results

Fig (5): Effect of different nitrogen sources on polygalacturonases production using P.citrinum under solid state fermentation.

4.2.3. Effect of inoculum size on polygalacturonases production by P.citrinum under solid state fermentation.

It is known that inoculum size has a marked effect on enzyme yields in fungal cultures in solid state conditions (Meyrath& Suchanex, 1972).The results showed that maximum polygalacturonase production took place using inoculum size of (1.8×105spores/ml) for solid state fermentation, but decrease subsequently with the increase in the inoculum size . Interestingly with the increase in the inoculum sizes, the enzyme production has been reduced

Results

rather drastically in the SSF. Apparently the conditions of the fermentation were adjusted according to the optimum conditions determined in the previous experiments.

Table (6): Effect of inoculum size on polygalacturonase production by P.citrinum under solid state fermentation.

Inoculum size Enzyme activity (Spores/ml) (U/gdfs)

9×104 81.2 ± 1.9 d

5.4×105 95.1 ± 1.8 c

3.6×105 115.1±1.9b

1.8×105 127.2±2a

* -gdfs:Units pectinase per gram dry fermented substrate.

*-Groups with different letters have siginificant between each other.

Results

Fig. (6): Effect of inoculum size on polygalacturonase production by P.citrinum under solid state fermentation.

4.2.4. Effect of different incubation periods on polygalacturonase enzyme production by Penicillium citrinum

The results represented in Table (7) and fig. (7) showed that P. citrinum started pectinases production from the second day of incubation period with enzyme activity (78.3U/gds), then started to increase significantly as the incubation period increased and reached its maximum activity in the seventh day of the incubation (129.2U/gds). Longer incubation period resulted in a significance decrease of the enzyme activity, especially in

Results

10 days of incubation (94.2U/gdfs).

Table (7): Effect of different incubation periods on production of the polygalacturonase enzyme by Penicillium citrinum

Incubation period(Days) Enzyme activity(U/gdfs) 2 78.3±2.3a

3 95.2±1.8b

4 98±2.2 b

5 108.2±1.9c

6 114.1±2.3d

7 129.2±2.2e

8 128.01±1.8 e

9 100.2±2c

10 94.2±2 b

Groups with same letters are non significant with each other.

Groups with different letters are significant with each other

Results

Fig (7): Effect of different incubation periods on polygalacturonase production by P.citrinum

4.2.5.Effect of different pH values on polygalacturonases production by P.citrinum under solid state fermentation.

Penicillium citrinum was allowed to grow at different pH values(3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5) under the conditions of the fermentation which adjusted according to the optimum condition determined in the previous experiments. The results in table (8) and fig. (8) showed that the fungal cultures were able to produce pectinases at all tested pH values but, it was obvious that at low pH range (3- 4.5), the production was low and the determined activities were (80.2, 87, 98.1, 100.9U/gds

Results

respectively) then began to increase gradually to reach its maximum production at pH range (5- 6) .The maximum activity was (126.1U/gds) at pH 5.5 , then the activity significantly decreased at pH range ( 6.0 -7.5) with the least recorded activity (90.5U/gds) was at pH 7.5 .

Table (8): Effect of different pH values on polygalacturonases production by P.citrinum under solid state fermentation .

pH Specific activity(U/gdfs)

3 80.2±2a

3.5 87±1.9b

4 98.1±1.8c

4.5 100.9±2.2c

5 114.2±2.1 d

5.5 126.1±1.8e

6 114±1.8 d

6.5 112.3±2.1 d

7 95.2±1.1f

7.5 90.5±2.0g

*-gdfs: Units pectinase per gram dry fermented substrate.

* Groups with different letters have significant difference between each other.

Results

Fig (8): Effect of different pH values on polygalacturonases production by P.citrinum

4.2. 6. Effect of different incubation temperatures on polygalacturonase production by P.citrinum under solid state fermentation.

The temperature is one of the major factors affecting the process of pectinases production under solid state fermentation. Results in Table (9) and fig. (9) showed that pectinases production started at 20 ºC with activity (100U/gds). It increased gradually by the rise in incubation temperature and reached its maximum activity at 25 ºC

Results

(127.3U/gds). The activity started to decrease with the increase in the incubation temperature and reached its minimal value at 40 ºC (82.3U/gds).

Table (9): Effect of different incubation temperatures on polygalacturonase production by Penicillium citrinum

Temperature(ºC) Enzyme activity(U/gdfs)

20 ºC 100± 2 d

25 ºC 127.1± 1.8 a

30 ºC 120.4± 2 d

35 ºC 92.3 ± 2.2 b

40 ºC 82.6 ± 2 c

*-gdfs: Units pectinase per gram dry fermented substrate.

* Groups with different letters have significant difference between each other.

Results

Fig (9): Effect of different incubation temperatures on polygalacturonase production by Penicillium citrinum

4.2.7. Effect of some surfactants on polygalacturonase production by P.citrinum:

Table (10) and fig. (10) showed the influence of different surfactants on pectinase production. Highest level of pectinase production has been obtained by the addition of Tween 40 (0.1%) to the culture medium (140.1 U/gds). While no effect on polygalacturonase production was observed upon using Triton X-100, Sunflower oil, Maize oil, Soybean oil, Olive oil, and Tween 80.Tween 20&60 produced polygalacturonases in a level similar to that of the control without surfactants. The lowest level of

Results

polygalacturonase has been observed when soybean oil was added to the fermentation medium (92.2U/gdfs).

Table (10): Effect of some surfactants on polygalacturonase production by P. citrinum under solid state fermentation.

surfactants Specific activity (U/gdfs)

Control 123.1 ± 2.07 a

Tween 40 140.1 ± 2.2 b

Tween 20 126.1 ± 1.9 a

Tween 60 128 ± 1.9 a

Tween 80 107.2 ± 2c

Olive oil 110.9 ± 2.3 d

Soybean oil 92.2 ± 2 e

Maize oil 104.2 ± 1.9 c

Sunflower oil 116.9± 2 f

Triton x100 115.2 ± 2.1 f

*-gdfs: Units pectinase per gram dry fermented substrate.

* Groups with different letters have significant difference between each other.

Results

Fig (10): Effect of some surfactants on polygalacturonase production by P.citrinum. 4.2.8. Application of factorial design for optimization of polygalacturonase production by P.citrinum under Solid state fermentation.

A factorial design has been applied to optimize polygalacturonase production by P.citrinum. Factorial design was used to study the effect of 5 variables (yeast extract, pH, Inoculum size, Incubation period, and Incubation temperature) on enzyme production. Only yeast extract, Inoculum size, and Incubation temperature had significant effect on pectinase production under the

Results

conditions of the assay, the interaction between them not being significant. So, a design of a total 32 experiments was generated, and Table (11) lists the high and low levels of each variable. The 32 experiments were carried out in triplicate. Table (11), (12) show the effect of each variable and its interactions on the enzyme production. As can be seen , high polygalacturonase production was obtained by using one gram of yeast extract in the fermentation medium incubated at 30ºC for 8 days at pH 5.5 ( 132 U/gds). Experimentally the obtained PGs yield is 132U/gds .A high degree of correlation between the experimental and predicted values of the exopolygalacturonase production was expressed by a high R2 value of 74 % (Fig. 12).

Results

Table (11) : Effect of the variables and their interactions in the 25 factorial designs for optimization of polygalacturonase production by P.citrinum under solid state fermentation.

Trials (Enzyme Factors production( U/gdfs*) N Incubation Inoculum pH Temperat content% period(day) size(spores/ml) -ure

(ºC)

1 86.6 - + + - +

2 103.7 + + + - +

3 113.6 - - + - +

4 70.3 + - + - +

5 100.8 - + - - +

6 111.5 + + - - +

7 65.9 - - - - +

8 119.4 + - - - +

9 60.9 - + + + +

10 73.5 + + + + +

11 55.6 - - + + +

12 122.4 + - + + +

13 88.9 - + - + +

14 132.0 + + - + +

15 81.9 - - - + +

Results

16 94.8 + - - + +

17 58.2 - + + - -

18 44.7 + + + + -

19 40.5 - - + - -

20 50.1 + - + - -

21 62.1 - + - - -

22 78.4 + + - - -

23 84.5 - - - - -

24 91.9 + - - - -

25 64.0 - + + + -

26 38.7 + + + + -

27 30.4 - - + + -

28 33.1 + - + + -

29 48.8 - + - + -

30 127.2 + + - + -

31 68.6 - - - + -

32 97.8 + - - - -

*U/gdfs: unit/gram dry fermented substrat

Results

Fig (11): Factorial design for the effects of yeast extract and inoculums size at pH5.5 on the polygalacturonase activity of Penicillium citrinum .One unit (U) of pectinase activity was defined as the amount of the enzyme, which catalysed the formation of 1 µmol of galacturonic acid per hour at 30ºC. Table (12). ANOVA table for the enzyme activity: effect of inoculums size, yeast extract and temperature on the activity of PGase.

Term Estimate Std Error t Ratio Prob>|t|

Intercept 78.552734 3.822781 20.55 <.0001*

Yeast extract(0.4,1) 8.1972656 3.822781 2.14 0.0488*

Incubation period(7,8) 2.3464844 3.822781 0.61 0.5485

Inoculm size(1.8,3.6) -12.25977 3.822781 -3.21 0.0059*

pH(5,5.5) -2.108984 3.822781 -0.55 0.5893

Temp(25,30) 14.958984 3.822781 3.91 0.0014*

Results

Yeast extract*Incubation period -0.383984 3.822781 -0.10 0.9213

Yeast extract*Inoculm size -7.427734 3.822781 -1.94 0.0710

Incubation period*Inoculm size -0.553516 3.822781 -0.14 0.8868

Yeast extract*pH 5.8589844 3.822781 1.53 0.1462

Incubation period*pH 1.2097656 3.822781 0.32 0.7560

Inoculm size*pH -3.608984 3.822781 -0.94 0.3601

Yeast extract*Temp 1.7410156 3.822781 0.46 0.6553

Incubation period*Temp 0.6777344 3.822781 0.18 0.8617

Inoculm size*Temp 6.3714844 3.822781 1.67 0.1163

pH*Temp -2.652734 3.822781 -0.69 0.4983

Fig (12): Plot of predicted versus actual polygalacturonase production

Results

4.2.9. Effect of Gamma irradiation doses on polygalacturonase production by P. citrinum under solid state fermentation using optimized conditions of factorial design:

Penicillium citrinum fungal spores were irradiated with increasing doses of gamma–rays, and then used for regular experiment for polygalacturonase production in sugar beet pulp solid medium. Data clearly indicated that maximum polygalacturonase production was observed when spores were irradiated at 0.7 KGy with an activity 152.2 U/gds as compared to the wild strain. Higher doses than 1kGy produced significant decrease in polygalacturonase activity (Table.13).

Results

Table (13): Effect of Radiation Dose on polygalacturonase production using Penicillium citrinum.

Radiation dose Enzyme activity (kGy) (U/gds)

Control (unirradiated) 132±1.9a

0.1 137.8±2.1b

0.2 142.2±1.3c

0.5 145.5±2.1d

0.7 152.2±2.2e

1 100.2±2.3f

1.5 95.5±2 g

2.0 N.D

*-gds: Units of pectinase per gram dry fermented substrate.

* Groups with different letters have significant between each other.

*N.D: not determined.

Results

Fig (13): Effect of different doses of gamma Radiation on polygalacturonase production using Penicillium citrinum.

4.3. Purification and characterization of the enzyme:

4.3.1. Purification steps:

Polygalacturonase produced by P.citrinum was purified using ammonium sulfate precipitation, and then underwent dialysis and gel filtration. Results observed in Table (14), indicate a decrease in total protein and total activity, whereas specific activity increased. Ammonium sulphate precipitation (salting out) is useful for concentrating dilute solutions of proteins. The ammonium- dialysate fractionated sample, 75% showed purification

Results

fold of 1.2 and the yield of 91%. In contrast, elution profile of the crude enzyme subjected to gel filtration on sephadex G-100 column chromatography showed purification fold of 1.6 and yield of 87 %. Both enzyme activity at 540 nm and protein content at 280 nm were determined for each fraction fig (14). The enzyme activity has been detected between the fractions No.16 to the fraction No.20.

Table (14): Purification of PGase secreted by P.citrinum

Purification Protein Total Specific Purification Yield step activity activity (mg) fold (%) (U) (U/mg) Crude 1300 2500 1.9 1 100 exract

(NH4)SO4 1000 2275 2.3 1.2 91 G-100 720 2192 3.0 1.6 87

Results

Absorbance(280nm) Enzyme activity(U/ml)

1.2 4.5

4 1

3.5 )

0.8 3

nm 280

( 2.5 0.6 2

0.4 1.5

Absorbance 1

0.2 Enzyme activity(U/ml) 0.5

0 0 1 6 11 16 21 26 31 36

Fraction Number

Fig.14.Gel filtration profile of polygalacturonase.

4.3.2. Characterization of the purified enzyme:

4.3.2.1. Effect of different pH values

4.3.2.1.1. On the activity of the enzyme.

The reaction was incubated at various pH range (4 to 8) using different pH buffers then the activity was measured under standard assay conditions. The effect of pH on the polygalacturonase activity is presented in Fig 15. As it can be observed, the enzyme was active over a broad pH range, displaying over 60% of its activity in the pH range of 4.0

Results

up to7.0 with an optimum pH of 6.0. Concerning to the PGase at pH 8, the relative activity decreased down up to 57%.

Table (15): Effect of different pH values on the activity of the partially purified polygalacturonase enzyme produced by P.citrinum.

pH Relative activity (%)

4 61

5 89

6 100

7 69

8 57

Results

Fig (15): Effect of different pH values on the activity of the partially purified polygalacturonase enzyme produced by P.citrinum.

4.3.2.1.2. On the stability of the enzyme.

The pH stability of the enzyme was determined by exposing the purified enzyme firstly to various pH values (4 to 8) using different pH buffers for 2 hours. Then the activity measured under standard assay conditions. The results presented in table (16) and fig. (16) revealed that the polygalacturonase enzyme was stable at the broad pH range of pH 4 up to 7, retaining more than 66% of its activity. PGase activity was more stable at pH 6.0. However, the stability was significantly reduced to 58% at pH 8.

Results

Table (16): Effect of different pH values on the stability of the partially purified polygalacturonase enzyme produced by P.citrinum.

pH Residual activity (%)

4 66

5 83

6 100

7 86

8 58

Results

Fig (16): Effect of different pH values on the stability of the partially purified polygalacturonase enzyme produced by P.citrinum.

4.3.2.2.Effect of different temperatures

4.3.2.2.1. On the activity of the enzyme

Different incubation temperatures ( 20 to 70 ºC) was investigated for their effect on the purified pectinase enzyme. The results illustrated in table (17) and Fig.(17) showed that the activity of P.citrinum polygalacturonase increased gradually at temperature ranged from 20°C up to 600 C. Moreover the optimum temperature was achieved at 56

Results

400 C; meanwhile the recorded relative activity was 49% at 700 C.

Table (17): Effect of the temperature on the activity of the partially purified polygalacturonase enzyme produced by P.citrinum.

Temperature(°C) Relative activity (%)

20 55

30 93

40 100

50 81

60 66

70 49

Results

Fig (17): Effect of the temperature on the activity of the partially purified polygalacturonase enzyme produced by P.citrinum

4.3.2.2. 2.On the stability of the enzyme:

The thermostability of the purified polygalacturonase was determined by measuring the residual activity of the enzyme after incubation at different ranges of temperatures (20°C - 70°C)after 30 minutes. Fig 18 showed that, the increase in temperature caused an overall increase in the stability up to 60°C; rising temprature above 60°C caused a decline in thermostability. It is worth mentioned that the maximum stability of 100% was observed at 50°C. However, the residual activity declined to 58% at 70°C respectively.

Results

Table (18): Effect of different temperatures on the stability of the partially purified polygalacturonase enzyme produced by P.citrinum.

Temperature(°C) Residual activity(%)

20 67

30 94

40 97

50 100

60 72

70 58

Results

Fig (18): Effect of different temperatures on the stability of the partially purified polygalacturonase enzyme produced by P.citrinum

4.3.2.3. Effect of different metal ions on the activity of the partially purified polygalacturonase enzyme produced by P.citrinum.

The effect of metal ions were examined by adding chlorides of Ca+2, Co+2, and Mg+2 ; sulphates of Cu+2, Zn+2,Cd+2 ; EDTA and nitrate of Ba+2 at concentration of 1mM to the buffer solution. Results in table 19 and Fig.19 revealed that the enzyme activity was enhanced in the presence of Mg+2 and Zn+2 to 12% and 5% respectively, whereas Ca+2 resulted in a reduction in the enzyme activity by 12%. Salts such as Ba (NO3), CoCl2.6H2O, CuSO4.5H2O and EDTA inhibited enzyme activity up to 50%.

Results

Table (19): Effect of different metal ions on the activity of the partially purified polygalacturonase enzyme produced by P.citrinum.

Metal ions (1mM) Relative activity (%)

Cacl2 88

Cu.SO4.5H2O 69.0

ZnSO4 105

CoCl2.6H2O 59.0

MgCl2 112.0

EDTA 50.0

CaSO4 88.1

CONTROL 100

Results

Fig (19): Effect of different metal ions on the activity of the partially purified polygalacturonase enzyme produced by P.citrinum.

4.4. Extraction and determination of pectic substances

Bioextraction of pectin from different agro-residues like sugar beet pulp, Bannana peels wastes, and Orange peels wastes by P.citrinum was markedly influenced by the previously mentioned factors obtained by factorial design system. As can be seen, SBP contains high amount of pectin as it weighed 2gm compared to both OPW and BPW that give 1.5 and 1.2gm respectively. The raw material extracted pectin has many applications in the pharmaceutical industry.

Results

Table (20): The different weights of pectin extracted from different agroindustrial by products inoculated with P.citrinum.

Agro-residues wastes Dry weight of extracted pectin(gm)

Sugar beet pulp waste 2

Orange peel waste 1.12

Banana peel waste 1.5

Discussion

Increasing population and industrialization has resulted in sudden increase in pollution. Because of the detrimental effects of pollution on humans, animals and plants, the ever inceasing pollution is causing concern all over the world.The microbial biodiversity is important on many grounds ranging from aesthetic considerations to its usefulness, particularly for biotechnology.The fastest growing segments are enzymes for feed and fuel production. Abundant amount of waste materials are produced by agricultural and fruit processing industries, which pose considerable disposal problems and ultimately leads to pollution.Vast varieties of microorganisms are present in the environment which can be exploited for the utilization of waste materials.For example in the processing of citrus fruits, a large proportion of the produced wastes are in the form of peel, pulp and seeds.Citrus peel is rich in carbohydrate, protein and pectin. Pectic substances are present in the pimary plant cell wall and the middle lamella. Besides these, other fruits like Mango(Mangifera indica), Avocado Pear (Avocado avocado), Guava (Psidium guajava), Banana (Musa sapientum), Papaya (Carica papaya), Cashew Apple (Anacardium occidentale),

Discussion

Garden-egg (Solanum nigrum Linn.), Star Apple (Crysophylum albidium), and Tomato (Lycopersicum esculentum) also contain substantial amounts of pectin having a high gelling grade. Sugar beet pulp, a by- product of sugar extraction, also contains pectin.Galacturonic acid (21%), arabinose(~21%), glucose(~21%), galactose(~5%) and rhamnose(~2.5%) are its main components (Micard et al.,1994).They are the constitutive monomers of cellulose and pectins.Pectin is a polymer of galacturonic acid residues connected by α-1, 4 glycosidic linkages.Pectin is hydrolysed by pectinase enzymes produced extracellularly by microflora available in our natural environment.With the help of these pectinase enzyme, micro-organisms can convert citrus wastes into sugars which can be used for food and value added products.These micro-organisms can also be exploited for production of pectinase which is an industrially important enzyme and have potential applications in fruit, paper, textile, coffee and tea fermentation industries.

Recently, a large number of microorganisms, isolated from different materials, have been screened for their ability to degrade polysaccharides present in vegetable biomass producing pectinases on solid-state culture (Soares et al., 2001). In the present study, fourteen species have

Discussion

been screened for thier pectinolytic activities. Penicillium citrinum has been found to be the best producer of pectinolytic enzymes (129.2±2U/gdfs). Fawole and Odunfa, 1992 reported that Aspergillus, Fusarium, Penicillium and Rhizopus showed high pectolytic activities. In a study by Spalding, and Abdul-Baki, (1973), Penicillium expansum, the causal agent of blue mould rot in apples, was shown to produce polygalacturonase in artificial media and when attacking apples. However, Singh et al., 1999 stated that the commercial preparations of pectinases are produced from fungal sources. According to Silva et al., 2002, PG production by P. viridicatum using orange bagasse and sugar cane bagasse was influenced by media composition. Aspergillus niger is the most commonely used fungal species for industrial production of pectinolytic enzymes (Naidu and Panda, 1998& Gummadi and Panda, 2003). Pectic substances are rich in negatively charged or methyl-estrified galacturonic acid. The esterification level and the distribution of esterified residues along the pectin molecule change according to the plant life cycle and between different species. Thus, the ability of some microorganisms to produce a variety of pectinolytic enzymes that differ in their characteristics, mainly in their substrate specifity, can provide them with

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more efficacy in cell wall pectin degradation and consequently more success in the plant infection (Pedrolli et al., 2009).This may explain that Polygalacturonase enzyme is the most abundant enzyme assayed in this study. In addition, Natalia et al., (2004) reported that higher production of PGase depended on the composition of the medium. . On the other hand, PL production depended on the strain used. More than 30 different genera of bacteria, yeasts and moulds have been used for the production of PGases. In the last 15 years, with strains of Aspergillus, Penicillium and Erwinia were reported to be the most effective in enzyme production (Torres et al., 2006).Pectin lyase (PL) and Polygalacturonase (PG) production by Thermoascus aurantiacus was carried out by means of solid-state fermentation using orange bagasse, sugar cane bagasse and wheat bran as a carbon sources(Martins et al., 2000). Commercial pectinase preparations are obtained mainly from Aspergillus and Penicillium (Said et al., 1991). Moreover, high activities of extracellular pectinase with viscosity-diminishing and reducing groups-releasing activities were produced by Penicillium frequentans after 48 h at 350C (Said et al., 1991), The selection of substrate for SSF depends upon several factors mainly the cost and availability and this may involve the screening for several

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agro-industrial residues which can provide all necessary nutrients to the micro organism for optimum function. The main objective of this study was to check the effect of physical and chemical components of the medium to find out the activators and inhibitors of pectinolytic activity from Penicillium citrinum. SSF is receiving a renewed surge of interest, for increasing productivity and using of a wide agro-industrial residue as substrate. The selection of the substrate for the process of enzyme biosynthesis is based on the following criteria

1) They should represent the cheapest agro-industrial waste. 2) They are available at any time of the year. 3) Their storage represents no problem in comparison with other substrate. 4) They resist any drastic effect of environmental conditions, e.g.temperature variation in the weather from season to season and from day to night. SSF are usually simple and could use wastes of agro-industrial substrates for enzyme production.The minimal amount of water allows the production of metabolites, less time consuming and less expensive. Solis-Pereyra et al., (1996) and Taragano, et al., (1997) came to the conclusion that production is higher under solid

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state fermentation than by submerged one. In this field many workers dealt with the main different factors that effect the enzyme productions such as temperature, pH, and aeration, addition of different carbon and nitrogen sources. In order to obtain high and commercial yields of pectinases enzyme, it is essential to optimize the fermentation medium used for growth and enzyme production. Sugar beet pulp has been shown to be the best used source for pectinase production from P.citrinum. Pectin acts as the inducer for the production of pectinolytic enzymes by microbial systems; this is in agreement with the results of Pandey et al., (2001) and Phutela et al., (2005). Since pectin can not enter the cell, it has been suggested that compounds structurally related to this substrate might induce pectic enzyme productions by microorganisms. Also, low levels of constitutive enzyme activities may attack the polymeric substrate and release low molecular products which act as inducers. Polygalacturonase and pectin transeliminase were not produced whenever the medium lacked a pectic substance, the production of polygalacturonase and pectin transeliminase is inductive. An adequate supply of carbon as energy source is critical for optimum growth affecting the growth of organism and its metabolism. Aguilar and Huitron, (1987) reported that the production of pectic

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enzymes from many moulds is known to be enhanced by the presence of pectic substrates in the medium. Fawole and Odunfa, (2003) found that pectin and polygalacturonic acid promoted the production of pectic enzyme and they observed the lack of pectolytic activity in cultures with glucose as sole carbon source, such observations reflect the inducible nature of pectic enzyme from a tested strain of Aspergillus niger.

In most microorganisms, both inorganic and organic forms of nitrogen are metabolized to produce amino acids, nucleic acid, proteins, and cell wall components. Recorded results showed that maximum polygalacturonase production by Penicillium citrinum was obtained in the presence of yeast extract; this result is in agreement with that reported by Bai et al., (2004) who found that high pectinase activities were obtained with (NH4)2SO4, yeast extract powder, soya peptone, soybean pulp powder and the monosodium glutamate water. Yeast extract served as the best inducer of exopectinase by Aspergillus sp (Mrudula and Anitharaj, 2011). Also, Thakur et al., (2010) reported that the best PGase production was obtained when casein hydrolysate and yeast extract were used together. It has been reported that nitrogen limitation decreases the polygalacturonase production. Also, Aguilar et al., (1991)

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showed that yeast extract (organic nitrogen source) was the best inducer of exopectinases by Aspergillus sp. Moreover, Kashyap et al., (2003) found that, yeast extract, peptone and ammonium chloride were found to enhance pectinase production up to 24% and addition of ammonium nitrate inhibited pectinase production. In this context, yeast extract proved to be the best nitrogen source likely because it provided other stimulatory components such as vitamins (Qureshi, 2012).Yeast extract has previously proved superior to other nitrogen sources in the production of pectinases by the thermophilic fungus Sporotrichum thermophile (Kaur et al., 2004). Bacillus shaericus produced maximum polygalactouronase when grown on mineral medium containing yeast extract as sole nitrogen source (Ranveer et al., 2010). Ammonium sulphate was also effective in the induction of polygalacturonase production. Galiotou-Panayotou and Kapantai (1993) observed that ammonium phosphate and ammonium sulphate did influence production of pectinase positively but also recorded an inhibitory effects of ammonium nitrate and potassium nitrate on pectinase production. Moreover, Patil and Dayanand (2006) revealed that both ammonium phosphate and ammonium sulphate did influence production of pectinase positively in both submerged and

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solid-state conditions. In addition, Sapunova (1990) found that ammonium salts stimulated the pectinolytic enzyme production in Aspergillus alliaceus. Moreover, Sapunova et al. (1997) has also observed that (NH4)2SO4 stimulated pectinase synthesis, as in its absence fungus did not produce extracellular pectinases. In addition, Fawole and Odunfa (2003) found ammonium sulphate and ammonium nitrate were good nitrogen sources for pectic enzyme production from Aspergillus niger. Also, Phutela et al.

(2005) found that presence of yeast extract + (NH4)2 SO4 in growth medium supported maximal production of pectinase followed by malt sprouts+ (NH4)2 SO4, which also supported maximal polygalacturonase activity. In addition, Rasheedha et al. (2010) found that ammonium sulphate has enhanced the production of Penicillium chrysogenum pectinase. On the contrary, Alcântara et al.( 2010) reported that the concentration of ammonium sulphate had a negative effect on enzyme activities .The observations of Hours et al. (1998) who suggested that lower levels of

(NH4)2SO4 or K2HPO4 added to the growth medium as inorganic nitrogen sources did not influence pectinase yield. In addition, Vivek et al. (2010) found that organic nitrogen sources showed higher endo, exo pectinases activities than inorganic nitrogen source. The nitrogen

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source can play an important role in affecting the pH changes in the substrate during the fermentation. The ammonium ion was taken up as ammonia, thereby releasing a proton into the medium and causing a decrease in pH (Qureshi et al., 2012). The size of inoculum added to the fermentation medium has significant effect on growth and enzyme production. Maximum polygalacturonase production took place at the inoculum size of (1.8 ×105 spores/ml) for SSF, but decrease subsequently with the increase in the inoculum size. Low inoculum density than the optimum may not be sufficient to initiate growth and to produce the required biomass, whereas highe inoculum can cause competition for nutrients (Jacob and Prema, 2008). Mrudula and Anitharaj (2011) reported that the optimum inoculum density is an important consideration for SSF process, since, over crowding of spores can inhibit growth and development. Higher inoculum levels besides increasing spores density increase water content of the medium as well. The inoculum size of 1×105ml-1 resulted the maximum production of endo- and exo-pectinases by Penicillium sp in submerged conditions and 1×107ml-1 had given maximum amount in solid-state condition (Patil, and Dayanand, 2006).Similar observations were made by

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Aguilar and Huitron(1987) for submerged condition and Pereira et al.( 1994) for solid-state condition.

pH stongly affects many enzymatic processes and transport of various components across the cell membrane (Moon & Parulekar, 1991). The effect of hydrogen ion concentration on the enzyme activity may be explained in part in terms of the relative molecular stability of the enzyme itself and in part on the ionizable groups (COO-, OH-) of the tertiary protein structure of the enzyme complex (Lehninger, 1973).In this study, the maximum production of polygalacturonase was recorded at a pH range of 5-6 with optimum production at pH 5.5. Boccas et al. (1994) also reported similar observations. The pH of the medium will also limit the growth of the culture or exert influence upon catalytic activity of the enzyme (Adeleke et al., 2012). Maximum polygalacturonase production was observed in the medium with acidic pH values within a range of 4 to 6 (Aminzadeh et al., 2007).Also, Ramanujam and Subramani (2008) reported that the optimum pH for Aspergillus niger was 6.0 using citrus peel and sugarcane bagasse, respectively for the production of pectinase in SSF. Observation in the study by Adeleke et al. (2012) showed optimum pH for enzymes production within 5 to 5.5. Banu et al. (2010) presented similar

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observations for polygalacturonase production by Penicillium viridicatum, Trichoderma longibrachiatum showed high production of glucose on the day 7,at pH 5 and 450C. Wide range of initial pH of the medium during the upstream bioprocess make the end product either acidic or alkaline, which tend to have varied applications (Hoondal et al., 2002). The pH regulates the growth and the synthesis of extracellular enzyme by several microorganisms particularly fungal strains (Suresh and Chandrasekaran, 1999). Fungi and yeasts produce mainly acidic PGases, whilst alkaline pectinases are mainly produced by bacteria.The highest titres of acidic PGase have been obtained with strains of Aspergillus, Penicillium and Candida (Torres., et al 2006) revealed that pH is the most significant factor that influence the enzyme production, and that the optimal value of 5, resulted in an increase in PGase production up to 6.67 fold.

Temperature is another critical parameter and must be controlled to get the optimum enzyme production. It has been found that temperature is a significant controlling factor for enzyme production (Kitpreechavanich et al., 1984). Temperature in solid state fermentation is maintained at 30-320C, as it cannot be precisely controlled due to the reason that solid-state fermentation has solid

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substances which limited heat transfer capacity. In the current study, the obtained results revealed that the highest polygalacturonase production has been achieved at 25°C during optimization using the classical methods (127.1U/gdfs) and at 30°C using the full factorial design (132U/gdfs). Most microorganisms are mesophiles which grow over a range of 25°C -300C while others are psychrophiles or thermophiles in nature. Akintobi et al. (2012) reported that the temperature of the medium also affected both growth and enzyme production by Penicillium variabile. Growth of the organism and production of pectinolytic enzymes were optimum at 30°C. According to Bailey and Pessa (1990), lower temperature slows down the hydrolysis of pectin. At low temperature (40C) there was no growth and at high temperature, generation of metabolic heat in solid state fermentation might be a reason for growth inhibition in microorganisms. Release of proteins into the medium was also optimum at 30°C, Growth and enzymes production were least supported at 20°C and 35°C. In general, temperature is believed to be the most important physical factor affecting enzyme activity (Dixon and Webbs, 1971). In contrast, Freitas et al. (2006) reported that the fungal species

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investigated for pectinase production showed optimum growth in the range of 45 to 600C.

Patil and Dayanand, (2006) stated that the period of fermentation depends upon the nature of the medium, fermenting organisms, concentration of nutrients and physiological conditions. Penicillium citrinum started polygalacturonase production from the second day of incubation period with low enzyme activity (78U/gds), which increased gradually as the incubation period was increased reaching its maximum activity on the seventh day of incubation (129.2U/gds),which decreased thereafter showing moderate increase on the ninth day of the incubation period and the activity reached (100.2U/gds) .These results are in agreement with that of Akhter et al., (2011) who demonstrated that the maximum pectinase production by A.niger was peaked on the seventh day of incubation . In contrast, Silva et al., (2002) reported that Polygalacturonase production by Penicillium viridicatum peaked between the 4th and the 6th days. Another study (Gupta et al., 1996) showed that the maximum production of polygalacturonase in SSF by Penicillium citrinum was at the 120th hour (i.e. the fifth day). Many results showed that PG activity increased during the primary metabolism and decreased when the secondary metabolism started. In

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Botrytis cinerea (Martinez et al., 1988) and Fusarium oxysporum (Martinez et al., 1991) the highest PG activities were obtained during the primary growth phase. In Trametes trogii (Ramos et al., 2010), the highest PGase activity was obtained when the biomass was at its highest level. The incubation period for maximum enzyme production was found to vary with different strains. Alternaria alternata (Kunte and Shastri, 1980) showed maximum polygalacturonase activity on the 4th day. The decrease in the activity can be due to the depletion of nutrients in the medium. The incubation period is generally dictated by the composition of the substrate and properities of the strain, such as its growth rate, enzyme production profile, initial inoculum and others (Lonsane and Ramesh, 1990).

Considering surfactants application, high level of polygalacturonase production was obtained upon addition of Tween 40 (0.1%) to the culture medium (140.1 U/gdfs). Also, Tween 20 and 60 126.1U/gdfs,128U/gdfs respectively slightly increased PGase activities than the enzyme produced in the surfactant free medium. These results are in agreement with Kapoor et al., 2000 and Zu- ming et al., 2008 who reported stimulation of pectinases when Tween-20 was supplemented to the medium. The

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reason is probably is due to the possibility that the surfactants might improve the turnover number of PGs by increasing the contact frequency between the active site of the enzyme and the substrate by lowering the surface tension of the aqueous medium(Kapoor et al., 2000). Moreover, Surfactants have been reported to affect the growth rate and enzyme production of many fungi. Similar finding have been recorded with respect to the action of surfactant on different microbial enzymes (Sukan et al., 1989). The mechanisms by which detergents enhance extracellular enzyme production were reported to be due to increased cell membrane permeability, change in lipid metabolism, and stimulation of the release of enzymes are among the possible modes of the action (Omar et al., 1988). Mrudula and Anitharaj , (2011) reported that production of pectinase is highest when Triton-X-100 was supplemented to the orange peel in SSF.

Full Factorial Statistical Design

Full factorial design was used in order to identify important parameters in the screening analysis. The factors were yeast extract, incubation period, inoculums size, pH, and temperature.. Selection of the best combination has been done using factorial design of 32 runs. Activities were

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measured after using sugar beet pulp as the best carbon source. The carbon substrate was determined for the screening study based on the results of the preliminary experiments. A significant model was obtained, in which yeast extract, Inoculum size, and Temperature had significant effects on the exo-PG activity, while incubation period and pH factors did not show significant variations. All interaction effects were also insignificant. Small p- values (p <0.0250) show that the parameters (yeast extract, inoculum size, and temperature) are significant on the response. The P-values used as a tool to check the significance of each of the coefficients, in turn indicate the pattern of interactions between the variables. Smaller value of P was more significant to the corresponding coefficient. According to the model, the highest exo-PG activity (132U/gds) has been obtained using 1.2% yeast extract as the best nitrogen source, inoculated with 1.8×105spores/ml incubated for 8 days at pH 5.5 and temperature 30°C. According to the results, the model predicts the experimental results well and estimated factors effects were real as indicated by R2 value (o.74). R2 value being the measure of the goodness to fit the model, indicated that 74% of the total variation was explained by the model, i.e., the good correlation between the experimental and

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predicted results verified the goodness of fit of the model (R2 = 0. 74). It is a known fact that the value of R2 varies from 0 to ±1. When R2 =0, there is no correlation between experimental and predicted activities. For R2= ±1, perfect straight line relationship exists between the experimental and predicted activities (Naidu and Panda, 1998). On the other hand, the conventional method (i.e., change-one- factor-at-a-time) traditionally used for optimization of multifactor experimental design had limitations because (i) it generates large quantities of data which are often difficult to interpret (ii) it is time consuming and expensive (iii) ignores the effect of interactions among factors which have a great bearing on the response. To overcome these problems, a full factorial design was applied to determine the optimal levels of process variables on pectinase enzyme production. The results indicated that (Full factorial design, FFD) not only helps us locate the optimum conditions of the process variables in order to enhance the maximum pectinase enzyme production, but also proves to be well suited to evaluating the main and interaction effects of the process variables on pectinase production from waste agricultural residues. There are few works in literature that report the effects of culture media on the optimization of PG activity.Tari et al., (2007) who evaluated the biomass,

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pellet size and polygalacturonase (PG) production by Aspergillus sojae using response surface methodology showing that concentrations of malt dextrin, corn steep liquor and stirring rate were significant (p<0.05) on both PG and biomass production.

Effect of gamma radiation on polygalacturonase production

Radiation effect on enzymes or on the energy metabolism was postulated.

Gamma irradiation potentiates the productivity of the enzyme to its maximum value (152.2U/gdfs) post exposure to 0.7 kGy. This enhancement of enzyme production might have been due to either, an increase in the gene copy number or the improvement in gene expression, or both (Meyrath et al., 1971; Rajoka et al., 1998; El- Batal et al., 2000 and El-Batal and Abdel-Karim, 2001). Also, induction of gene transcriptions or proteins has been found after low dose irradiation (Wolff, 1998 and Saint- Georges 2004), indicating that the induction of gene transcription through the activation of signal transduction may be involved in the low dose effects. A gradual decrease in the enzyme activity after exposure to the different doses of 1, 1.5kGy was observed. The complete

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inhibition of growth and consequently on enzyme production has been obtained at a level of 2kGy dose. This could be explained by damage or deterioration in the vitality of the microorganism as radiation causes damage to the cell membrane. This major injury to the cell allows the extracellular fluids to enter into the cell. Inversely, it also allows leakage out of essential ions and nutrients which the cell brought inside. El-Batal and Khalaf, (2002) evidenced that production of pectinases increased by gamma irradiated interspecific hybrids of Aspergillus.sp using agroindustrial wastes.

Enzyme purification

Pectinase enzyme was purified from crude sample by ammonium sulfate fractionation and further dialysis was carried out. The 75% ammonium-dialysate fractionated sample showed 1.2 purification fold and a yield of 91% .Elution profile of the crude enzyme subjected to gel filtration on sephadex G-100 column chromatography showed 1.6 purification fold and 87 %yield. Enzyme activity at 540 nm and protein content at 280 nm were determined for each fraction. The enzyme activity has been detected between the fractions No.16 to the fraction No.20, while fraction No.10 to the fraction No.13 had no enzyme

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activity suggesting a number of isoforms of PGase. According to Viniegra-Gonzalez and Favela-Torres (2006) and Torres et al. ( 2006), variation in the isoforms of extracellular enzymes obtained by SSF can be attributed to alteration of the water activity (aw) that results in changes in the permeability of fungal membranes, limitation of sugar transport and presence or absence of inducer. It is even reported that pectinases produced by the same microorganism have exhibited different molecular weights, degrees of glycosylation and specificities. These variations may be due to the post transitional modification of a protein from a single gene or may be the products of different genes (Cotton et al., 2003 and Serrat et al., 2002).

Enzyme characterization

Effect of pH on polygalacturonase activity and stability

The enzyme of P.citrinum was active over a broad pH range, displaying over 60% of its activity within the pH range of 4.0 to7.0 with an optimum pH at 6.0. Optimum pH for different pectinases has been reported to vary from 3.8 to 9.5 depending upon the type of enzyme and the source (Joshi et al., 2011). Meanwhile, P.viridicatum showed an optimum pH at 6.0 as mentioned by Silva et al., (2007), Moniliella sp showed its maximum activity at pH 4.5 and at

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pH 4.5-5.0 for Penicillium sp (Martin et al., 2004). The maximum activity of Monascus sp. and Aspergillus sp for exo-PGase was obtained at pH 5.5 (Freitas et al., 2006). Also, Silva et al.,( 2002) and Zhang et al. (2009 ) reported that optimum pH for pectinase activity was 5.0 for both Penicillium viridicatum and Penicillium oxalicum respectiviely.Similarily, PGases of Aspergillis niger were shown to possess maximum catalytic activity at pH 5.0 (Shubakov and Elkina , 2002). However, the optimal pH of polymethylploygalacturonase was found to be 4.0 (Kollar, 1966 and Kollar and Neukom, 1967). Dixon and Webbs, (1971) & Conn and Stump (1989) separately reported that the changes in pH have an effect on the affinity of the enzyme for the substrate. The effect of pH on the structure and activity of polygalacturonase from A.niger was described by Jyothi et al. (2005). They reported that the active conformation of PGase was favored at pH between 3.5 and 4.5, alterations in the secondary and tertiary structures resulted at pH (from 5.0 to 7.0). This could be attributed to Histidine residues that have ionizable side-chains, increasing the net negative charge on the molecule in the neutral-alkaline pH range and leading to repulsion between the strands, resulting in a destabilization

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of the hydrogen-bond structure of the enzyme (Jyothi et al. 2005).

Stability of the enzyme when incubated at pH in suitable buffer systems for 2hs at 30°C was also investigated during this work. The results revealed that the polygalacturonase enzyme of P.citrinum was stable at a broad pH range 4 -7, retaining more than 66% of its activity. PGase activity was more stable at pH 6.0. However, the stability was significantly reduced to 58% at pH 8. It was reported that the inactivation process was found to be faster at high alkaline pHs due to disulfide exchange, which usually occur at alkaline condition (Dogan and Tari, 2008). In this sense, Gadre et al. (2003) reported that PGase activity show higher stability in the range from 2.5 to 6.0; however, at pH 7.0 the stability was 60% lower. On the other hand, Hoondal et al. (2002) evaluated a PGase from Aspergillus fumigates that kept their activity in a range of pH from 3 to 9.

Effect of temperature on polygalacturonase activity and stability

The results showed that the activity of P.citrinum polygalacturonase increased gradually within temperature range from 200C up to 600C. Moreover, the optimum

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temperature was achieved at 40oC; and a relative activity of 49% was attained at 700C. This is supported by results of Juwon et al. (2012), who reported a decline in the enzyme activity at temperatures more than 400C. Similar observation had been reported by Palaniyappan et al. (2009) by Aspergillus niger. Also, PGase produced by Aspergillus flavus, Aspergillus fumigatus and Aspergillus repens exhibited maximum activity at 350C, 400C and 450C respectively (Arotupin, 2007). Similarly, Barthe et al. (1981) and Yoon et al. (1994) documented temperature of 400C for the maximum PGase activity from Colletotrichum lindemuthianum and Ganoderma lucidum. The same optimum temperature was implicated for the PGase obtained from Aspergillus niger, Botryodiplodia theobromae and Penicillium variabile and Aspergillus alliaceus(Juwon et al., 2012). On the other hand, other studies conducted by several authors using different strains revealed that optimum temperature of an exopolygalacturonase from Aspergillus niger was 60°C (Sakamoto et al. ,2002).Furthermore, the partially purified polygalacturonase from Sporotrichum thermophile apinis was optimally active at 55°C (Jayani et al., 2005; Kashyap et al., 2001).These variations in the optimum temperature of fungal PGase suggested a broad range of

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temperature tolerable by the enzyme. In addition, nature, source and differences in the physiological activities of fungi may be responsible for these variable observations (Arotupin, 1991).

Thermostability is the ability of the enzyme to tolerate against thermal changes in the absence of substrates (Bhatti et al., 2006). The thermostability of the purified polygalacturonase was determined by measuring the residual activity of the enzyme after incubation at different ranges of temperatures (20°C - 70°C) after 30 minutes. The increase in temperature caused an overall increase in the stability up to 600C of PGase from P.citrinum; rising temperature above 60°C caused a decline in thermostability. It is worth mentioned that the maximum stability of 100% was observed at 500C. Similarly, the optimum temperatures for PGase of Aspergillus niger and Penicillium dierckii were shown to be 500 C and 600C, respectively (Shubakov and Elkina, 2002). However, the residual activity declined up to 58% at 700C. Also, Exo-PG of Monascus sp and Aspergillus sp showed stability at temperature up to 500C (Freitas et al., 2006).

A loss in PGase activity percentage obtained at 700 C from Aspergillus niger,Botryodiplodia theobromae and

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Penicillium variabile was reported by Oyede, (1998) and Ajayi et al.( 2003). Daniel et al., 1996 who also reported the thermal inactivation of the enzymes at high temperature. It was reported that extremely high temperature lead to deamination, hydrolysis of the peptide bonds, interchange, and destruction of disulphide bonds and oxidation of the amino acids side chains of the enzyme protein molecules (Creighton, 1990 and Daniel et al., 1996).

The study conducted by Maciel et al. (2011) is not in agreement with our study, they recorded that exo-PGase was stable at 80°C and showed 60% residual activity remaining after 1 h at this temperature.

Effect of metal ions on polygalacturonase activity.

Results in the present study revealed that the enzyme activity was enhanced in the presence of Mg+2 and Zn+2 by 12% and 5% respectively, whereas Ca+2 resulted in a reduction in the enzyme activity by 12%. The cations may affect protein stability by electrostatic interaction with a negatively charged protein surface, by induction of dipoles, changes in the inter-strand dispersion forces and by their ability to modify the water structure in the vicinity of the protein and thus influence its hydration environment (Zarei

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et al., 2011). Salts such as Ba (NO3), CoCl2.6H2O,

CuSO4.5H2O and EDTA inhibited enzyme activity up to 50%. Jurick et al., (2009) reported that there was an increase in PG enzyme activity by adding magnesium and iron whereas a decrease in activity occurred when calcium and manganese were included in the PGase assay. Also,

Banu et al., (2010) reported that HgCl2, CoCl2 and CuSO4 caused inhibition of pectinase activity by P.chrysogenum up to 60%. Thus, Hg+2 and Cu+2 block thiol groups on the protein (Skrebsky et al., 2008 and Tabaldi et a.,l 2007). Besides this effect,Cu+2 induces protein polymerization by forming Histidine-Cu-Histidine bridges between adjacent peptide chains(Follmer and Carlini, 2005) and can interfere in the structure of some proteins through its coordination geometry (Pauza et al., 2005). Similarly

BaCl2 and EDTA resulted in the maximum inhibition of pectinases activity up to 40% (Banu et al., 2010). Also, Oyede (1998) reported the stimulatory role of K+2, Na+2 and Mg+2 on PGase activity from Penicillium sp, while concentrations of Ca+2 beyond 15mM inhibited the enzyme activity. This variation in degrees of stimulation and inhibition could be a function of the sources of enzyme from different mould genera. Also, Murray et al. (1990) showed that the formation of a chelate compound between

124

Discussion

the substrate and metal ions could form a more stable metal-enzyme-substrate complex and stabilizing the catalytically active protein conformation. Also, Brown and Kelly (1993) affirmed the ability of metal ions often acting as salt or ion bridges between two adjacent amino acids. Famurewa et al. (1993) and Sakamoto et al. (1994) confirmed the inhibitory activity of EDTA on enzyme. The metal building reagent like EDTA can inactivate enzyme either by removing the metal ions from the enzyme forming coordination complex or by building inside enzyme as a ligand ( Schmid, 1979).

125

Concluding Remarks

5-Concluding remarks

Pectinases are among the first enzymes to be used at homes. Their commercial application was first observed in 1930 for the preparation of wines and fruit juices. As a result, pectinases are today one of the upcoming enzymes of the commercial sector. It has been reported that microbial pectinases account for 25% of the global food enzymes sales (Jayani et al., 2005).

Higher cost of the production is the major problem in commercialization of new sources of enzymes. Though, using high yielding strains, optimal fermentation conditions and cheap raw materials as a carbon source can reduce the cost of enzyme production for subsequent applications in industrial processes. So, the production of pectinases from agro-wastes is promising and required further investigations.

In the coming times, it should increase attention toward the study of the molecular aspects of pectinases, the impact effect of radiation exposure on pectinase as well as developing the mutant of the superior pectinase producing strains. Also, further studies should be devoted to the understanding of the regulatory mechanism of the enzyme secretion at the molecular level.

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جحسيي االًحاج الفطري لالًزيوات الوحللة للبكحيي باسحخدام اشعة جاها جحث ظروف الحخور شبه الجافة.

(شيواء عبد الوحسي ابراهين(

قسن الٌبات والويكروبيولوجي-كلية العلوم-جاهعة حلواى

الوسحخلص العربي

رى في ْذِ انذراصخ فحص نًغػًٕخ يٍ انفطزيبد انزي رؼطي اػهي اَزبط يٍ اَزيًبد انجكزيُيز ٔ قذ ٔعذ اٌ فطز انجُضهيٕو صيززيُى يؼطي اػهي اَزبط يٍ اَزيى انجٕني عبالكزٕريُيز ٔ قذ رى دراصخ ربصيز انؼٕايم انزي رؤصز ػهي اَزبط االَزيى حيش ٔعذ اٌ يبدح نت انجُغز رؼطي اػهي اَزبط نالَزيى كًصذر ٔحيذ نهكزثٌٕ. ٔيٍ ثيٍ انًصبدر انًخزهفخ نهُيززٔعيٍ ٔعذاٌ خالصخ انخًيزح رؼطي اػهي قيًخ يٍ اَزبط االَزيى. ٔ يٍ انؼٕايم االخزي انزي رى دراصخ ربصيزْب ػهي اَزبط االَزيى كًيخ انهقبػ حيش ٔعذ اٌ رزكيز 8.1×815 يؼطي اػهي اَزبط. ٔ كبَذ فززح انزحضيٍ يٍ اْى انؼٕايم انًؤصزح حيش ٔعذ اٌ اػهي اَزبط نالَزيى يحذس في انيٕو انضبثغ يٍ انزحضيٍ. ٔ رًذ دراصخ ربصيزانزقى انٓيذرٔعيُي ٔرجيٍ اٌ االس انٓيذرٔعيُي 5.5 يؼطي اػهي اَزبط نالَزيى.ٔ اٌ درعخ حزارح انزحضيٍ )55( درعخ يئٕيخ رؼطي اػهي اَزبط نالَزيى. ٔاخيزا رًذ دراصخ ربصيز يخزصبد انزٕرز انضطحي ٔرجيٍ اٌ يبدح رٕي01ٍ رؼطي اػهي َضجخ اَزبط. ٔ قذ رى اصزخذاو اصهٕة انًُذعخ االحصبئي نذراصخ ربصيز خًش يزغيزاد )خالصخ انخًيزح، انزقى انٓيذرٔعيُي، فززح انزحضيٍ ، درعخ حزارح انزحضيٍ، ٔكًيخ انهقبػ )ػهي اَزبط اَزيى انجٕني عبالكزٕريُيز.ٔقذ اصفزد انُزبئظ ػهي االري: رى انحصٕل ػهي اػهي اَزبط الَزيى انجٕني عبالكزٕريُيزثؼذ صًبَي ايبو في درعخ حزارح C°30 ٔاالس انٓيذرٔعيُي 5.5 يغ خالصخ انخًيزح كبفضم يصذر نهُيززٔعيٍ ثززكيز 1.5% يحزٕي َيززٔعيُي.ٔ ثبصزخذاو ْذِ انظزٔف انجيئيخ انًضهي ثبالضبفخ اني اصزخذاو االشؼبع انغبيي ثغزػخ 1.0% كيهٕعزاي رى انحصٕل ػهي اَزبط يزرفغ َضجيب يٍ اَزيى انجٕني عبالكزٕريُيز. ٔقذ اعزيذ ػًهيبد رُقيخ عزئيخ الَزيى انجٕني عبالكزٕريُيز ثؼذ رزصيجّ ثٕاصطخ اصزخذاو 05% يٍ كجزيزبد االيَٕيٕو ، صى انذيهزِ صى انفصم انكزٔيبرٕعزافي ثٕاصطخ صيفبدكش 811.ٔقذ ٔعذ اٌ انظزٔف انًضهي نُشبط االَزيى يكٌٕ ػُذ درعخ انحزارح C°40 ٔاس ْيذرٔعيُي 1-0 انضجبد انٓيذرٔعيُي ثيٍ 51-01 درعخ يئٕيخ.ػُٔذ دراصخ ربصيز ايَٕبد انؼًبدٌ ػهي انُشبط االَزيًي ٔعذ اٌ انًغُضيٕو ٔانزَك يحفزح نهُشبط االَزيًي.