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Optimization of Glucoamylase Production by Mucor Indicus, Mucor Hiemalis, and Rhizopus Oryzae Through Solid State Fermentation

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Optimization of Glucoamylase Production by Mucor Indicus, Mucor Hiemalis, and Rhizopus Oryzae Through Solid State Fermentation Turkish Journal of Biochemistry – Türk Biyokimya Dergisi; 2016; 41 (4): 250-256 Biotechnology Research Article – 78700 Sanaz Behnam*, Keikhosro Karimi, Morteza Khanahmadi, Zahra Salimian Optimization of glucoamylase production by Mucor indicus, Mucor hiemalis, and Rhizopus oryzae through solid state fermentation Mucor indicus, Mucor hiemalis, ve Rhizopus oryzae tarafından üretien glukoamilazın katı hal fermantasyonu ile optimizasyonu DOI 10.1515/tjb-2016-0036 Özet: Amaç: Glukoamilaz, çok çeşitli endüstriyel uygu- Received August 5, 2015; accepted February 12, 2016; lamaları olan bir hidrolizleme enzimidir. Glukoamilaz, published online August 1, 2016 doğal zygomycetes küflerinden olan Mucor indicus, Mucor hiemalis, and Rhizopus oryzae kullanılarak buğday kepeği Abstract: Objective: Glucoamylase is a hydrolyzing enzyme üzerinde katı hal fermentasyonu ile üretilmiştir. with several industrial applications. Glucoamylase was produced via a solid state fermentation by three naturally Metod: Enzim üretimi için kültür sıcaklığı, ortalama nem occurring zygomycetes fungi of Mucor indicus, Mucor hie- miktarı ve kültür süreleri çalışılmıştır. Deneyler, üç değiş- malis, and Rhizopus oryzae on wheat bran. ken için merkezi birleşik kompozit tasarımı üzerinde tepki yüzeyi metodolojisi (RSM) kullanılacak şekilde tasarlan- Methods: The effects of cultivation temperature, medium mıştır. moisture content, and cultivation time on the enzyme pro- duction were investigated. Experiments were designed Bulgular: Glukoamilaz üretimi için kullanılan üç küf için with an orthogonal central composite design on the three optimum sıcaklık ve ortalama nem miktarı, sırası ile, variables using response surface methodology (RSM). 26.6oC ve %71.8 olarak bulunmuştur. Optimum kültür süresi ise M. hiemalis and R. oryzae için 33.1 s, M. indicus Results: For glucoamylase production, the optimum tem- için ise 66.8 s olarak bulunmuştur. Optimum koşullarda, perature and medium moisture content for the three fungi M. indicus, M. hiemalis, ve R. oryzae’nin glukoamilaz üre- were 26.6oC and 71.8%, respectively. The optimum cultiva- timleri sırası ile, 255.3, 272.3, ve 1545.3 U per g kuru subst- tion time for M. hiemalis and R. oryzae was 33.1 h, while rat olarak elde edilmiştir. it was 66.8 h for M. indicus. At optimum conditions, glu- coamylase production by M. indicus, M. hiemalis, and R. Sonuç: R. oryzae, glukoamilazın endüstriyel üretimi için oryzae was respectively 255.3, 272.3, and 1545.3 U per g dry uygun bir adaydır. substrate. Anahtar Kelimeler: Glukoamilaz, Mucor hiemalis, Mucor Conclusion: R. oryzae is a suitable candidate for industrial indicus, Optimizasyon, Rhizopus oryzae, Katı hal fermen- production of glucoamylase. tasyonu Keywords: Glucoamylase, Mucor hiemalis, Mucor indicus, Optimization, Rhizopus oryzae, Solid state fermentation *Corresponding author: Sanaz Behnam: Department of Chemical Morteza Khanahmadi: Isfahan Agriculture and Natural Resources Engineering, Isfahan University of Technology, Isfahan 84156-83111, Research Centre, Isfahan 81785-199, Iran, Iran, e-mail: [email protected] e-mail: [email protected] Keikhosro Karimi: Department of Chemical Engineering, Isfahan Zahra Salimian: Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran, University of Technology, Isfahan 84156-83111, Iran, e-mail: [email protected] e-mail: [email protected] Sanaz Behnam et al.: Optimization of glucoamylase production through SSF 251 1 Introduction 2 Materials and Methods Glucoamylase is a hydrolyzing industrial enzyme which 2.1 Microorganisms and fermentation can degrade both amylose and amylopectin and produce conditions glucose [1,2]. Glucoamylase is commercially used in the production of glucose and fructose juice, baking for Three different species of fungi: Mucor indicus (CCUG improving bread quality, beverage pharmaceuticals, and 22424), Mucor hiemalis (CCUG 16148), and Rhizopus oryzae detergents industries [3–5]. (CCUG 28958) were obtained from the Culture Collec- Solid state fermentation (SSF) and submerged fer- tion, University of Göteborg, Sweden. The strains were mentation are common ways of enzymes production [6]. maintained on plates containing 40 g l–1 glucose, 10 g In solid- state fermentation, the microorganisms (mainly l–1 peptone, and 15 g l–1 agar and kept at 32oC for 5 days fungi) grow on moist solid materials in the absence of to grow and then stored at 4oC until use. Sterile distilled free-flowing water. The water, which is needed for micro- water containing 0.1% (v/v) Tween 80 (Polyoxyethylene bial activities, is available in the solid matrix of the sub- Sorbitan Monooleate) was used to separate spores. An strate [7–9]. Solid state fermentation is more advanta- amount of 10 g moistened wheat bran was added to differ- geous than submerged fermentation, since its capital and ent Erlenmeyer flasks and autoclaved at 121oC for 20 min. operating costs are lower; less space is needed, simpler After cooling, the flasks were inoculated with the spore equipment and media are used, and less wastewater is suspension and kept at 30oC for 7 days to grow the spores produced [6,10–12]. Among the microorganisms used in on the bran surface. Afterwards, 50 ml sterile distilled SSF, filamentous fungi can grow on complex solid sub- water containing 0.1% (v/v) Tween 80 and sterile glycerol strates and produce a wide variety of extracellular enzyme were added to the flasks to separate the spores from the [13]. The amount of enzyme production depends on the bran to be kept in micro tubes at -20oC. fermentation conditions; therefore, optimization of the Fermentation was carried out in 250-ml flasks con- conditions is essential to decrease the cost of enzyme pro- taining 10 g dry wheat bran. They were autoclaved, cooled, duction [14]. and inoculated with spore suspensions (1000 spores per g This work was aimed to investigate the ability of three dry bran). Medium was moistened by addition of water. fungal strains of Mucor indicus, Mucor hiemalis, and Rhizo- The amount of added water was adjusted such that the pus oryzae to produce glucoamylase on wheat bran via the bed moisture after addition of spore suspension was equal solid state fermentation. Wheat bran is an ideal substrate to that required for each experiment. The flasks were put for solid state fermentation whose bed has an appropriate in an incubator at experiment’s temperature. A dish filled porosity for fungal growth and air stream. These fungal with water was put in the incubator to moisten the air strains have several industrial applications. They are sep- in the incubator and reduce water evaporation from the arated from edible sources with high abilities for ethanol substrate. Under such conditions the substrate moisture production from cellulosic sources. They can convert remains nearly constant during the fermentation course. substrate containing inhibitors. Their biomass has a high nutritional value and contains considerable amounts of chitosan and glucosamine as valuable components 2.2 Enzyme extraction and assay [15–17]. To our knowledge, there is no investigation on optimization of glucoamylase production by these strains One unit of the enzyme activity (U) was defined as the on wheat bran. Furthermore, solid state fermentation as amount of enzyme required to liberate 1 μmol of the reduc- an advantageous method for enzyme production than ing sugar (glucose) per min under the assay conditions. The submerged fermentation was applied. Response Surface yields were expressed as U per gram dry substrate (gds). Methodology (RSM) was used to design the experiments At the desired fermentation time, the enzyme produced at various medium temperatures, moisture contents, and in each flask was extracted by adding 90 ml distilled water cultivation times. A quadratic model as a function of the and mixing in a rotary shaker (100 rpm) at room tempera- three mentioned parameters was evaluated for glucoam- ture for 30 min. Then the samples were filtered and the fil- ylase production by the fungi. Finally, the parameters trates were analyzed for glucoamylase activities. The filtrate effects on the enzyme production were studied and their was diluted by citrate buffer. Dilution should be such that optimum values to obtain the highest enzyme amount at the end of following stages, glucose concentration will be were reported. around 1 g/l. In these experiments, dilution was performed 252 Sanaz Behnam et al.: Optimization of glucoamylase production through SSF two times (one ml of the filtrate was mixed with one ml Table 1: The experimental domain (α=1.682). citrate buffer.) Afterwards, one ml of distilled water and 0.1 ml of the diluted sample were mixed in a 10-ml tube. Then Independent variables Symbol Range and level one ml of the substrate solution (1 w/w % of soluble starch (unit) in 50 mM citrate buffer) was added, mixed, and placed in -α -1 0 +1 +α a water bath at 60°C for 15 min. Afterwards, the reaction Temperature T (oC) 26.59 30 35 40 43.4 tube was immediately placed in boiling water for 5 min Moisture content (W/W) W (%) 38.18 45 55 65 71.81 to inactivate the enzyme and terminate the reactions. The Time I (h) 33.18 40 50 60 66.81 concentration of the released glucose was measured using 3,5-dinitrosalicylic acid (DNS) method of Miller [18]. Table 2: Experimental design and results for glucoamylase produc- tion (U/gds) by the fungi. 2.3 Experimental design and statistical NO. Uncoded level Fungus analysis TWI M. indicus M. hiemalis R. oryzae In order to study the effect of temperature, medium mois- 1 30.00 45.00 40.00 96.00 77.93 910 ture content, and cultivation time on the enzyme produc- 2 30.00 45.00 60.00 121.0 13.09 885 3 30.00 65.00 40.00 49.50 143.3 1158 tion and to find the optimum values to get the highest 4 30.00 65.00 60.00 161.5 66.60 645 amount of glucoamylase, Response Surface Methodology 5 40.00 45.00 40.00 64.50 28.84 825 (RSM) was used.
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