Alpha Amylase from Bacillus Pacificus Associated with Brown Algae

processes

Article Alpha Amylase from Bacillus paciﬁcus Associated with Brown Algae Turbinaria ornata: Cultural Conditions, Puriﬁcation, and Biochemical Characterization

Mona Alonazi 1 , Aida Karray 2 , Ahmed Yacine Badjah-Hadj-Ahmed 3 and Abir Ben Bacha 1,4,*

1 Biochemistry Department, Science College, King Saud University, P.O. Box 22452, Riyadh 11495, Saudi Arabia; [email protected] 2 Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS Route de Soukra, University of Sfax, Sfax 3038, Tunisia; [email protected] 3 Chemistry Department, Science College, King Saud University, P.O. Box 22452, Riyadh 11495, Saudi Arabia; [email protected] 4 Laboratory of Plant Biotechnology Applied to Crop Improvement, Faculty of Science of Sfax, University of Sfax, Sfax 3038, Tunisia * Correspondence: [email protected]; Tel.: +00966-115-0129; Fax: +00966-114-769-137

Abstract: We aimed in the current study, the identification of a marine bacterial amylase produced by Bacillus pacificus, which was associated with Turbinaria ornata. Cultural conditions were optimized for the highest amylase production on Tryptic soy broth media supplemented with starch 1% at initial pH 9, 55 ◦C for 24 h. The newly purified amylase was characterized for a possible biotechnological application. Data indicated that the obtained amylase with a molecular weight of 40 kD and the N-terminal sequence of the first 30 amino acids of amBp showed a high degree of homology with known alpha amylase, and was stable at 60 ◦C of pH 11. Among the tested substrate analogs, amBp was almost fully active on Alylose and Alylopectine (97%), but moderately hydrolyzed glycogen < sucrose < maltose < lactose. Therefore, the current amylase mainly generated maltohexaose from starch. Mg2+ and Zn2+ improved amylase activity up to 170%. While ethylenediamine tetraacetic Citation: Alonazi, M.; Karray, A.; Badjah- acid (EDTA) similarly induced the greatest activity with purified amylase, PCMB had the least Hadj-Ahmed, A.Y.; Ben Bacha, A. Al- effect. Regarding all these characteristics, amylase from marine bacterial symbionts amBp has a new pha Amylase from Bacillus pacificus promising feature for probable therapeutic, industrial, and nutritional applications. Associated with Brown Algae Turbina- ria ornata: Cultural Conditions, Pu- Keywords: α-amylase; Bacillus pacificus; purification; characterization rification, and Biochemical Character- ization. Processes 2021, 9, 16. https://dx.doi.org/10.3390/pr9010016 1. Introduction Received: 23 November 2020 Accepted: 20 December 2020 Starch is a glucose polymer containing of two types of α-glucans, amylose and amy- Published: 23 December 2020 lopectin, Amylose is a linear water insoluble polymer of glucose joined by α-1, 4 glycosidic bonds, while amylopectin is branched water soluble polysaccharide with short α-1, 4 and Publisher’s Note: MDPI stays neu- α-1, 6 [1]. Based on the mode of action, starch hydrolyzing enzymes may be endo-acting tral with regard to jurisdictional claims or exo-acting. α-Amylases (EC 3.2.1.1), categorized in GH-13 family of glyosyl hydro- in published maps and institutional lases, are the extracellular endo-acting enzymes that hydrolyse α-1,4 glycosidic linkages affiliations. of starch randomly, while bypassing the branch points and liberating α-limit dextrins as products [2], Sivaram, 1993 #271. Classified according to the similarity of the primary structure of the well-defend amino acid sequence of the catalytic domains, α-amylase mainly belongs to the GH13 family, together with the GH57, GH119, and eventually GH126 Copyright: © 2020 by the authors. Li- families (http://www.cazy.org/Glycoside-Hydrolases.html)[3]. censee MDPI, Basel, Switzerland. This article is an open access article distributed Amylases are universally distributed throughout the animal, plant, and microbial [4]. α under the terms and conditions of the Despite the large difference in the characteristics of microbial -amylases, their molecular Creative Commons Attribution (CC BY) weights are generally in the same range of 40 to 70 kDa [1]. It has been reported that license (https://creativecommons.org/ the α-amylase from Chloroflexus aurantiacus have the highest molecular weight with 210 licenses/by/4.0/).

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Processes 2021, 9, 16 2 of 13 reported that the α-amylase from Chloroflexus aurantiacus have the highest molecular weight with 210 kDa [5]. Whereas, much lower molecular weight of about 10 kDa, was noted from Bacillus caldolyticus [2]. kDaThe [5 industrial]. Whereas, application much lower of molecular amylolytic weight enzymes of about requires 10 kDa, thermostable was noted fromenzymesBacillus includingcaldolyticus the optimum[2]. temperature greater than or equal to 60 ° C. In recent years, several researchesThe industrial have been application done on of the amylolytic production enzymes of amylases requires by microorganisms thermostable enzymes that including the optimum temperature greater than or equal to 60 ◦C. In recent years, several are well appropriate for new several industrial applications thanks to their stability to researches have been done on the production of amylases by microorganisms that are well extreme conditions including increased salt, acidic or alkaline pH and higher appropriate for new several industrial applications thanks to their stability to extreme temperature. Among them α -amylases from marine Bacillus subtilis [6], Bacillus sp. conditions including increased salt, acidic or alkaline pH and higher temperature. Among dsh19-1 [7], and Bacillus aquimaris BaqA [8]. Their use in the industrial plants processes them α -amylases from marine Bacillus subtilis [6], Bacillus sp. dsh19-1 [7], and Bacillus offers the advantages of reducing the risk of contamination, reducing the reaction, the aquimaris BaqA [8]. Their use in the industrial plants processes offers the advantages of cost of cooling the exterior [9–11] and increase the diffusion rate [12]. The main reducing the risk of contamination, reducing the reaction, the cost of cooling the exte- amylolytic enzymes used in the starch industry are α-amylase, β-amylase, glucoamylase, rior [9–11] and increase the diffusion rate [12]. The main amylolytic enzymes used in the pullulanase, maltase, and α-1,6 glucosidase. Most of the commercial amylases are of starch industry are α-amylase, β-amylase, glucoamylase, pullulanase, maltase, and α-1,6 bacterial origin [13] for their interest in starch saccharification for, glucose, maltose, glucosidase. Most of the commercial amylases are of bacterial origin [13] for their interest maltotriosein starch, and saccharification dextrins production for, glucose, [13,14 maltose,]. maltotriose, and dextrins production [13,14]. Thus,Thus, improving improving the themicrobial microbial enzyme enzyme production production involves involves generally generally optimization optimization of of environmental environmental parameters parameters including mainly mainly pH, pH, temperature, temperature, substrate, substrate, andnutrients. and nutrients.α-amylase α-amylase is the mostis the critical most critical industrial industrial enzyme enzyme mainly mainly known known for degrading for degrading starch into starchsimple into sugar, simple and sugar, one of and the leading one of biocatalysts the leading in biocatalysts food processing in food industries, processing wastew- industries,ater treatment, wastewater and detergent treatment, production, and detergent in addition production, to its recent in addition clinical to and its medicinal recent clinicalapplications. and medicinal Thus, applications. in this study aThus, new inα-amylase this study from a newBacillus α-amylase pacificus fromassociated Bacillus with pacificusthe brown associated alga withTrubinaria the brown ornata algawas Trubinaria purified and ornata biochemically was purified characterized. and biochemically Cultural characterized.conditions Cultural were prior conditions optimized were for amylaseprior optimized production. for amylase production.

2. Results2. Results 2.1. 2.1.Cultivation Cultivation Conditions Conditions of Marine of Marine Bacterial Bacterial Amylase Amylase OptimizationOptimization of the of theFermentation Fermentation Media Media AmongAmong the theculture culture media media tested, tested, the theproduction production of the of theamylase amylase by the by theB. pacificus B. paciﬁcus strainstrain reached reached maximum maximum on onTSB TSB medium medium (16U (16U/mL)./mL). Whereas Whereas only only 4U 4U/mL/mL and and 6U 6U/mL/mL were found when NB and Pw were used, respectively. The composition media weakly were found when NB and Pw were used, respectively. The composition media weakly affected bacterial growth (Figure1). Furthermore, two inoculum sizes (0.5% and 1%) affected bacterial growth (Figure 1). Furthermore, two inoculum sizes (0.5% and 1%) from the overnight culture of the isolated strain were used to determine their effects on from the overnight culture of the isolated strain were used to determine their effects on enzyme production. It was noted that the inoculum size did not signiﬁcantly affect amylase enzyme production. It was noted that the inoculum size did not significantly affect production. (Data not shown). amylase production. (Data not shown).

Figure 1. Effect of culture media on Bacillus paciﬁcus growth (OD600) and amylase activity (U/mL). Starch soluble in 250 mL ﬂasks were incubated with 1 mL of bacterial overnight culture

(2 × 106 CFU/mL) at 37 ◦C for 48 h.

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Figure 1. Effect of culture media on Bacillus pacificus growth (OD600) and amylase activity (U/mL). Processes 2021, 9, 16 Starch soluble in 250 mL flasks were incubated with 1 mL of bacterial overnight culture (2 × 106 3 of 13 CFU/mL) at 37 °C for 48 h.

2.2. Effect of Physical Parameters 2.2. Effect of Physical Parameters 2.2.1.2.2.1. The The Effe Effectct of Incubation of Incubation Time Time on onCell Cell Growth Growth and and Amylase Amylase Prod Productivityuctivity InformationInformation from from Figure Figure 2 show2 showeded that that enz enzymeyme production production and and cell cell growth growth at atthe the samesame time time increased increased with with incubation incubation time time to to reach reach an an optimal optimal activity activity of of 18U 18U/mL/mL (at (at 24 24 h h of of incubationincubation time) and maximum cellcell growthgrowth nearlynearly inin 37 37 h h of of time time culture. culture. Then, Then, after after two twodays days of fermentation of fermentation at 37 at◦ C, 37 bacterial °C , bacter growthial growth and amylase and amylaseactivitydeclined activity dramatically. declined dramatically.Notably, bacterial Notably, growth bacterial and enzyme growth activity and enzyme are nonreciprocal. activity are This nonreciprocal. could be explained This couldby thebe explained exhaustion by of the nutrients exhaustion and of accumulation nutrients and of accumulation byproducts inof thebyproducts medium, in such the as medium,toxins, such protease as toxins, enzymes protease hydrolyzing enzymes amylases, hydrolyzing and couldamylases, be that and the could cells be have that reached the cellstheir have decline reached phase their and decline diminished phase and their diminished amylase synthesis their amylase [15,16 ].synthesis [15,16].

Figure 2. The time courses cell growth of Bacillus pacificus (filled square) and amylase production Figure(filled 2. circle).The time The courses culture cell was growth conducted of Bacillus at 55 ◦pacificusC in shaking (filled at square) 150 rpm. and Cell amylase growth production was monitored (filledby measuringcircle). The theculture absorbance was conducted at 600 nm. at 55 °C in shaking at 150 rpm. Cell growth was monitored by measuring the absorbance at 600 nm. 2.2.2. Effect of Initial pH, Temperature, and Substrate Concentration on Bacterial 2.2.2.Amylase Effect of Activity Initial pH, Temperature, and Substrate Concentration on Bacterial AmylaseThe Activit effecty of initial pH ranging from 4.0 to 11.0 on amylase production by Bacillus ◦ pacificusThe effect. cultivated of initial on pH TSB ranging media, from at 37 4.0C to for 11.0 24 on h wasamylase studied. production The highest by Bacillus amylase pacificusproductivity. cultivated was obtainedon TSB media, at pH9.0, at 37 indicating °C for 24 the h alkaliphilic was studied. nature The of highest strain, andamylase reaches productivity25U/mL. Moreover,was obtained a considerable at pH 9.0, reduction indicating of amylasethe alkaliphilic titer was nature noted in of acid strain pH, and values reach(Figurees 25U3A)./mL. Previous Moreover, work a considerable proved that thereduction optimum of amylase pH for amylase titer was production noted in acid from pHBacillus values cereus (Figurewas 3A 9). under Previous solid statework fermentationproved that [17 the]. Figureoptimum3B illustrates pH for amylase the effect ◦ ◦ productionof fermentation from Bacillus temperature cereus(30 wasC–70 9 underC) on solid the amylase state fermentation produced by[17]Bacillus. Figure pacificus 3B illustratesgrown inthe TSB effect fermentation of fermentation medium tem atperature pH 9.0 for(30 60 °C h.–70 Observably, °C ) on the theamylase enzymatic produced activity by improvedBacillus pacificus at incubation grown temperaturein TSB fermentation and maximum medium amylase at pH 9.0 production for 60 h. (30U/mL)Observably, was observed at 55 ◦C. Beyond 55 ◦C, amylase activity was reduced to 21U/mL and 7U/mL the enzymatic◦ activity◦ improved at incubation temperature and maximum amylase productionat 66 C and(30U 70/mL)C, was respectively. observed at This 55 is°C due. Beyond to decrease 55 °C , amylase in microbial activity growth was atreduced elevated to temperature. 21U/mL and For 7U/mL further at 66 comparison, °C and 70 the °C highest, respectively. amylase This secretion is due from to decreaseBacillus cereus in Bacillus ◦ microbial(IND4) growth and at elevatedsp. (WA21) temperature. was obtained For further at 45 C[ comparison,17,18]. the highest amylase

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(A) (B)

Amylase Activity (U/ml) (U/ml) Activity Amylase

(U/ml) Activity Amylase

(C)

Amylase Activity (U/ml) (U/ml) Activity Amylase

Figure 3. Effect of initial pH, temperatureFigure 3. Effect and of substrate initial pH, temperature concentration and substrat on bacteriale concentration amylase on bacterial activity. amylase (A): activity. Effect of initial pH on amylase production at 37 ◦C(A): for Effect 24 hof ininitial TSB pH medium. on amylase The production experiments at 37 °C werefor 24 h performed in TSB medium. in triplicate.The experiments Error bars are shown for the standard deviation.were (B): performed Influence in oftriplicate. the fermentation Error bars are shown temperature for the standard on amylase deviation. productivity (B): Influence of on the TSB and at fermentation temperature on amylase productivity on TSB and at pH 9.0 for 24 h. The experiments pH 9.0 for 24 h. The experiments werewere performed performed in triplicate. in triplicate. Error bars Error are barsshown are for shownthe standard for thedeviation. standard (C): Effect deviation. of starch (C): Effect of starch concentration (0–2%) onconcentration amylase activity (0%–2%) from on amylaseBacillus activity pacificus fromwhen Bacillus incubated pacificus when in aincubated shaking in incubatora shaking at 55 ◦C, incubator at 55 °C, initial pH of 9.0 for 24 h. The experiments were performed in triplicate. initial pH of 9.0 for 24 h. The experiments were performed in triplicate. 2.3. Purification of Amylase from Bacillus pacificus amB.p On theExtracellular other hand, α-amylase, the enzymatic namely, amB.p, activity was produced of the α -amylaseby Bacillus pacificus increased grown with an increase in theon substrate TSB media, (starch) supplemented concentration with 1% starch (Figure soluble3C) at to pH attain 9 and a55 maximum °C for 24 h. ofSuch 35 U/mL with 1% starch.conditions The are same optimal hydrolysis for amylase rate (35U/mL)activity. It was was purified observed by ammonium with 1.5% sulfate starch, and slightly fractionation (40%–85%) followed by Mono Q Sepaharose chromatography. The deceasedhomogeneity beyond. of Indeed,α-amylase itamB.p has beenwas eluted reported into four that separate carbon peaks sources with stronglya linear affected the amylasegradient production of NaCl (0.15 whereas, to 0.5 M) the with most a specific commonly activity of used 105 units/mg substrate protein is starch and fold [19 ]. purification of 8.07 (Figs. 4 and Table 1). The molecular mass of the purified α-Amylase 2.3. Purificationwas approximately of Amylase 40 kDa from by SDS-PAGE Bacillus pacificus (Figure 4B). amB.p AmB.p N-terminal sequencing allowed the αclear identification of 25 residues of the pure enzyme: Extracellular(DAILHAFNWKYSRVTANAEQKAAAG).-amylase, namely, amB.p,The resu waslts presented produced in Table by Bacillus2 show the pacificus grown ◦ on TSBalignment media, of supplementedthe N-terminal sequence with 1%of amylase starch from soluble Bacillus at pacificus pH 9 (present and 55 work)C for 24 h. Such conditions are optimal for amylase activity. It was purified by ammonium sulfate fractionation (40–85%) followed by Mono Q Sepaharose chromatography. The homogeneity of α-amylase amB.p was eluted into four separate peaks with a linear gradient of NaCl (0.15 to 0.5 M) with a specific activity of 105 units/mg protein and fold purification of 8.07 (Figure4 and Table1). The molecular mass of the purified α-Amylase was approximately 40 kDa by SDS-PAGE (Figure4B). AmB.p N-terminal sequencing allowed the clear identification of 25 residues of the pure enzyme: (DAILHAFNWKYSRVTANAEQKAAAG). The results presented in Table2 show the alignment of the N-terminal sequence of amylase from Bacillus pacificus (present work) with those from Tepidimonas fonticaldi, from unclassified Vibrio and Vibrio sp. A8-1 with an accession number QBN20693.1, WP161425494.1, and WP_170905550.1, respectively. Processes 2020, 8, x; doi: FOR PEER REVIEW 6 of 15

with those from Tepidimonas fonticaldi, from unclassified Vibrio and Vibrio sp. A8-1 with Processes 2021, 9, 16 5 of 13 an accession number QBN20693.1, WP161425494.1, and WP_170905550.1, respectively.

0.8 (A) 1 2 0.7 NaCl M

0.6 0.5 M 0.5

0.45 M 0.4 (B)

0.3 0.35 M

0.2

0.25 M 0.1

0.15 M

Figure 4. Purification of amB.p (A) ion exchange chromatography on Mono Q Sepaharose. Column Figureof 4. Mono Purification Q (20 cm of amB.p× 2.5 cm)(A) ion was exchange equilibrated chromatography with 0.1 M Tris-HCl on Mono buffer, Q Sepaharose. pH 8. Non-adsorbed Column of Monoproteins Q (20 were cm removed× 2.5 cm) bywas rinsing equilibrated the column with within0.1 Μ theTris-HCl same buffer.buffer, pH The 8. bound Non-adsorbed proteinswere proteinsthen were eluted removed into four by separaterinsing the peaks column with within a linear the gradient same bu offfer. NaCl The (0.15 bound to 0.5 proteins M) at a were flow rate then ofeluted 30 mL/h, into four and separate 2.0 mLfractions peaks with were a linear collected. gradient (B) SDS-PAGE of NaCl (0.15 for toBacillus 0.5 M) pacificus at a flowα -amylase.rate of 1: 30 mL/h, and 2.0 mL fractions were collected. (B) SDS-PAGE for Bacillus pacificus α-amylase. 1: Molecular markers (14kD, 20kD, 30kD, 43kD, 67kD, and 97Kd. 2: Mono Q Sepaharose amB.p. Molecular markers (14kD, 20kD, 30kD, 43kD, 67kD, and 97Kd. 2: Mono Q Sepaharose amB.p.

Table 1. Flow sheet of amBp. puriﬁcation.

Total Specific Activity Protein Purification Purification Step Activity Activity Recovery (Mg) Factor (Units) (U/Mg) (%) Crude extract 6210 477.7 13 100 1 Heat treatment (70 4937 133.4 37 79.5 2.8 ◦C, 10 min) Ammonium sulfate Fractionation 3160 58.5 54 58 4.1 (40–85%) Mono Q-Sephadex 1585 15.1 105 25.5 8.07

Table 2. N-terminal sequence analysis of amB.p.

DAILHAFNWKYSRVTANAEQKAAAG (Present Work) DAIVHAFNWKYSRVTANAEHKAAAG (α-amylase from Tepidimonas fonticaldi QBN20693.1) DAILHAFNWKYSDVTANAEHIAAAG (α-amylase from unclassiﬁed Vibrio WP161425494.1) DAILHAFNWKYSDVTANAEHIAAAG (α-amylase from Vibrio sp. A8-1 WP_170905550.1)

2.4. Pure Amylase amB.p Characterization The Effect of pH and Temperature on Amylase Activity and Stability Pure amB.p was characterized in terms of pH and temperature; activity and stability. Regarding pH, it is as well a critical parameter that must be controlled for optimal amylase activity. Figure5A illustrates both the effect of pH on activity and stability of the complexes’ enzyme-substrate catalysis reaction. When we ﬁrst measured the amylase activity at different pH values, pure amylase was found to be active in the pH range of 6.0–12.0, with maximal activity at pH 9.5. For pH stability, while α-Amylase from Bacillus paciﬁcus had sharp pH stability ranging from 6.0 to 11.0 for 1 h, enzyme stability was relatively low in

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Pure amB.p was characterized in terms of pH and temperature; activity and stability. Regarding pH, it is as well a critical parameter that must be controlled for optimal amylase activity. Figure 5A illustrates both the effect of pH on activity and stability of the Processes 2021, 9, 16 complexes’ enzyme-substrate catalysis reaction. When we first measured the amylase 6 of 13 activity at different pH values, pure amylase was found to be active in the pH range of 6.0–12.0, with maximal activity at pH 9.5. For pH stability, while α-Amylase from Bacillus pacificus had sharp pH stability ranging from 6.0 to 11.0 for 1 h, enzyme stability was acidicrelatively pH ranges low in (3.0–7.0). acidic pH ranges Interestingly, (3.0–7.0). Interestingly, amylase was amylase found was to found be thermostable to be since it thermostable since it maintains 98.5% ◦ of its activity at 60 °C and with an optimum ◦ maintainstemperature 98.5% activity of its at activity55 °C . At 80 at °C 60 theC enzyme and with lost most an optimum of its activity. temperature activity at 55 C. At 80 ◦C the enzyme lost most of its activity.

(A) (B)

(%)

Residual Activity Residual

Residual Activity (%) Activity Residual

Figure 5. (A) Effect of pH on pure amylase activity and stability. For pH activity, the relative activity was measured by incubating pure enzyme with starchFigure substrate 5. (A) Effect at different of pH on pH pure values amylase (6-12). activity Buffers and usedstability are. For as follows: pH activity, A phosphate the relative buffer, pH 6.0–8; glycine–NaOH buffer, pHactivity 9.0–12.0. was pH measured stability by of incubating pure amylase pure enzyme was determined with starch substrate by incubation at different for pH 1 h values at specified pH (6-12). Buffers used are as follows: A phosphate buffer, pH 6.0–8; glycine–NaOH buffer, pH values (with buffers cited above).9.0 Results–12.0. pH are stability the average of pure amylase of triplicate was determined experiments. by incubation (B) Effect for 1 of h temperatureat specified pH onvalues pure amylase activity. The enzyme was incubated(with with buffers the substrate cited above). for Results1 h at different are the average temperatures of triplicate for 1 experiments.h. Thermostability (B) Effect of amylaseof was determined by prior incubating soletemperature pure enzyme on pure at amylase temperature activity. forThe 1 enzyme h. Results was incubated are the average with the of substrate triplicate for experiments.1 h at different temperatures for 1 h. Thermostability of amylase was determined by prior incubating sole pure enzyme at temperature for 1 h. Results are the average of triplicate experiments. 2.5. Substrate Speciﬁcity of B. paciﬁcus α-Amylase amB.p 2.5. Substrate Specificity of B. pacificus α-Amylase amB.p The ability of amB.p to catalyze the hydrolysis of several substrates’ analogs to starch was The ability of amB.p to catalyze the hydrolysis of several substrates’ analogs to starch studiedwas studied to characterize to characterize the enzyme the enzyme biochemically. biochemically. Figure Figure6 6shows shows that that amB.p preferentially catalyzespreferentially the hydrolysis catalyzes ofthe starch. hydrolysis Pure ofamB.p starch.remained Pure amB.p active remained (up toactive 97% (up and to 92%)97% at the analogs structurallyand 92%) related at the to analogs starch, structurally Alylose and related Alylopectine, to starch, Alylose respectively. and Alylopectine, Therefore, weakening the enzymerespectively. catalyzed Therefore, the hydrolysis weakening ofthe substratesenzyme catalyzed in the the order hydrolysis of glycogen of substrates < sucrose in < maltose < the order of glycogen < sucrose < maltose < lactose. These findings clearly indicate that lactose.the substrates These findings with clearly high molecular indicate that weight the have substrates high withaffinity high toward molecular enzyme. weight have high Processes 2021, 9, x FOR PEER REVIEW affinityNevertheless, toward enzyme.α-amylase Nevertheless,was not able to digestα-amylase directly was the notraw ablestarch. to digest7 of directly 14 the raw starch.

Residual activity (%) activity Residual

FigureFigure 6. Hydrolysis 6. Hydrolysis efficiency efﬁciency of amylase of amylase produced produced by Bacillus by Bacilluspacificus toward paciﬁcus differenttoward different substrates: substrates:Starch, alylose,Starch, alylose, alylopectine, alylopectine, glycogen, glycogen, sucrose, sucrose, maltose,maltose, and and lactose. lactose. The The different different polysaccharides polysaccharides and disaccharides (1 mg/mL) were mixed with pure amylase enzyme and reaction wasand incubated disaccharides at 55 °C at (1pH mg/mL) 9. were mixed with pure amylase enzyme and reaction was incubated at 55 ◦C at pH 9. 2.6. Analysis of the Starch Hydrolysis Products The HPLC profile of the starch hydrolysis products at an E/S ratio of 10 was examined for the purified enzyme. Clearly, the maltohexaose was the preponderant hydrolysis product (Figure 7), compared with standards.

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2.6. Analysis of the Starch Hydrolysis Products The HPLC proﬁle of the starch hydrolysis products at an E/S ratio of 10 was examined Processes 2021, 9, x FOR PEER REVIEW for the puriﬁed enzyme. Clearly, the maltohexaose was the preponderant8 of 14 hydrolysis product (Figure7), compared with standards.

(A) (B)

(C)

Figure 7. HPLC Profiles of the starch hydrolysis products of amBP under E/S ratio of 10U/g. (A) HPLC profile of a solution containing 10Figure g/L soluble7. HPLC starch. Profiles (B) of Standards the starch HPLC hydrolysis profile ofproducts a solution of amBP containing under 8 g/LE/S ratio glucose of 10U/g. and 5 g/L (A) for each of maltose, maltotriose,HPLC profile maltotetraose, of a solution maltopentaose, containing maltohexaose 10 g/L soluble (DP6), starch. and (B maltoheptaose) Standards HPLC (DP7). profile (C) HPLC of a profiles of the starch hydrolysissolution containing products relative 8 g/L to glucose amBP. and 5 g/L for each of maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose (DP6), and maltoheptaose (DP7). (C) HPLC profiles of the starch hydrolysis products relative to amBP. 2.7. Effect of Metal Ions and Chemical Reagents on Pure α-Amylase Activity 2.7. Effect of Metal IonsFrom and Figure Chemical8A, Reagents the enzyme on Pu activityre α-Amylase increased Activity withthe addition of some tested metal ions (0.1%) to reach 187.9%, 176.63%, 157.95%, and 146.7% in the presence of MgCl , MgSO , From Figure 8A, the enzyme activity increased with the addition of some tested 2 4 ZnSO , and ZnCl , respectively. These findings support Mg2+ and Zn2+ ions enhanced metal ions (0.1%) to4 reach 187.9%,2 176.63%, 157.95%, and 146.7% in the presence of amylase activity. Indeed, similar results confirmed that Mg2+ acts as an activator of α- MgCl2, MgSO4, ZnSO4, and ZnCL2, respectively. These findings support Mg2+ and Zn2+ amylase from B. licheniformis. Figure8A also shows that most tested metals have moderate ions enhanced amylase activity. Indeed, similar results confirmed that Mg2+ acts as an inhibitory effect on amylase activity such as BaCl and BaSO . Many amylases had Ca2+ activator of α-amylase from B. licheniformis. Figures 8A also 2 shows that4 most tested cation in the active site, therefore, they are metal ion-dependent [20,21]. Concerning the metals have moderate inhibitory effect on amylase activity such as BaCL2 and BaSO4. effect of some chemical reagents, while N-Ethylmaleimide and urea poorly inhibited amBp 2+ Many amylasesactivity had Ca (96% cation residual in the activity), active site, PCMB therefore, strongly they affected are metal the amylase ion-dependent hydrolysis (Figure8B). [20,21]. Concerning the effect of some chemical reagents, while N-Ethylmaleimide and urea poorly inhibited amBp activity (96% residual activity), PCMB strongly affected the amylase hydrolysis (Figures 8B).

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(A) (B)

Figure 8. Influence of metal ions (0.1%) (A) and chemical reagents (B) (5 mM) on Bacillus pacificus α-amylase activity. Figure 8. Influence of metal ions (0.1%) (A) and chemical reagents (B) (5 mM) on Bacillus pacificus α-amylase activity. 3. Discussion 3. Discussion Macroalgae offer a rich substrate and safe habitat for bacteria to grow and reproduce novel secondary metabolites of potential activity and bioapplication. Identifying and estab- Macroalgae offer a rich substrate and safe habitat for bacteria to grow and reproduce lishing algae-bacterial association is now possible using new microbiological, molecular, novel secondary metabolites of potential activity and bioapplication. Identifying and and microscopic techniques. However, the rate of bacterial occurrence, distribution, and establishing algae-bacterial association is now possible using new microbiological, presence associated with brown algae along with their ecological contribution has not yet molecular, and microscopic techniques. However, the rate of bacterial occurrence, been determined. Screening and identification of bacteria inhabiting marine macroalgae distribution, andare presence now the associated main objective with of brown research algae involving along taxonomy with their and ecological ecology. In this study, the contribution has ability not yet of Bacillus been determined. pacificus associated Screening with the and macroalga identificationTurbinaria of bacteria ornata recently identified inhabiting marine(unpublished macroalgae data) are to now produce the α main-amylase objective is studied. of research involving taxonomy and ecology.In In the this literature, study, the several ability works of Bacillus have been pacificus focused associated on the with production the of α-amylase macroalga Turbinariausing ornat variousa recently media identified compositions (unpublished with different data) to substrates produce byα-amylaseBacillus sp. Therefore, in is studied. current study, the initial screening of isolated Bacillus sp. associated with the macroalga In the literature,Turbinaria several ornata worksrevealed have been its capability focused toon produce the productionα-amylase of α and-amylase was first demonstrated using various mediausing compositions the iodine test with on different starch agar substrates plates. by Bacillus sp. Therefore, in current study, the initialOptimization screening of theisola detectedted Bacillus marine sp. associated bacterial α -amylasewith the macroalga production started by deter- Turbinaria ornata revealedmining the its phasecapability of the to cell produce growth α in-amylase which amylaseand was secretion first demonstrated is initiated, the biomass and using the iodine testincubation on starch time agar effects plates. on amylase activity for 0–72 h. Our findings demonstrated that the Optimizationhighest of theα -amylase detected activity marine (16.04 bacterial± 3 U/mL)α-amylase was production recorded at 24started h of incubation,by although determining the the phase bacterial of the growth cell growth was slightly in which less amylase compared secretion with 36 is hinitiated incubation, the time. Prolonged biomass and incubationincubation time time effects increased on the amylase bacterial activity count but for affected 0–72 h. the Ourα-amylase findings activity negatively. demonstrated thatThese the highest findings α-amylase were similar activity to Singh(16.04 et± 3 al. U [/mL)22] and was Paul recorded et al. [at23 24] who h of reported that an incubation, althoughextended the incubation bacterial growth period beyond was slightly 48 h did less not compared increase the with enzyme 36 h production from incubation time. ProlongedBacillus sp. incubation strain B-10 time and increasedBacillus sp. the MB6, bacterial respectively. count but As affected for the the inoculum size, this α-amylase activitystudy negatively. showed These no significant findings were alteration similar in αto-amylase Singh et productional. [22] and regardingPaul et the volume of al. [23] who reportedthe bacterialthat an extended suspension incubation used. The period inoculum beyond size 48 plays h did anot notable increase role the in the fermentation enzyme productiorate.n from However, Bacillus the sp. volume strain B could-10 and range Bacillus from sp. 0.5% MB6, for respectively.B. amyloliquefaciens As for [24] to 2.95% [25] the inoculum size,for thisBacillus studysp. showed and 8%no forsignificantB. cereus alteration[26]. Temperature in α-amylase was production also controlled because it regarding the volumegreatly of the affected bacterial enzyme suspension production. used. Notably, The inoculum temperature size plays can a affect notable enzyme availability role in the fermentationby directly rate. affecting However, the reaction the volume constant could rate or range by thermal from denaturation 0.5% for B. of the enzyme at amyloliquefaciens [24]elevated to 2.95% temperatures [25] for Bacillus [27]. Data sp. and from 8% this for study B. cereus indicated [26]. Temperature that the maximal α-amylase ◦ was also controlledactivity because was it obtainedgreatly affected at initial enzyme pH 9 underproduction. shaking Notably, for 24 temperature h at 55 C. Therefore, media can affect enzymecontained availability TSB (Tryptic by directly soy broth), affecting and the supplemented reaction constant with 1% rat starche or allowed by to reach the thermal denaturationhighest of the enzyme enzyme activity at elevated (35U/mL). temperatures Our study [27] indicates. Data from alkaliphilic this study nature of strain since indicated that thethe maximalα-amylase α-amylase production activity and was the obtained microbial at growth initial werepH 9 optimalunder shaking at pH 9.0. Similar result for 24 h at 55 °C . wasTherefore, described media for contained amylase producingTSB (TrypticBacillus soy broth), cereus anunderd supplemented solid state fermentation [26]. with 1% starch allowed to reach the highest enzyme activity (35U/mL). Our study

Processes 2021, 9, 16 9 of 13

Under optimal conditions, the α-amylase produced by Bacillus pacificus was purified by ammonium sulfate fractionation (40–85%), a brief heat treatment followed by Mono Q Sepaharose chromatography. The eluted enzyme with a linear gradient of NaCl (0.15 to 0.5 M) was found to have a specific activity of 105 units/mg protein and fold purification of 8.07. The molecular mass of α-Amylase amB.p was approximately 40 kDa by SDS- PAGE. A similar molecular weight was detected in T. matsutake [28]. Moreover, the higher molecular weight of α-amylase was detected for T. harzianum (70 kDa) [29]. Biochemical characterization of pure enzyme included the effect of pH and temperature on the activity and stability. Our results indicated that pH 9 was the optimal value for maximal marine bacterial amylase activity. Similar results were obtained with Saxena et al. [14] where alkaline conditions (pH 7.5–11.0) improve α-amylase production in Bacillus sp. Maximal activity of amBp was recorded at 55 ◦C. Similar result was obtained with marine α-amylase from Bacillus subtilis [6]. For pH stability, α-Amylase from Bacillus pacificus had sharp pH stability ranging from 6.0–11.0 for 1 h. Conversely, enzyme stability relatively decreased in acidic pH ranging from 3.0 to 7.0. The current findings clearly showed the industrially interesting characteristic alkaliphilic nature of amBp and thus making it a suitable additive. In a previous study, B. methylotrophicus amylase was found to be stable at pH 6.0–9.0 for 1 h, with a significant decrease of its initial activity at pH 4.0 and 5.0. Interestingly, α-amylase was found to be thermostable since it maintains 98.5% of its activity at 60 ◦C. At 80 ◦C, the enzyme lost most of its activity. Our findings showed an alkali-tolerant and a high thermal nature of the enzyme, a dual extreme characteristic, since amBp was 100% stable up to 9.0 pH and 65 ◦C. In 2013, an acid-alkali stable α-amylase from a marine bacterium Bacillus subtilis S8–18 was isolated and was found to be highly stable for 24 h over a wide range of pH from 4.0 to 12.0 by showing 84–94% activity [6]. For the substrate specificity, α-amylase from Bacillus pacificus hydrolyzes preferentially soluble starch followed by Alylose and Alylopectine. amBp was not able to display any enzymatic activity with potato starch. Therefore, the current amylase mainly generated maltohexaose from starch. Conversely, various metallic salts with the chemical reagents affected the activity of the α-amylase enzyme. Divalent cations Mg2+ increased the activity of the purified α-amylase. EDTA similarly induced the greatest activity with purified α-amylase, whereas PCMB had the least effect. Observably, throughout this study, bacterial growth and enzymatic productivity or stability or both are nonreciprocal, this could be explained by the fact that Bacillus isolate was identified living together with Turbinaria ornata and that this latter is rich in different kind of starch [30] thus this bacterium utilized the starch present in the medium as the single carbon source and then produced its extracellular α-amylase where it reached its maximal activity followed by a decrease in the bacterial growth due to the depletion of the carbon source in the medium.

4. Materials and Methods 4.1. Microorganism and Cultivation Conditions Bacterial strains associated with the brown alga Turbinaria ornata (bacterial symbiont) were isolated from the surface of the macroalga collected from shallow surface of the Red Sea, Jeddah, Saudi Arabia. A direct swab from the algal surface onto Tryptone Soy agar (TSA) (Oxoid, Lenexa, KS, USA) and Marine agar (MA) (Oxoid, USA) plates were incubated aerobically at 37 ◦C for 18–24 h. Bacterial strains, isolated, identiﬁed, and stored in the Botany and Microbiology Department-College of Science at King Saud University (Riyadh, Saudi Arabia) were screened for amylase production in starch liquid medium. The media included, separately: TSB (Tryptic soy broth), NB (nutrient broth) and PW (peptone water) and supplemented with 1% starch soluble in 250 mL ﬂasks were incubated with 1 mL of the bacterial overnight culture (2 × 106 CFU/mL). The media were incubated in a shaking incubator at 37 ◦C for 3 days. Then, cultures were tested and screened based on the highest α-amylase productivity. Samples were collected at a regular interval of 24 h, and growth was determined spectrophotometrically (OD) at 600 nm. Processes 2021, 9, 16 10 of 13

Data were presented as average ± standard deviation (SD) for three independent determinations. Each culture broth was centrifuged at 10,000 rpm for 10 min at 4 ◦C and the collected supernatant was used as an enzyme source for further biochemical analysis of α-amylase activity.

4.2. α-Amylase Assay α-Amylase activity was measured at the indicated pHs and temperatures by determining the liberated reducing sugars as end products according to Nelson’s method (1944). One unit of enzyme activity was deﬁned as the amount; which catalyzed 1 mmol formation of maltose under the assay conditions.

4.3. Inoculum Size The inoculum size effect on both α-amylase activity and cell growth was investigated as follows: 50 mL of selected fermentation medium was inoculated with 2 × 106 and 2 × 109 CFU/mL (v/v) of overnight bacterial. After 60 h of incubation, the α-amylase activity was investigated in collected culture ﬁltrate. Media were incubated in a shaking incubator at 37 ◦C for an interval of 2 days and checked for cell growth and α-amylase activity.

4.4. The Effect of Incubation Time The effect of various incubation times on microbial growth and enzyme production was checked in the appropriate selective medium. Samples were collected at a regular interval of 12 h. Growth (OD) and α-amylase activity was measured using a spectropho- tometer.

4.5. Inﬂuence of Initial pH and Temperature Both temperature and pH are crucial parameters for the determination of the enzyme activity. Indeed, amylase activity was measured after incubation at initial pH values ranging from 4.0–11.0 and a temperature range 40 ◦C–70 ◦C, separately. First, 10% (v/v) inocula was used to inoculate the enzyme production medium. Buffers at pH values ranging from 4 to 11 were used to test the α-amylase activity at 37 ◦C. Indeed, ﬂasks at the appropriate pH values were incubated in a shaking incubator at 37 ◦C for 24 h. Whereas, for temperature optimization, activity was determined by testing the enzyme activity at temperatures ranging from 40 to 70 ◦C and the pH was adjusted to 9.0. α-amylase activity was determined after centrifugation for 10 min at 10,000 rpm and 10 ◦C, as previously described.

4.6. Effect of Substrate Concentration Effect of starch concentration (0–2%) on α-amylase activity from Bacillus paciﬁcus was tested in triplicate with incubation at 55 ◦C for 24 h, and at initial pH of 9.0.

4.7. α-Amylase Puriﬁcation The supernatant containing extracellular α-amylase produced in the optimized medium was incubated for 10 min at 70 ◦C, then rapidly cooled and centrifuged (at 12,000 rpm, 30 min) to discard insoluble material. The resulting supernatant containing approximately 80% of the initial α-amylase activity was subjected to ammonium sulfate fractionation (40–85%). After centrifugation for 30 min at 12,000 rpm, the precipitate obtained was resus- pended in 25 mM Tris-HCl buffer, pH 8 and dialyzed overnight at 4 ◦C against repeated changes after 6 and 12 h in the same buffer. The clear supernatant was then loaded on a 20 cm × 2.5 cm column of Mono Q Sepaharose, pre-equilibrated with the Tris-HCl buffer, pH 8. Non-adsorbed proteins were removed by rinsing the column within the same buffer. The bound proteins were then eluted into four separate peaks with a linear gradient of NaCl (0.15 to 0.5 M). Every 6 min, fractions of 2.0 mL were collected and assayed for α-amylase activity and protein content. The sample with the highest amylase activity were gathered, lyophilized, and stored at 4 ◦C until further use. Processes 2021, 9, 16 11 of 13

4.8. Protein Analysis The protein content was checked by the Bradford method [31] using crystalline bovine serum album as a standard. The purity of the isolated α-amylase and estimation of its molecular mass were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 15% polyacrylamide gels in the presence of β-mercaptoethanol as a reducing agent [32] while its N-terminal sequence was determined by Edman’s degradation technique according to Hewick et al. [33].

4.9. HPLC Analysis of the Starch Hydrolysis Products The puriﬁed α-amylase reacted separately on starch at enzyme/substrate ratio (E/S) of 10 U/g. In a screwcapped tube, 5 mL of the reaction medium containing 10 g/L starch was incubated at 60 ◦C for 24 h. An Aminex HPX-42A (Bio Rad Laboratories S.A., Marnes La Coquette, France) column packed with a sulfonated polystyrene divinylbenzene resin in silver ionic form was used for HPLC analysis of the taken samples. The column (300 × 7.8 mm) was used in ion-moderated partition involving the separation of oligosac- charides (up to DP11). Elution of the hydrolysis products were performed at a ﬂow rate of 0.4 mL/min with water and a Smartline Refractive Index Detector 2300 (Knauer GmbH, Berlin, Germany) was used for detection.

4.10. Metal Ions and Chemical Reagents Effects on α-Amylase Activity The effect of several metal ions and compounds on the α-amylase activity was investigated by pre-incubating the enzyme solution for 5 min in the presence of the respective compounds or ion at room temperature. Then, aliquots were withdrawn at the end of the incubation time and tested under optimal conditions. The α-amylase activity was expressed as a percentage of the control enzyme activity (100%).

4.11. Substrate Specificity Amylase substrate specificity was determined in an assay mixture containing numer- ous disaccharides and polysaccharides (1 mg/mL) at 55 ◦C. To estimate the amount of released maltose, aliquots were withdrawn at the time interval. The ability of α-amylase to digest raw starch was investigated using the native insoluble starches (corn starch and potato starch). [34] Briefly, the starches (5%) were suspended in 500 mL of 100 mM Tris buffer (pH 9.0). The reaction was started by adding 25 µl of α-amylase followed by incubation at 55 ◦C.

5. Conclusions This study described the optimization of cultural conditions for the production of α-amylase from the identiﬁed marine Bacillus paciﬁcus associated with the brown alga Turbinaria ornata. The highest amylase titer was observed when grown in TSB medium supplemented with starch 1% at 24 h incubation time at 55 ◦C and pH 9. This study provided a promising outline for marine microbial α-amylase and opened new horizons for its exploration, engineering, and various applications since the protein remains 100% stable up to 9.0 pH and 65 ◦C.

Author Contributions: Conceptualization, A.K. and A.B.B.; methodology, M.A.; formal analysis, A.Y.B.-H.-A.; investigation, M.A. and A.Y.B.-H.-A.; resources, M.A.; data curation, A.K.; writing— original draft preparation, A.K.; writing—review and editing, A.B.B. and A.K.; visualization, A.Y.B.- H.-A.; supervision, A.B.B.; project administration, M.A. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statements: Data is contained within the article. Processes 2021, 9, 16 12 of 13

Acknowledgments: The authors extend their appreciation to the researchers Supporting Project number (RSP-2020/237), King Saud University, Riyadh, Saudi Arabia, for funding this work. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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