Purification, Biochemical Characterization, and Gene Cloning of a New Extracellular Thermotolerant...

Purification, Biochemical Characterization, and Gene Cloning

of a New Extracellular Thermotolerant and Glucose Tolerant

Maltooligosaccharide-Forming �-Amylase from an Endophytic

Ascomycete Fusicoccum sp. BCC4124

Verawat CHAMPREDA,y Pattanop KANOKRATANA, Rutchadaporn SRIPRANG,

Sutipa TANAPONGPIPAT, and Lily EURWILAICHITR

Molecular and Enzyme Screening Laboratory, BIOTEC Central Research Unit, National Center

for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Paholyothin Road,

Klong Luang, Patumthani 12120, Thailand

Received April 5, 2007; Accepted May 15, 2007; Online Publication, August 7, 2007

[doi:10.1271/bbb.70198]

An endophytic fungus, Fusicoccum sp. BCC4124,

showed strong amylolytic activity when cultivated on

multi-enzyme induction enriched medium and agro-

industry substrates. �-Amylase and �-glucosidase activ-

ities were highly induced in the presence of maltose

and starch. The purified target �-amylase, Amy-FC1,

showed strong hydrolytic activity on soluble starch

(kcat/Km ¼ 6:47� 103 min�1(ml/mg)) and selective ac-

tivity on �- and �-cyclodextrins, but not on �-cyclo-

dextrin. The enzyme worked optimally at 70 �C in a

neutral pH range with t1=2 of 240min in the presence of

Ca2þ and starch. Maltose, matotriose, and maltotetraose

were the major products from starch hydrolysis but

prolonged reaction led to the production of glucose,

maltose, and maltotriose from starch, cyclodextrins, and

maltooligosaccharides (G3–G7). The amylase showed

remarkable glucose tolerance up to 1M, but was more

sensitive to inhibition by maltose. The deduced protein

primary structure from the putative gene revealed that

the enzyme shared moderate homology between �-

amylases from Aspergilli and Lipomyces sp. This

thermotolerant, glucose tolerant maltooligosaccharide-

forming �-amylase is potent for biotechnological appli-

cation.

Key words: Fusicoccum sp.; �-amylase; thermostability;

glucose tolerant; gene cloning

In today’s biotechnological industry, starch-convert-ing enzymes play essential roles in a number ofapplications, including the starch processing, food, anddetergent industries.1) The enzymatic hydrolysis ofstarch involves the synergistic action of many endo-and exo-acting amylolytic enzymes. The key amylolyticenzyme, �-amylase (endo 1,4-�-D-glucan glucohydro-

lase: EC 3.2.1.1) randomly cleaves the �-1,4 glycosidiclinkages, giving oligosaccharides of varying chainlength with an �-configuration and �-limited dextrinsas products, which are further hydrolysed to glucose byexo-acting amylases and other amylolytic enzymes.Among the diverse microorganisms producing amy-

lases, �-amylases from Bacillus sp. have been the mostextensively studied, partly due to their extreme thermo-stability,2,3) while other bacterial amylases with specialproperties are also continually reported.4) However,mesophilc and thermophilic fungi are also importantsources of amylolytic enzymes due to their differentproperties as to bacterial enzymes, including optimalworking conditions and reaction specificity. Theseenzymes are widely used in many applications, includ-ing the food, feed, and fermentation industries. Detailedstudies on amylolytic enzymes have been largely limitedto a few species of mesophilic and thermophilicfungi,2,3) especially Aspergillus sp., which have beenstudied in terms of theory and application.5) In addition,a number of new fungal isolates producing amylolyticenzymes have also been reported recently, e.g., Rhizo-mucor pusillus6) and Trichoderma sp.,7) accentuatingacademic and industrial interest on amylases. Driven bythe search for new amylolytic enzymes with desirableindustrial properties, including improved thermostabil-ity, novel reaction specificity, and low inhibition byglucose, the end product of the saccharification process,the exploration of new fungi as a source of potent starchconverting enzymes is thus of interest.Endophytic fungi are considered potent microbes

producing hydrolytic enzymes targeting plant-derivedmacromolecules. Considerable interest has been devotedto ascomycete in the family Botryosphaeriaceae as toenzyme production and characterization.8,9) Amylolytic

y To whom correspondence should be addressed. Tel: +66-2564-6700 ext. 3473; Fax: +66-2564-6707; E-mail: [email protected]

Biosci. Biotechnol. Biochem., 71 (8), 2010–2020, 2007

http://dx.doi.org/10.1271/bbb.70198

enzyme production from Botryodiplodia theobromae(Lasiodiplodia theobromae) grown on different agricul-tural residues has also been reported.10,11) The BIOTECCulture Collection (BCC) holds one of the world’slargest fungal collections, which is a promising sourcefor industrial enzyme discovery.12,13) From an extensivescreening of the fungal isolates collected in BCC, anendophyte Fusicoccum sp. BCC4124, an asexual state ofBotryosphaeria sp., was identified for its strong thermo-tolerant amylolytic activity. In this study, the production,inducibility, biochemical characterization and putativegene cloning of the new thermotolerant and glucosetolerant maltooligosaccharide forming extracellular �-amylase (Amy-FC1) from this fungus is reported. Thisshould contribute to understanding of the diversity offungal �-amylases and their applications, accentuatingthe potential of biodiversity wealth as the source of newenzymes of industrial and academic interest.

Materials and Methods

Microorganism strain and growth conditions. Anendophyte Fusicoccum sp. BCC4124 was obtained fromBIOTEC Culture Collection in Thailand (www.biotec.or.th/bcc). The fungus was maintained on PDA (potato-dextrose agar) for experimental use. Fusicoccum sp.BCC4124 was grown aerobically in a 50-ml sub-merged liquid culture in 250ml conical flasks at 30 �Cwith rotary shaking at 200 rpm. Inoculum was preparedfrom the culture on PDA by plunging four agar piecescovered with a profuse mycelial mat using cock borernumber 2 inoculated into 50ml of medium. In enzymeproduction study, the fungus was cultivated in (i) MX(2% soybean meal, 1.5% wheat flour, 1% celluloseavicel, 0.5% maltodextrin 15, 1% birchwood xylan and0.3% bacto peptone in distilled water); (ii) wheat bran-soybean (WS) (4% wheat bran and 3% soybean meal);(iii) cassava pulp-soybean (CS) (4% dried cassava pulpand 3% soybean meal) and (iv) cassava pulp (C) (4%dried cassava pulp). Samples were collected at timeintervals for amylolyic enzyme activity assay. Forenzyme inducibility study, the fungus was cultivated inminimal medium (0.2% Na2CO3, 0.2% KCl, 0.1%KH2PO4, 0.1% NH4NO3, 0.1% (NH4)H2PO4, 0.05%MgSO4

.7H2O, and 0.05% yeast extract, pH 6.0)14)

supplemented with different C-sources (0.5%), includingglucose, xylose, arabinose, maltose, cellobiose, starch,birchwood xylan, avicel, and carboxymethyl cellulose.The culture was then grown for 7 d and the supernatantwas collected for activity assay. For preparation ofenzyme for biochemical characterization, starter inoc-ulum was cultivated in 50ml MX medium as describedabove for 3 d. The starter was then inoculated into400ml MX medium in a 2-liter flask and incubated at30 �C with shaking (200 rpm) for 2 d. Crude enzyme(culture filtrate) was obtained by filtration through a0.5mm diameter mesh, centrifuged at 7;500� g for15min, and the supernatant was stored at �20 �C.

Chromatographic purification of Amy-FC1. The crudeenzyme was clarified by centrifugation at 48;300� g at4 �C for 1 h and the supernatant was used for enzymepurification. For small-scale purification, an equalvolume of 100mM MOPS buffer, pH 6.8, was addedto 30ml of the clarified crude enzyme and loaded onto aself-packed 3-ml DEAE-Sepharose Fast Flow column(GE-Healthcare Biosciences, Uppsala, Sweden) equili-brated with 50mM MOPS buffer, pH 6.8, at 1ml/min.After the column was washed with 5 bed-volumes ofequilibrating buffer, the enzyme was eluted in a stepgradient containing 50mM, 100mM and 250mM NaCl in50mM MOPS buffer, pH 6.8, at 1ml/min for 2 bed-volumes at each step. Fractions of 1ml were collectedand amylolytic activity determined. Fractions containingactivity were then pooled and stored at �20 �C.

Enzymatic activity assay. �-Amylase activity wasmeasured spectrophotometrically by determining theamount of liberated reducing sugars by the 3,5-dinitro-salicylic acid method.15) The standard reaction containedthe appropriate dilution of the enzyme (approximately0.075 units) in 50mM MOPS buffer, pH 7.0, with 10mg/ml soluble starch (or as otherwise specified) as thesubstrate in a 100-ml total reaction volume. The reactionwas incubated at 60 �C for 10min and the level ofreducing sugars was determined from the absorbance at540 nm and compared to a standard curve of glucose.One unit (U) of �-amylase activity was defined as theamount of the enzyme that produced the equivalence of1 mmole of reducing sugar under the optimal conditionsin 1min. Alternatively, �-amylase activity was assayedusing a chromogenic substrate, AZCL-amylose (Mega-zyme, Wicklow, Ireland). The assay reactions containedappropriate enzyme dilutions in 25mM MOPS buffer,pH 7.0, and 0.125% AZCL-amylose in a total volume of200 ml. The reactions were incubated at 50 �C for 20minand terminated by incubation in a boiling water bath for5min. Release of the azo-dye was determined by meas-uring absorbance at 620 nm. �-Glucosidase activity wasanalysed in 50mM sodium acetate buffer, pH 5.0, using10mM p-nitrophenyl-�-D-glucopyranoside (PNP�G) asthe substrate in a total reaction volume of 100-ml. Thereaction was incubated at 50 �C for 60min and thenstopped by adding 100 ml of 0.2 M Na2CO3. The amountof released p-nitrophenolate was measured spectropho-tometrically at 405 nm and calculated using a specificabsorbance of 16,900 M�1 cm�1. The linearity of theenzymatic activity measured at different time intervalswas ensured for accuracy of the assay. Experimentswere carried out in triplicate and the values reportedrepresent averages.

Enzyme characterization.Optimum temperature and thermostability. The opti-

mal working temperature was evaluated by measuringamylase activity in 50mM MOPS buffer, pH 7.0, atdifferent temperatures (15–80 �C). Thermostability was

�-Amylase from Fusicoccum sp. BCC4124 2011

determined by pre-incubating the purified enzyme at thecorresponding temperatures (in the presence or absenceof 1.25% soluble starch) for 1 h and then it was rapidlycooled on ice. The residual activity was then assayed forreducing sugars under standard conditions.

Optimum pH and pH stability. The pH optimum of theenzyme was determined by varying the pH of thereaction mixtures using the following buffers (50mM):sodium citrate (pH 3–6), MOPS (pH 6–8), and Tris–HCl(pH 8–10). Stability at different pH levels was deter-mined by pre-incubating the purified enzyme in theabove buffers at 50 �C for 1 h. Residual activity was thenanalyzed for reducing sugars under the standard con-ditions.

Effects of external stabilizing factors. Enzyme ther-mostability was determined by pre-incubating theenzyme at specified temperatures for specified periodsof time in the presence or absence of stabilizing factors(2mM metal ion and/or 1.25% substrate). Residualactivity was then assayed for reducing sugars under thestandard conditions.

Kinetic analysis. The kinetic parameters were deter-mined by varying the substrate concentrations (0.155–40mg/ml for soluble starch and 0.078–40mM for �- and�-cyclodextrin) in the reactions containing 50mM

MOPS buffer, pH 7.0, at 60 �C. The data obtained werefitted to the Michaelis–Menten equation by non-linearleast square regression.

Product specificity profile analysis. An appropriateamount of substrates (10mg/ml starch, 12.5mg/mlcyclodextrin or 2mM maltooligosaccharides) was incu-bated with the enzyme at 50 �C for 24 h or as otherwiseindicated. The reactions were then heated at 100 �C for10min and spotted onto a siliga 60 aluminum plate(Merck, NJ). The product specificity was analyzed bythin-layer chromatography.16) Maltooligosaccharides(G3–G7), maltose and glucose were used as standards.

Protein electrophoresis and enzyme activity detectionin gel. SDS–PAGE was done with 12% polyacrylamidegel according to Laemmli and stained with Coomassiebrilliant blue.17) For enzymatic activity detection in gel,the crude enzyme was subjected to electrophoresis undernon-denaturing conditions. The gel was then equilibrat-ed in 50mM MOPS buffer, pH 7.0, for 1 h. The gel wasplaced onto a 1.5% agar plate containing 0.1% AZCL-amylose (Megazyme, Wicklow, Ireland) in 50mM

MOPS buffer, pH 7.0, and incubated at 50 �C until theprotein band with amylase activity became clearlyvisible.

Identification of the gene encoding Amy-FC1.BCC4124 mycelia were frozen in liquid nitrogen andthen ground up in an ice-cold mortar until a powderyconsistency was achieved. Tri-Reagent (MolecularResearch Center, Cincinnati, OH) was used for totalRNA extraction from powdered BCC4124 mycelia,

following the manufacturer’s instructions. The qualityand integrity of RNA was determined by agarose gelelectrophoresis.First-strand cDNA from BCC4124 was synthesized

with a ReverseAid H Minus First Strand cDNA Syn-thesis Kit (Fermentas, Vilnius, Lithuania) according tothe supplier’s protocol. First-strand cDNAs were used asthe template for partial gene sequence amplificationusing degenerate primers designed based on conservedregions of fungal amylases: amyC-F1 (50-ATGGGNT-TYACNGCNATHTGG-30) and amyC-R (50-RAANCK-NGGRTTATCRTGRTTYTC-30) (N = A/G/C/T; H =A/C/T; Y = C/T; K = G/T; R = A/G). PCR wasperformed with 1 U of DyNAzyme DNA polymerase(Finnzymes, Espoo, Finland) in a total volume of 50 mlcontaining 1 mM of the forward and reverse primers,1 � DyNAzyme buffer, 0.2mM dNTPs, 2mM MgCl2and 4 ml cDNA. The PCR condition was 1 cycle of 4minat 94 �C, 30 cycles of 1min at 94 �C, 1min at 40 �C(cycles 1–10) or 45 �C (cycles 11–30), and 1min at72 �C, and finally 10min at 72 �C. The downstreamregion was obtained by 30-RACE. The upstream regionwas obtained by amplification using a degenerateprimer, amyN-DF (50-GGGGGCCIGCICARTGGMG-30) (I = Inosine; M = A/C), designed based on the N-terminal amino acid sequence and a primer designedbased on the internal sequence of the partial gene firstlyamplified with the conserved primers. The full-lengthgene was then amplified using amyN-F (50-CGGCG-CAATGGCGCTCGCGCTC-30), designed based on theobtained upstream sequence and amy-R (50-TCACTT-ACCACAAATCCCCGACCCCGTCAG-30), designedbased on the downstream sequence obtained from 30-RACE. The PCR products were analyzed by agarose gelelectrophoresis. The amplicon of the expected size wasgel-purified using a QIAquick gel extraction kit (Qiagen,Hilden, Germany) and cloned into pTZ57R/T vector(Fermentas, Vilnius, Lithuania). Sequencing was per-formed by Macrogen (Seoul, South Korea) using M13Forward and Reverse primers.The nucleotide and deduced amino acid sequences

were searched against the GenBank database. Multiplesequence alignment was performed using ClustalXwith defaulted parameters (http://bips.u-strasbg.fr/fr/Documentation/ClustalX/). The deduced amino acidsequence was analyzed with primary structure analysistools at www.expasy.org. The nucleotide sequence ofthe full-length amy-FC1 was submitted to GenBank (accno. DQ663472).

Other molecular and biochemical techniques. Proteinconcentration was determined with a Bio-Rad proteinassay reagent (Bio-Rad, Hercules, CA) based onBradford’s method using bovine serum albumin (BSA)as the standard. The N-terminal amino acid sequencewas analyzed from a protein sample blotted onto aPVDF membrane according to the standard protocol anddetermined by Edman’s degradation method at the

2012 V. CHAMPREDA et al.

Biomolecular Research Center, University of Newcastle,Australia.

Results

From an extensive screening of amylolytic enzymesfrom over 700 fungal isolates in BIOTEC CultureCollection, an endophyte BCC4124 was selected due toits high amylolytic activity and thermostability based onformation of a blue zone on AZCL-amylose agar plates.The fungus produced natural dark-blue color pigmentwhen grown for a few days on a PDA plate or in MXliquid medium. Since the fungus did not sporulate underthe culturing conditions, BCC4124 was identified byanalyzing the internal transcribed spacer (ITS) sequenceas a Fusicoccum sp. with 100% identity to Fusicoccumsp. isolate CMW7023 (GenBank accession no.,AY615189).

Production of amylolytic enzymes from Fusicoccumsp. BCC4124

Production of amylases from BCC4124 was opti-mized in different media. Extracellular amylolyticactivity from the fungus was studied by cultivation ineither an enriched defined medium for multi-carbohy-drate degrading enzyme induction (MX) or complexmedia containing inexpensive agricultural substrates(WS, wheat bran-soybean; CP, cassava pulp; andCP+S, cassava pulp-soybean) (Fig. 1A). In MX andCP+S, amylolytic activity was remarkably increased at48 h after incubation under aerobic conditions, while ittook 72 h in WS. Enzyme productivity in CP was verylow and was enhanced by addition of soybean meal tosupplement the protein of the cassava pulp, whichgenerally contains only 1–2%. In MX, WS, and CP+S,the amylolytic activity reached a stationary level after3 d of incubation. The highest volumetric productivity,at 9.8U/ml (106mg glucose/h/ml), was obtained in WSafter 120 h of cultivation. The specific activity of �-amylase and �-glucosidase in WS was highest at 25.9 U/mg and 62.11mU/mg at 96 h of incubation (Fig. 1B).However, maximum specific activity (90U/mg) wasobtained in MX at day 2 after incubation, which mightbe due partially to a lower protein background from themedium (data not shown). BCC4124 was then cultivatedin MX for further enzyme purification.

Induction and repression of amylolytic activitySynthesis of catabolic hydrolytic enzymes is con-

trolled by induction and catabolite repression. Sincestarch has a high molecular weight and does not passthrough cell membrane, signals for accelerated amylasesynthesis are usually low-molecular mass fragmentsresulting from starch hydrolysis and repressed by simplecarbon sources. To study the induction and repression ofamylolytic activities in BCC4124, the fungus wascultivated in minimal medium containing 0.5% of theindicated carbon substrate as the sole carbon and energy

sources at 30 �C for 7 d, and amylolytic activities in thecrude extracellular enzyme were analyzed using solublestarch and PNP�G as substrates for �-amylase and �-glucosidase respectively (Table 1). BCC4124 grew on arange of substrates, including glucose, maltose, cello-biose, and starch while it showed only very low growthon non-starch polysaccharides, including carboxy-meth-ylcellulose and avicel, while no detectable growth wasobserved on birchwood xylan. This corresponded to theremarkable �-glucosidase activity and to relatively lowcellulase and xylanase activity in the extracellular crudeenzyme of this fungus grown in enriched medium (WS)and in minimal medium containing the correspondingcarbon substrates, though it is an endophytic fungus(data not shown). Analysis of the amylolytic activity inthe crude enzymes showed that a low level of �-amylasewas present in the culture grown on glucose, a common

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Fig. 1. Production of Amylolytic Enzymes in Multi-Enzyme Induc-

tion Enriched Medium and in Agricultural Derived Substrates.

A, Amylase activity in different media: �-amylase activity of the

crude extracellular enzymes was analyzed on 10mg/ml soluble

starch in 50mM MOPS buffer, pH 7.0, at 60 �C under standard

conditions. B, Amylolytic enzyme activities in WS medium: �-

amylase activity was analyzed as described above, and �-glucosi-

dase activity was assayed on 10mM PNP�G in 50mM sodium

acetate buffer, pH 5.0, at 50 �C. Amy, �-amylase activity; a-glu, �-glucosidase activity�. �mU, milli unit.


catabolic repressor, and on some other mono- anddisaccharides. Active synthesis of amylolytic enzymeswas achieved during growth in maltose and starch.Maltose was the best inducible substrate for �-amylaseamong carbon substrates as determined from bothspecific activity (9:97� 0:64U/mg) and volumetricactivity (0:312� 0:020U/ml). However, �-amylaseactivity was lower than when the fungus was cultivatedin WS. In contrast, �-glucosidase specific activity washigher when BCC4124 was grown in minimal mediumthan in WS. This was partly due to the higher proteinconcentration in crude enzyme from the enrichedmedium. The specific activity of �-glucosidase was alsohighest when grown on maltose (0.34U/mg). Lessrepression of �-glucosidase activity by other carbonsubstrates was observed than of �-amylase activity. Theamylase induction profile was comparable to thosereported for certain filamentous fungi e.g., Aspergillussp.18–20) and Trichoderma sp.7)

Chromatographic purification of Amy-FC1Fusicoccum sp. BCC4124 was found to produce only

one major extracellular amylolytic enzyme, since in-geldetection of amylase activity of the fungal filtrate ofBCC4124 cultivated in MX on native PAGE yieldedonly one protein band. The target enzyme was purifiedto high homogeneity by a single step of anion exchangechromatography (Table 2). The fractions eluted at100mM NaCl in the step gradient were found to containthe target enzyme. The purity of the enzyme wasapproximately more than 90% as determined by SDS–PAGE analysis with a molecular mass of approximately50 kDa (Fig. 2).

Biochemical characterizationOptimal working conditionsPurified Amy-FC1 functioned optimally at high

temperature, between 60–70 �C, with highest activityat 70 �C, in the neutral pH range as assayed on solublestarch (Fig. 3A and B). In the absence of any stabilizingfactors, the enzyme retained more than 95% activity

after incubation at 50 �C for 1 h, but hardly any activitywas observed at 60 �C pre-incubation. However, in thepresence of substrate (soluble starch), enzyme thermo-stability increased remarkably. The purified enzymeretained more than 90% residual activity after prolongedincubation at 60 �C.

Kinetics and action mechanismsAmy-FC1 showed high activity on soluble starch,

with Vmax and Km of 640:8� 55:9U/mg protein and5:20� 1:16mg/ml respectively (Table 3). The enzymealso showed high activity selectively on �-cyclodextrinand �-cyclodextrin, but only trace activity on �-cyclo-dextrin, while it showed no hydrolysis on pullulan.Reaction efficiency was highest with starch, followed by�- and �-cyclodextrins, as shown by the respectivedecreases in kcat/Km. Even though it also possessed

Table 2. A Typical Purification Profile of Amy-FC1

Purification step Specific activity (U/mg) Total activity (U) Yield (%) Purification fold

Crude enzyme 90.8 787.5 100 1

DEAE Sepharose 838.9 381.9 48.5 9.24

Amy-FC1

1 2 3

Fig. 2. Chromatographic Purification of Amy-FC1.

Crude enzyme from culture filtrate of Fusicoccum sp. BCC4124

grown in MX medium was purified with a DEAE-Sepharose Fast

Flow anion exchanger using 50mM MOPS buffer, pH 6.8, and a

NaCl elution gradient. lane 1, Protein M.W. marker; lane 2, culture

filtrate; lane 3, purified Amy-FC1.

Table 1. Induction and Repression of Amylolytic Activity of Fusicoccum sp. BCC4124 Cultivated in Different Carbon Substrates

C-substrate Growth Amylase (U/mg) Alpha-glucosidase (U/mg)

Glucose ++++ 0:85þ=� 0:16 0:31þ=� 0:01Xylose ++ 0:47þ=� 0:05 0:21þ=� 0:09

Arabinose + nd nd

Maltose ++++ 9:97þ=� 0:64 0:34þ=� 0

Cellobiose +++ 0:35þ=� 0:16 0:06þ=� 0:01Starch ++ 1:59þ=� 0:17 0:11þ=� 0:01

nd, not detectable.


high cyclodextrinase activity, Amy-FC1 was classifiedas an �-amylase according to substrate specificity andgene similarity.

When Amy-FC1 was incubated with soluble starch,maltotetraose, maltotriose and maltose were the pre-dominant hydrolysis products in the early stage withtrace amount of longer maltooligosaccharides e.g.,maltopentaose. These maltooligosaccharides were fur-ther hydrolysed into smaller products, including glucose,maltose, and maltotriose as the final products fromcomplete hydrolysis over prolonged incubation. (Fig. 4).Similar product profiles were obtained from completehydrolysis of cyclodextrins. The enzyme cleaved sub-strates as short as maltotriose and released sugars(glucose and maltose) and short maltooligosaccarides(� G3) as the major final products from completehydrolysis. The minimum substrate length was con-firmed by assaying activity on maltooligosaccharides ofdifferent chain lengths (G2–G7). The rate increasedaccording to the increase in substrate length, suggestinghigher affinity of the enzyme on longer substrates(Table 4). The hydrolysis profiles thus suggested thatAmy-FC1 is an endo-acting amylase. This is similar tothe product specificity of some fungal �-amylases e.g.,from Cryptococcus S221) and A. flavus22) but differs tothat of L. starkeyi which gave longer products.23) Notransglycosylation activity of Amy-FC1 was detectedunder the experimental conditions. Amy-FC1 was hence

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Fig. 3. Optimal Working Conditions and Stability of Amy-FC1.

The reactions contained Amy-FC1 in 50mM buffer with 10mg/

ml starch as a substrate and were incubated at the corresponding

temperature and pH levels for 10min. The optimal temperature

experiment (A) was performed at pH 7, and the optimal pH (B) was

determined at 60 �C. Thermostability in the presence (+S) and

absence (�S) of starch at pH 7 and pH stability (at 50 �C) were

determined by pre-incubating the purified enzyme at the corre-

sponding temperature or pH for 1 h before assay for residual activity

under the standard conditions. Buffer: pH 3–6, sodium citrate;

pH 6–8, MOPS; pH 8–10, Tris–HCl. The activity at 60 �C and pH 7

represents 100% relative activity.

Table 3. Kinetic Parameters of Amy-FC1

Substrate Vmax (U/mg) Km (mg/ml) kcat (min�1) kcat/Km (min�1(ml/mg))

Soluble starch 640:8� 55:9 5:20� 1:16 3:37� 104 6:47� 103

�-Cyclodextrin nd nd nd nd

�-Cyclodextrin 327:7� 38:5 14:46� 3:30 1:70� 104 1:18� 103

�-Cyclodextrin 553:3� 25:4 9:18� 1:20 2:91� 104 3:17� 103

nd, no detectable activity.

Fig. 4. Product Specificity Profile of Amy-FC1.

Purified Amy-FC1 was incubated with the corresponding sub-

strate in 50mM MOPS buffer, pH 7.0, at 50 �C for the indicated

times. Lane 1, G1–G7 standard; Lanes 2–4, soluble starch, incubated

for 2, 8 and 24 h respectively; Lanes 5–7, �, � and �-cyclodextrin

respectively, incubated for 24 hours.


classified as a liquefying enzyme which can directlyconvert soluble starch to sugars and small maltooilogo-saccharides with varying chain lengths depending on theincubation period.

Effect of product inhibition on hydrolysis reactionGlucose inhibition on amylolytic enzymes is consid-

ered to be one of the limiting factors on their industrialapplications. To determine product inhibition on Amy-FC1 activity, the enzyme was incubated with 0.125%AZCL-amylose in 25mM MOPS buffer, pH 7.0, in thepresence of different concentrations of glucose ormaltose at 50 �C for 20min, and the residual activityas compared to the control reaction in the absence ofglucose/maltose was analyzed. Amy-FC1 was tolerantto inhibition by glucose at up to 1M, as shown inTable 5, but the enzyme was more sensitive to inhibitionby maltose. This remarkable tolerance to glucose iscomparable to that of reported �-amylases from thethermophilic fungus Scytalidium thermophilum24) andAspergillus tamarii.25)

Extrinsic stabilizing factors of Amy-FC1�-Amylases are metalloenzymes, which can be

activated and stabilized by several divalent metal ions.Their roles in activation and stabilization have beenwell-documented, but details of metal activation andstabilization may be different among �-amylases fromdifferent sources. Among all metal ions, Ca2þ isrecognized as the most common stabilizing ion, thoughinactivation by Ca2þ has also been reported.2,26) Aconcentration of Ca2þ on Amy-FC1 activity was found

to cause the same activation effect, with no significantdifference from 1mM–40mM Ca2þ under the standardassay conditions and no inhibitory effect at high Ca2þ

concentrations was observed (data not shown). Whenadded directly to the reaction at 2mM concentration,Ca2þ significantly increased the enzyme activity (117%relative activity) while Sr2þ, Mg2þ and Ba2þ had nosignificant effect on it (Fig. 5). The transition metal ionswere found to decrease activity of the enzyme, espe-cially Fe2þ and Zn2þ. An experiment on the EDTA-treated (20mM) and dialyzed ion-depleted Amy-FC1added with the same concentration of metal ions showedsimilar activation and inactivation effects, of thespecified metal ions. The change in relative activitysuggests different effects of binding and displacement bydifferent metal ions on the structural and catalyticproperties of the enzyme.To determine further the effect of metal ions on

enzyme stability, the purified enzyme was pre-incubatedwith metal ions for different durations at higher temper-atures. In the presence of Ca2þ and Sr2þ, Amy-FC1retained full activity after 1 h of pre-incubation at 60 �C,and more than 80% activity after 5 h, while Ba2þ

showed no stabilization effect on the enzyme (Fig. 6A).The addition of EDTA to chelate metal ions from Amy-FC1 led to a decrease in enzyme thermostability. Theaddition of either Ca2þ or Sr2þ, but not Ba2þ, to the ion-depleted enzyme resulted in a partial regaining ofthermostability (55.0% relative activity for Sr2þ after 5 hof pre-incubation at 60 �C), confirming the importanceof the metal ions in the thermostabilization of the nativeenzyme.The presence of natural substrates such as starch

showed a strong stabilizing effect, since Amy-FC1retained almost 100% activity even after incubation at60 �C for 5 h, while relative activity was lower atincreasing temperature (Fig. 6B). In addition to starch,smaller substrates, including, maltodextrin 15 andmaltooligosaccharides, also stabilized the enzyme, albeitwith a lesser stabilizing effect (data not shown). This

Table 4. Relative Activity and Product Specificity Profile of Amy-

FC1 on Maltooligosaccharides

The reactions were incubated at 50 �C for 20min for relative activity

assay and for 24 h for product specificity analysis.

Substrate Specific activity (U/mg) Product specificity

G2 nd nd

G3 5.4 G2 G1

G4 9.6 G2 G3 G1

G5 43.5 G2 G3 G1

G6 45.0 G2 G3 G1

G7 80.0 G2 G3 G1


Table 5. Effect of Glucose and Maltose on Amylolytic Activity of

Amy-FC1

ConcentrationResidual activity (%)

Glucose Maltose

Control 100 100

0.125M 100 100

0.25M 99.8 31.1

0.5 M 85.9 2.6

1M 65.6 nd


0

20

40

60

80

100

120

140

Mg Ca Sr Ba Fe Mn Ni Zn

ion

% r

elat

ive

acti

vity

Fig. 5. Effect of Metal Ions on Amy-FC1 Activity.

The reaction contained purified Amy-FC1 in 50mM MOPS buffer,

pH 7.0, 2mM metal ions and 10mg/ml starch. The activity of the

enzyme in the absence of metal ion was defined as 100%.


strong stabilization by substrates might be due tostabilization of the active site by substrate binding,which results in lowering of the potential energy of theenzyme.27) The presence of both stabilizing metal ionsand substrate led to a distinct enhancement of enzymethermostability at 70 �C (Fig. 6C). The synergistic co-stabilization by metal ions and substrate which stabi-lized enzyme thermostability by different mechanismsled to a maximal increase in the enzyme half-life at70 �C, to 4 h, which suggests an elevation of enzymethermostability up to 20 �C as compared to that in theabsence of any stabilizing factor.

Analysis of the gene encoding Amy-FC1The putative gene amyFC1 contained 1,428 bps

encoding a mature �-amylase of 475 amino acid

residues with no secretion signal. The translated N-terminal sequence showed exact identity to the N-terminal amino acid sequence analyzed by Edman’sdegradation (PAQWRSRXIYQVL, where X indicatesan unidentified amino acid residue), indicating that thegene sequence identified encoded the target amylase.From the translated sequence, the calculated molecularmass of the enzyme was 52,581.27Da, with a theoreticalpI of 4.56 (http://au.expasy.org/tools/pi tool.html),which is in good accordance with the estimated MWof the protein from SDS–PAGE analysis. Amy-FC1 wasfound to contain conserved amino acid residues at thecorresponding specified positions (K207, H208, V229,D231, and G232) characteristic of �-amylases (whichact solely on the �-1,4 linkage) according to MacGregoret al.28)

A B

C

0

20

40

60

80

100

120

0 1 2 43 65

Incubation time (h)

% r

elat

ive

acti

vity

Ca

Sr

Ba

EDTA/CaEDTA/Sr

EDTA/Ba

Control/ EDTA control

0

20

40

60

80

100

120

0 654321

Incubation time (h)

% r

elat

ive

acti

vity

Starch 60 C

Starch 65 C

Starch 70 C

Control 60 C

0

20

40

60

80

100

120

0 1 65432

Incubation time (h)

% r

elat

ive

acti

vity

Ca

Sr

Ca+Starch

Sr+Starch

Fig. 6. Extrinsic Stabilizing Factors of Amy-FC1.

A, stabilization by metal ions at 60 �C; B, stabilization by substrate at different temperatures; C, co-stabilization by metal ions and substrate at

70 �C. Amy-FC1 was pre-incubated in 50mM MOPS buffer, pH 7.0, containing stabilizing factors (2mM metal ions and/or 1.25% soluble starch)

at the specified temperatures for the indicated times. The residual activity was then analyzed under standard assay conditions. Control reactions

using the native enzyme treated and untreated with EDTA with no external stabilizing factors were included.


From analysis of the protein primary structure, Amy-FC1 shares moderate homology to TAKA-amylase Afrom Aspergillus oryzae (6TAA) (65% identity and 78%positives), as well as several other �-amylases fromAspergilli and to �-amylase from Lipomyces starkeyi(63% identity and 78% positives to Amy-FC1 and 62%identity to TAKA amylase A, not including the extendedN-terminus).23) The putative amyFC1 gene also showeda lesser extent of similarity (76–78% positives) to thosefrom Emericella nidulans (AAF17103.1), Neosartoyafisheri NRRL 181 (EAW23731.1), Ophiostoma flocco-sum (ABF72530.1), and Debraryomyces occidentalis(CAA51912.1). Multiple sequence alignment of thededuced amino acid sequence of Amy-FC1 with theclosely related �-amylases clearly showed that Amy-FC1 exhibited relative similarity to both related en-zymes (Fig. 7). Amylase conserved regions present incatalytic domains of a ð�=�Þ8 structure, which arepresent in �-amylase, CGTase, maltase, isoamylase,pullulanase, oligo-1,6-�-glucosidase, and branching en-zymes, were identified in Amy-FC1.29) Conserved aminoacids forming disulphide bridges (C28-C36, C148-C162,C238-C281, C438-C473) characteristic of fungal �-amylases were identified. The essential catalytic resi-dues were the catalytic nucleophile D204, the protondonor E228 and the second conserved aspartic acidD295. The two histidine residues important for transi-tion state stabilization (H120 and H294) were alsopresent, as well as the conserved primary structural Ca2þ

binding site (N119, E160, and D173). These stabilizinginteractions, characteristic of fungal amylases,30) con-tributed to the thermostability of Amy-FC1, in additionto the extrinsic stabilizing factors. However, furtherstudy of the recombinant protein encoded by theputative gene is needed to elucidate the detailed mo-lecular characteristics of the enzyme.

Discussion

An endophyte Fusicoccum sp. BCC4124 producedstrong �-amylase activity and also significant �-gluco-sidase activity in extracellular crude enzyme. Highamylolytic activity was obtained by cultivating thefungus in agricultural substrates. It was comparable tothe expression level of �-amylases from industrialenzyme producers, Aspergilli,19) but much higher thanthe productivity of �-amylase reported in the relatedBotryodiplodia theobromae cultivated in cassava starchresidue.11) Besides the reported amylolytic enzymeproduction, enzymatic study of endophytic fungi in thefamily Botryosphaeria has been limited. One studyincluded the production and induction of laccases from alignolytic and pectinolytic Botryosphaeria sp.9,31) ForFusicoccum sp. BCC4124, a range of hydrolytic enzymeactivities has been detected. In addition to strongdextrinizing and significant saccharifying amylolyticactivities, the fungus also produced some extracellularnon-starch hydrolytic enzymes, including �-glucosidase

Fig. 7. Primary Protein Structure Comparison of Amy-FC1 with Related �-Amylases.

This multiple sequence alignment was generated using ClustalX with defaulted parameters. The four conserved regions in amylases are

indicated (underlined). Cysteine residues contributing to the conserved disulphide bond formation are indicated with closed triangles. Sources of

the �-amylases and their GenBank accession numbers are given. AmyFC1 (DQ663472), Ao (A. oryzae TAKA-amylase A, 6TAA), Ls

(Lipomyces starkeyi, AAN75021).


(0.23U/mg) and �-xylosidase (0.042U/mg), whengrown in WS, which probably involved in the degrada-tion of plant cell components, cellulose, and hemi-cellulose respectively. But only low activities of theupstream non-starch polysaccharide hydrolyzing en-zymes were detected as compared to strong amylolyticactivity under the experimental conditions (unpublisheddata). Thus the study of enzymatic profiles of theendophytes grown on living plants may be of interest inorder to elucidate the enzymatic mechanisms of thisfungus under natural conditions. Amylolytic activities,especially �-amylase, in Fusicoccum sp. BCC4124 wereinducible by maltose and starch. In Aspergilli, amylo-lytic enzyme expression was controlled by catabolicrepression via CreA and induced by maltose, which isconverted to isomaltose, which acts as a physiologicalinducer of amylolytic genes.32,33) Whether the regulationof amylolytic enzymes in Fusicoccum sp. and relatedendophytic fungi shows any similarity to those reportedfor Aspergilli requires further investigation.

Compared to many reported fungal �-amylases,2,3)

Amy-FC1 worked optimally at relatively high temper-atures (60–70 �C), and catalyzed starch hydrolysis in theabsence of any additional metal ion with high stability.The optimal pH was in the neutral range, which is higherthan the enzymes from Aspergilli, which usually workoptimally in a mild acidic pH range.34) The substrateaffinity of Amy-FC1 as determined from Km wascomparable to those reported for other �-amylases, inthe range of 0.2–5mg/ml2) for soluble starch, while themaximum velocity was comparable to the enzyme fromAspergilli.25) The short maltooligosaccharide productspecificity profiles of Amy-FC1 suggest that the enzymeworks synergistically with the co-expressed �-glucosi-dase to completely hydrolyze and saccharify starchdirectly to glucose.

Amy-FC1 showed apparent tolerance to glucose,which is considered a desirable characteristic of amy-lolytic enzymes in industrial saccharification process. Todate, only very few �-amylases have been reported toshow product tolerance.24,25) Compared to the �-amylasefrom the thermophilic S. thermophilum, Amy-FC1showed comparable glucose tolerance, but with appa-rently much higher specific activity and higher thermo-stability, though it originated from a mesophilic fungus.This result holds great interest for further studies on thispotential amylolytic enzyme and also for the applicationof the enzyme in the biotechnological saccharificationprocess. The enzyme is promising for application inglucose syrup and short maltooligosaccharide produc-tion from starch in the pharmaceutical and biotechno-logical industries, especially where highly purified �-amylase is needed.

In conclusion, in this work, we report the inductionand biochemical and molecular characterization of anovel thermotolerant and glucose tolerant maltooligo-saccharide-forming fungal �-amylase with potential forfurther biotechnological applications that is of academic

interest. This indicates the wealth of unexplored bio-diversity as a valuable source for potential enzymediscoveries.

Acknowledgment

This project was supported by the National Center forGenetic Engineering and Biotechnology, Thailand(Grant BT-B-02-NM-BC-4801). The authors would alsolike to thank Dr. Piyanun Harnpichanchai for her kindassistance in the revision of this paper.

References

1) Van der Maarel, M. J. E. C., Van der Veen, B.,Uitdehaag, J. C. M., Leemhuis, H., and Dijkhuizen, L.,Properties and applications of starch-converting en-zymes of the �-amylase family. J. Biotech., 94, 137–155 (2002).

2) Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K.,and Chauhan, B., Microbial �-amylases: a biotechno-logical perspective. Process Biochem., 38, 1599–1616(2003).

3) Pandey, A., Nigam, P., Soccol, C. R., Soccol, V. T.,Singh, D., and Mohan, R., Advances in microbialamylases. Biotechnol. Appl. Biochem., 31, 132–152(2000).

4) Kaneko, T., Ohno, T., and Ohisa, N., Purification andcharacterization of a thermostable raw starch digestingamylase from a Streptomyces sp. isolated in a millingfactory. Biosci. Biotechnol. Biochem., 69, 1073–1081(2005).

5) Spier, M. R., Woiciechowski, A. L., Vandenberghe, L. P.D. S., and Soccol, C. R., Production and characterizationof amylases by Aspergillus niger under solid statefermentation using agro-industrial products. Int. J. FoodEng., 2, Article number 6 (2006).

6) Silva, T. M., Attili-Angelis, D., Azevedo Carvalho, A.F., Da Silva, R., Boscolo, M., and Gomes, E., Productionof saccharogenic and dextrinogenic amylases by Rhizo-mucor pusillus A 13.36. J. Microbiol., 43, 561–568(2005).

7) Pacheco Chavez, R. A., Carvalho, J. C. M., Converti, A.,Perego, P., Tavares, L. C., and Sato, S., Production of �-amylase and glucoamylase from different starches by anew Trichoderma sp. isolate. Ann. Microbiol., 54, 169–180 (2004).

8) Rezende, M. I., Barbosa, A. M., Vasconcelos, A. F.,Haddad, R., and Dekker, R. F., Growth and productionof laccases by the lignolytic fungi, Pleurotus ostreatusand Botryosphaeria rhodina, cultured on basal saltmedium containing the herbicide, Scepter (Imazaquin).J. Basic Microbiol., 45, 460–469 (2005).

9) Da Cunha, M. A. A., Barbosa, A. M., Giese, E. C., andDekker, R. F. H., The effect of carbohydrate carbonsources on the production of constitutive and induciblelaccases by Botrysphaeria sp. J. Basic Microbiol., 43,385–392 (2003).

10) Nwufo, M. I., and Faljola, A. O., Production ofamylolytic enzymes in culture by Botryodiplodia theo-bromae and Sclerotium rolfsii associated with the cormrots of Colocasia esculenta. Acta Microbiol. Hung., 35,


371–377 (1988).11) Ray, R. C., Extracellular amylase(s) production by fungi

Botryodiplodia theobromae and Rhizopus oryzae grownon cassava starch residue. J. Environ. Biol., 25, 489–495(2004).

12) Asano, K., Sriprang, R., Gobsuk, J., Eurwilaichitr, L.,Tanapongpipat, T., and Kirtikara, K., Endo-1,4-�-xyla-nase B from Aspergillus cf niger BCC 14405 isolated inThailand: purification, characterization and gene isola-tion. J. Biochem. Mol. Biol., 38, 17–23 (2005).

13) Ratanachomsri, U., Sriprang, R., Sornlek, W.,Buaban, B., Champreda, V., Tanapongpipat, S., andEurwilaichitr, L., Thermostable xylanase from Maras-mius sp.: purification and characterization. J. Mol. Biol.Biochem., 39, 105–110 (2006).

14) Riou, C., Salmon, J. M., Vallier, M.-J., Gunata, Z., andBarre, P., Purification, characterization and substratespecificity of a highly glucose-tolerant �-glucosidasefrom Aspergillsu oryzae. Appl. Environ. Microbiol., 64,3607–3614 (1998).

15) Miller, G. L., Using dinitrosalicylic acid for determi-nation of reducing sugar. Anal. Chem., 31, 426–428(1959).

16) Tang, K., Utairungsee, T., Kanokratana, P., Sriprang, R.,Champreda, V., Eurwilaichitr, L., and Tanapongpipat,S., Characterization of a novel cyclomaltodextrinaseexpressed from environmental DNA isolated from BorKleung Hotspring in Thailand. FEMS Micrbiol. Lett.,260, 91–99 (2006).

17) Sambrook, J., Fritsch, E. F., and Maniatis, T., ‘‘Molecu-lar Cloning, a Laboratory Manual’’ 2nd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor (1989).

18) Carlsen, M., and Nielsen, J., Influence of carbon sourceon alpha-amylase production by Aspergillus oryzae.Appl. Microbiol. Biotechnol., 57, 346–349 (2001).

19) Goto, C. E., Barbosa, E. P., Kistner, L. C., Moreira, F.G., Lenartovicz, V., and Peralta, R. M., Production ofamylase by Aspergillus fumigatus utilizing alpha-meth-yl-D-glycoside, a synthetic analogue of maltose, assubstrate. FEMS Microbiol. Lett., 167, 139–143 (1998).

20) Morkeberg, R., Carlsen, M., and Nielsen, J., Inductionand repression of alpha-amylase production in batch andcontinuous cultures of Aspergillus oryzae. Microbiology,141(Pt 10), 2449–2454 (1995).

21) Iefuji, H., Chito, M., Kato, M., and Iimura, Y., Raw-starch-digesting and thermostable alpha-amylase fromthe yeast Cryptococcus sp. S2: purification, character-ization, cloning and sequencing. Biochem. J., 318(Pt3),989–996 (1996).

22) Khoo, S. L., Amirul, A. A., Kamaruzaman, M., Nazalan,N., and Azizan, M. N., Purification and characterizationof alpha-amylase from Aspergillus flavus. Folia Micro-biol. (Praha), 39, 393–398 (1994).

23) Kang, H. K., Lee, J. H., Kim, H., Day, D. F., Robyt, J. F.,Park, K.-H., and Moon, T.-W., Cloning and expressionof Lipomyces starkeyi �-amylase in Escherichea coli anddetermination of some of its properties. FEMS Micro-biol. Lett., 233, 53–64 (2004).

24) Aquino, A. C. M. M., Jorge, J. A., Terenzi, H. F., andPolzeli, M. L. T. M., Studies on a thermostable �-amylase from thermophilic fungus Scytalidium thermo-philum. Appl. Microbiol. Biotechnol., 61, 323–328(2003).

25) Moreira, F. G., Lenartovicz, V., and Peralta, R. M., Athermostable maltose-tolerant �-amylase from Aspergil-lus tamarii. J. Basic Microbiol., 44, 29–35 (2004).

26) Laderman, K. A., Davis, B. R., Krutzsch, H. C., Lewis,M. S., Griko, Y. V., Privalov, P. L., and Anfinsen, C. B.,The purification and characterization of an extremelythermostable �-amylase from hyperthermophilic arch-aebacterium Pyrococcus furiosus. J. Biol. Chem., 268,24394–24401 (1993).

27) Vielle, C., and Zeikus, G. J., Hyperthermophilic en-zymes: sources, uses and molecular mechanisms forthermostability. Microbiol. Mol. Biol. Rev., 65, 1–43(2001).

28) MacGregor, E. A., Janecek, S., and Svensson, B.,Relationship of sequence and structure to specificity inthe �-amylase family of enzymes. Biochim. Biophys.Acta, 1546, 1–20 (2001).

29) Kuriki, T., and Okada, S., A new concept of the criteriafor classification of various amylolytic enzymes andrelated enzymes; similarities in specificities and struc-tures. In ‘‘Enzyme Chemistry and Molecular Biology ofAmylases and Related Enzymes,’’ ed. Takehiko, Y.,CRC Press, Boca Raton, pp. 87–92 (1995).

30) Feller, G., Lonhienne, T., Deroanne, C., Libioulle, C.,Van Beeumen, C., and Gerday, C., Purification, charac-terization, and nucleotide sequence of the thermolabilealpha-amylase from the antarctic psychrotroph Alter-omonas haloplanctis A23. J. Biol. Chem., 267, 5217–5221 (1992).

31) Dekker, R. H. F., and Barbosa, A. M., The effects ofaeration and veratryl alcohol on the production of twolaccases by the ascomycete Botryosphaeria sp. EnzymeMicrob. Technol., 28, 81–88 (2001).

32) Kato, N., Murakoshi, Y., Kato, M., Kobayashi, T., andTsukagoshi, N., Isomaltose formed by alpha-glucosidas-es triggers amylase induction in Aspergillus nidulans.Curr. Genet., 42, 43–50 (2002).

33) Nakamura, T., Maeda, Y., Tanoue, N., Makita, T., Kato,M., and Kobayashi, T., Expression profile of amylolyticgenes in Aspergillus nidulans. Biosci. Biotechnol. Bio-chem., 70, 2363–2370 (2006).

34) Godfry, T., and West, S., ‘‘Industrial Enzymology’’ 2nded., Macmillan Press, London (1996).


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