Production of α-amylase by Myxococcus coralloides D

M.E. FÁREZ-VIDAL, A. FERNANDEZ-VIVAS AND J.M. ARIAS. 1992. Myxococcus coralloides D secreted amylase into a liquid growth medium containing 1% starch. Amylase activity was highest at the end of the exponential growth phase. Of the nitrogen sources tested, the greatest growth and amylase production were obtained with trypticase peptone, casitone, probion L and probion F. When starch was replaced by other carbon sources, amylase production was reduced; trisaccharide produced better results than disaccharide while monosaccharide reduced amylase production to basal levels. Maltose repressed amylase production. Amylase production was greater in stirred flasks, at pH between 6.5 and 7.5, and at temperatures from 28C to 33C. The activity of partially purified M. coralloides D amylase was used to determine the products released from the hydrolysis of starch with thin-layer chromatography, paper chromatography and nuclear magnetic resonance. These products were maltose and glucose and limit dextrins.     

Production and partial purification of α-amylase from a novel isolate <Emphasis Type="Italic">Streptomyces gulbargensis</Emphasis>

Extracellular amylase production by a newly isolated alkali-thermotolerant strain Streptomyces gulbargensis DAS 131 was optimized and characterized. The highest amylase production was achieved by growing S. gulbargensis DAS 131 in media with 1% starch. Strain exhibited maximal activity at pH 9.0 and 45 degrees C and relatively stable in alkaline conditions (pH 11). Starch and peptone were found to be the good source of carbon and nitrogen with a yield of 2,216.6 and 2,156.1 U, respectively. Maltose and maltotriose were the main end products of starch hydrolysis, indicating alpha-amylase activity. SDS-PAGE analysis revealed a monomeric form with a molecular weight of 55 kDa.     

Culture Conditions for Production of Thermostable Amylase by Bacillus stearothermophilus

Bacillus stearothermophilus grew better on complex and semisynthetic medium than on synthetic medium supplemented with amino acids. Amylase production on the complex medium containing beef extract or corn steep liquor was higher than on semisynthetic medium containing peptone (0.4%). The synthetic medium, however, did not provide a good yield of extracellular amylase. Among the carbohydrates which favored the production of amylase are, in order starch > dextrin > glycogen > cellobiose > maltohexaose-maltopeptaose > maltotetraose and maltotriose. The monosaccharides repressed the enzyme production, whereas inositol and d-sorbitol favored amylase production. Organic and inorganic salts increased amylase production in the order of KCI > sodium malate > potassium succinate, while the yield was comparatively lower with other organic salts of Na and K. Amino acids, in particular isoleucine, cysteine, phenylalanine, and aspartic acids, were found to be vital for amylase synthesis. Medium containing CaCl(2) 2H(2)O enhanced amylase production over that on Ca -deficient medium. The detergents Tween-80 and Triton X-100 increased biomass but significantly suppressed amylase synthesis. The amylase powder obtained from the culture filtrate by prechilled acetone treatment was stable over a wide pH range and liquefied thick starch slurries at 80 degrees C. The crude amylase, after (NH(4))(2)SO(4) fractionation, had an activity of 210.6 U mg. The optimum temperature and pH of the enzyme were found to be 82 degrees C and 6.9, respectively. Ca was required for the thermostability of the enzyme preparation.     

The use of sorghum for thermostable amylase production from Thermoactinomyces thalpophilus

Thermoactinomyces thalpophilus isolated from flour mill wastes was found to produce extracellular amylase in shake flask cultures using sorghum as carbon source. Sorghum in mineral salts medium significantly supported a higher rate of amylase synthesis by the organism than soluble starch, giving peak amylase activity at the stationary phase. The optimum temperature and pH of the enzyme was 90°C and 5·0, respectively, with more than 50% enzyme activity retained at 100°C (30 min).     

Production of amylase by Arthrobacter psychrolactophilus

Arthrobacter psychrolactophilus ATCC 700733 grew with a doubling time of 1.5–2.3 h (22°C) and produced up to 0.2 units/mL (soluble starch assay) of extracellular amylase in tryptic soy broth without dextrose (TSBWD) containing 0.5% or 1.0% (w/v) soluble starch or maltose as the fermentable substrate. Time-course experiments in media containing soluble starch as substrate showed that amylolytic activity appeared in cultures at 24 h (after exponential growth had ceased), reached peak levels in 72–96 h, and declined rapidly after reaching peak levels. Peak levels were highest in TSBWD containing 1.0% soluble starch. Proteolytic activity appeared at about the same time as amylolytic activity and increased during the period of amylase production. Significant amylase production was not observed in cultures in TSBWD with 0.5% glucose or in cultures grown at 28°C, but low levels of amylase were observed in TSBWD cultures grown at 19–23°C which contained no added carbohydrate. A single band of activity was observed after electrophoresis of supernatant fractions in non-denaturing gels, followed by in situ staining for amylolytic activity. The amylase possessed a raw starch-binding domain and bound to uncooked corn, wheat or potato starch granules. It was active in the Phadebas assay for -amylase. Activity was maximum on soluble starch at a temperature between 40°C and 50°C. The amylase after purification by affinity chromatography on raw starch granules exhibited two starch-binding protein bands on SDS gels of 105 kDa and 26 kDa.     

Isolation, optimization, and partial purification of amylase from Chrysosporium asperatum by submerged fermentation

A potent fungus for amylase production, Chrysosporium asperatum, was isolated from among 30 different cultures obtained from wood samples collected in the Junagadh forest, India. All of the isolated cultures were screened for their ability to produce amylase by submerged fermentation. Among the selected cultures, C. asperatum (Class Euascomycetes; Onygenales; Onygenaceae) gave maximum amylase production. In all of the different media tested, potato starch was found to be a good substrate for production of amylase enzyme at 30 degrees C and pH 5.0. Production of enzyme reached the maximum when a combination of starch and 2% xylose, and organic nitrogen (1% yeast extract) and ammonium sulfate were used as carbon and nitrogen sources, respectively. There was no significant effect of metal ions on enzyme activity. The enzyme was relatively stable at 50 degrees C for 20 min, and no inhibitory effect of Ca+2 ions on amylase production was observed.     

Production of amylase by newly isolated moderate halophile,Halobacillus sp. strain MA-2

Production of extracellular amylase was demonstrated under stress conditions of high temperature and high salinity in aerobically cultivated culture of a newly isolated moderately halophilic bacterium of spore-forming Halobacillus sp. strain MA-2 in medium containing starch, peptone, beef extract, and NaCl. The maximum amylase production was secreted in the presence of 15% (w/v) Na(2)SO(4) (3.2 U ml(-1)). The isolate was capable of producing amylase in the presence of NaCl, NaCH(3)COOH, or KCl, with the results NaCl>NaCH(3)COOH>KCl. Maximum amylase activity was exhibited in the medium containing 5% (w/v) NaCl (2.4 U ml(-1)). Various carbon sources induced enzyme production. The potential of different carbohydrates in the amylase production was in the order: dextrin>starch>maltose>lactose>glucose>sucrose. In the presence of sodium arsenate (100 mM), maximum production of the enzyme was observed at 3.0 U ml(-1). Copper sulfate (0.1 mM) decreased the amylase production considerately, while lead nitrate had no significant enhancement on amylase production (p<0.05). The pH, temperature, and aeration optima for enzyme production were 7.8, 30 degrees C, and 200 rpm, respectively, while the optimum pH and temperature for enzyme activity was 7.5-8.5 and 50 degrees C, respectively.     

Amylolytic activity of Thermomonospora fusca

Amylases which produce maltotriose as the major end-product from starch are relatively rare. The thermophilic actinomycete, Thermomonospora fusca, produced an extracellular -amylase which generated maltotriose as 61% of the identified products. The addition of maltotriose to a glucose-adapted exponential phase culture at 55°C in mineral salts medium caused rapid induction of amylase biosynthesis. Addition of glucose to cells growing on starch did not repress amylase biosynthesis because the actinomycete had a marked preference for maltotriose over glucose. The pH and temperature optima for the amylase activity of concentrated, washed extracellular protein were 6.0 and 65°C, respectively, with an energy of activation of 59kJ/mol. The thermostability of the concentrated, washed amylase was increased by the presence of its starch reaction products, but not by added Ca2+.     

Pearl millet, a source of alpha amylase production by Bacillus licheniformis

The present study is concerned with the selection of economically available agricultural starchy substrate for the production of alpha amylase by Bacillus licheniformis. Different agricultural starchy substrates such as soluble starch, hordium, pearl millet, rice, corn, gram and wheat starch were tested for the production of alpha amylase by parental and its mutant derivatives. The production of alpha amylase was 10-folds better by the mutant strain B. licheniformis GCUCM-30 than the parental strain when pearl millet starch at 1.5% level and nutrient broth concentrations at the level of 0.25% was supplemented to the fermentation medium.     

Production of surfactant and detergent-stable,halophilic, and alkalitolerant alpha-amylase by a moderately halophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. Strain <Emphasis Type="Italic">TSCVKK</Emphasis>

A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl₂ at pH 8.0 at 30 °C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 °C. The amylase that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose, and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase.     

Biochemical characterization of alpha-amylase from the yeast Cryptococcus flavus

During our screening of amylolytic microorganisms from Brazilian fruits, we isolated a yeast strain classified as Cryptococcus flavus. When grown on starch-containing medium this strain exhibited the highest amylase production after 24 h of cultivation. The extracellular amylase from C. flavus was purified from the culture broth by a single step using chromatography on a Sephacryl S-100 column. The enzyme was purified 16.14-fold with a yield of 50.21% of the total activity. The purified enzyme was a glycoprotein with an apparent molecular mass of 75 and 84.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The enzyme lost approximately 50% of the molecular mass after treatment with glycosidases. The major end products of starch, amylose, amylopectin, pullulan and glycogen were maltose and maltotriose. The K(m) value for the pure enzyme was 0.056 mg ml(-1) with soluble starch as the substrate. Enzyme activity was optimal at pH 5.5 and 50 degrees C. The enzyme retained 90% of the activity after incubation at 50 degrees C for 60 min and was inhibited by Cu(2+), Fe(2+) and Hg(2+).     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Purification and characterization of an extracellular alpha-amylase from Bacillus subtilis AX20

A Bacillus subtilis AX20 from soil with ability to produce extracellular alpha-amylases was isolated. The characterization of microorganism was performed by biochemical tests as well as 16S rDNA sequencing. Maximum amylase activity (38 U/ml) was obtained at stationery phase when the culture was grown at 37 degrees C. The enzyme was purified to homogeneity with an overall recovery of 24.2% and specific activity of 4133 U/mg. The native protein showed a molecular mass of 149 kDa composed of a homodimer of 78 kDa polypeptide by SDS-PAGE. The optimum pH and temperature of the amylase were 6 and 55 degrees C, respectively. The enzyme was inhibited by Hg(2+), Ag(2+), and Cu(2+) and it did not show an obligate requirement of metal ions. The enzyme was not inhibited by EDTA or EGTA, suggesting that this enzyme is not a metalloenzyme. The end products of corn starch and soluble starch were glucose (70-75%) and maltose (20-25%). Rapid reduction of blue value and the end products suggest an endo mode of action for the amylase. The purified amylase shows interesting properties useful for industrial applications.     

Production of amylase by a submerged culture ofAspergillus wentii

Soluble starch was hydrolysed to maltose byAspergillus wentii Wehmer (IMI 17295). Studies on nutritional requirements ofAspergillus wentii for production of amylase revealed that the optimum conditions were achieved in fermentation culture medium containing 1% starch, and incubated at 20 °C for 3 days at pH 6.0. Tryptophan was the best nitrogen source. The amylase activity was completely inhibited when 1 mm sodium iodoacetate was incorporated into the medium. With 10 mm sodium citrate the amylase activity was increased from 3.51 to 6.0 mg/ml.     

General Biochemical Characterization of Thermostable Extracellular beta-Amylase from Clostridium thermosulfurogenes

Clostridium thermosulfurogenes, an anaerobic bacterium which ferments starch into ethanol at 62 degrees C, produced an active extracellular amylase and contained intracellular glucoamylase but not pullulanase activity. The extracellular amylase was purified 2.4-fold, and its general physicochemical and catalytic properties were examined. The extracellular amylase was characterized as a beta-amylase (1,4-alpha-d-glucan maltohydrolase) based on demonstration of exocleavage activity and the production of maltose with a beta-anomeric configuration from starch. The beta-amylase activity was stable and optimally active at 80 and 75 degrees C, respectively. The pH optimum for activity and the pH stability range was 5.5 to 6 and 3.5 to 6.5, respectively. The apparent [S](0.5V) and V(max) for beta-amylase activity on starch was 1 mg/ml and 60 U/mg of protein. Similar to described beta-amylase, the enzyme was inhibited by p-chloromercuribenzoate, Cu, and Hg; however, alpha- and beta-cyclodextrins were not competitive inhibitors. The beta-amylase was active and stable in the presence of air or 10% (vol/vol) ethanol. The beta-amylase and glucoamylase activities enabled the organism to actively ferment raw starch in the absence of significant pullulanase or alpha-amylase activity.     

Continuous alpha-amylase production using Bacillus amyloliquefaciens adsorbed on an ion exchange resin

Summary A method for the continuous production of extracellular alpha amylase by surface immobilized cells of Bacillus amyloliquefaciens NRC 2147 has been developed. A large-pore, macroreticular anionic exchange resin was capable of initially immobilizing an effective cell concentration of 17.5 g DW/1 (based on a total reactor volume of 160 ml). The reactor was operated continuously with a nutrient medium containing 15 g/l soluble starch, as well as yeast extract and salts. Aeration was achieved by sparging oxygen enriched air into the column inlet. Fermentor plugging by cells was avoided by periodically substituting the nutrient medium with medium lacking in both soluble starch and yeast extract. This fermentor was operated for over 200 h and obtained a steady state enzyme concentration of 18700 amylase activity units per litre (18.7 kU/l), and an enzyme volumetric productivity of 9700 amylase activity units per litre per hour (9.7 kU/l-h). Parallel fermentations were performed using a 2 l stirred vessel fermentor capable of operation in batch and continuous mode. All fermentation conditions employed were identical to those of the immobilized cell experiments in order to assess the performance of the immobilized cell reactor. Batch stirred tank operation yielded a maximum amylase activity of 150 kU/l and a volumetric productivity of 2.45 kU/l-h. The maximum cell concentration obtained was 5.85 g DW/l. Continuous stirred tank fermentation obtained a maximum effluent amylase activity of 6.9 kU/l and a maximum enzyme volumetric productivity of 2.73 kU/l-h. Both of these maximum values were observed at a dilution rate of 0.345 l/h. The immobilized cell reactor was observed to achieve larger volumetric productivities than either mode of stirred tank fermentation, but achieved an enzyme activity concentration lower than that of the batch stirred tank fermentor.     

The evaluation of amylolytic activity of strains of coagulase negative staphylococci

Amylase activity of 30 strains of Staphylococcus spp. was determined by Tryptic Soy Agar on supplemented with 1.0% starch as the substrate. After incubation (time incubation 24 h or 168 h), the plates were flooded with Lugol solution. A clear zone around the colonies indicated amylase activity. The 23 (76.7%) strains CNS demonstrated the amylase activity. It was observed that 17 (80.9%) strains of S. epidermidis, and 6 (66.7%) strains non-S. epidermidis, starch hydrolyzed. Amylase production depends of time incubation (frequently 168 h) and growth atmosphere (frequently oxygen atmosphere)     

Heterologous expression of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> thermostable alpha-amylase in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> and its application in boiling stable resistant sago starch preparation

A gene encoding the thermostable α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced, and cloned into Yarrowia lipolytica P01g host strain using the vector pYLSC1 allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 730 U/l in the Hinton flask culture broth. It is higher than that observed in P. pastoris expression system and E. coli expression system. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 72-h treatment, the DP _w of raw sago starch obviously decreased from 830,945 to 237,092. The boiling stable resistant starch content of the sago starch increased from 8.3 to 18.1%. The starch recovery rate was 71%.     

嗜盐碱性淀粉酶产生条件和性质的初步研究

从我国内蒙古自治区察汗淖碱湖分离到一株能产胞外嗜盐碱性淀粉酶的极端嗜盐嗜碱杆菌(Natronobacterium sp.)C-212,该菌产酶的最适pH和NaCl浓度分别为9.5和20%,最适碳源为可溶性淀粉,氮源为复合蛋白胨.酶反应最适温度为50℃,pH为8.5,NaCl浓度为2.6mol/L,该酶在pH9.5最稳定,NaCl可增加酶的热稳定性,酶降解可溶性淀粉的主要产物为葡萄糖、麦芽糖、麦芽三糖及其他寡糖.