Production, purification and characterization of an α-amylase produced by Saccharomycopsis fibuligera

Saccharomycopsis fibuligera ST 2 produced high levels of extracellular amylase during the stationary phase of growth. Glucose or other low molecular weight metabolizable sugars did not repress the synthesis of the amylase, indicating the lack of catabolite repression in this organism. Of the nitrogen sources examined, yeast extract and corn steep liquor stimulated the highest yield of amylase. Ammonium sulphate inhibited α-amylase synthesis. The enzyme was purified 118-fold from the culture supernatant fluid by isopropanol precipitation and DEAE-Sephadex A50 chromatography. The purified enzyme was characterized as an α-amylase. The α-amylase had the following properties: molecular weight, 40900 ± 500; optimum temperature, 60°C; activation energy, 1600 cal/mol; optimum pH, 4·8–6·0; range of pH stability, pH 4·0–9·4; K_m (50°C, pH 5·5) for soluble starch, 0·572 mg/ml; final products of starch hydrolysis—glucose, maltose, maltotriose and maltotetraose.     

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+.     

Characterization of an organic solvent-tolerant α-amylase from a halophilic isolate, <Emphasis Type="Italic">Thalassobacillus</Emphasis> sp. LY18

A halophilic isolate Thalassobacillus sp. LY18 producing extracellular amylase was isolated from the saline soil of Yuncheng Salt Lake, China. Production of the enzyme was synchronized with bacterial growth and reached a maximum level during the early stationary phase. The amylase was purified to homogeneity with a molecular mass of 31 kDa. Major products of soluble starch hydrolysis were maltose and maltotriose, indicating an α-amylase activity. Optimal enzyme activity was found to be at 70°C, pH 9.0, and 10 % NaCl. The α-amylase was highly stable over broad temperature (30–90°C), pH (6.0–12.0), and NaCl concentration (0–20 %) ranges, showing excellent thermostable, alkalistable, and halotolerant nature. The enzyme was stimulated by Ca²⁺, but greatly inhibited by EDTA, indicating it was a metalloenzyme. Complete inhibition by diethyl pyrocarbonate and β-mercaptoethanol revealed that histidine residue and disulfide bond were essential for enzyme catalysis. The surfactants tested had no significant effects on the amylase activity. Furthermore, it showed high activity and stability in the presence of water-insoluble organic solvents with log P _ow ≥ 2.13.     

Synthesis and localization of α-amylase and α-glucosidase in Bacillus licheniformis grown in batch and continuous culture

In batch and continuous cultures of Bacillus licheniformis NC1B 6346 α-amylase was invariably extracellular and could not be detected in the cytoplasm or cell surface. α-Glucosidase however, was largely intracellular but at the end of exponential growth and during slow growth under Mg²⁺ limitation it was detected in the culture fluid. Both enzymes were susceptible to catabolite repression and glucose totally inhibited their synthesis in batch culture. In maltose-limited chemostat culture, synthesis of both enzymes was maximal at D = 0.2/h and declined at higher growth rates. α-Amylase synthesis was constitutive but α-glucosidase synthesis was induced by maltose and maltotriose but not by methyl-α-D-glucoside or phenyl-α-D-glucoside. α-Amylase was synthesized at pH 6.5 and above in maltose-limited chemostat culture but not below this pH. Intracellular α-glucosidase synthesis varied little with pH. Increasing temperature decreased the synthesis of both enzymes in chemostat culture to the extent that α-glucosidase was undetectable at 50° C. Polar lipid composition varied with pH and temperature but there was no correlation between this and enzyme secretion. Moreover cerulenin, an antibiotic that inhibits protein secretion in some bacteria by interacting with the membrane had no effect on α-amylase secretion but decreased the release of α-glucosidase upon protoplast formation.     

Repeated batch fermentation from raw starch using a maltose transporter and amylase expressing diploid yeast strain

We successfully demonstrated batch ethanol fermentation repeated ten times from raw starch with high ethanol productivity. We constructed a yeast diploid strain coexpressing the maltose transporter AGT1, α-amylase, and glucoamylase. The introduction of AGT1 allows maltose and maltotriose fermentation as well as the improvement of amylase activities. We also found that α-amylase activity during fermentation was retained by the addition of 10 mM calcium ion and that the highest α-amylase activity was 9.26 U/ml during repeated fermentation. The highest ethanol productivity was 2.22 g/l/h at the fourth batch, and after ten cycles, ethanol productivity of more than 1.43 g/l/h was retained, as was α-amylase activity at 6.43 U/ml.     

Starch Utilization by Bacteroides ovatus Isolated from the Human Large Intestine

Starch supported growth of continuous cultures of Bacteroides ovatus when this carbohydrate provided the sole source of carbon and energy. Inducible amylase and α-glucosidase activities were inversely related to dilution rate in starch-limited and starch-excess chemostats over the dilution rate (D) range D = 0.03/h to D = 0.20/h, and were partly repressed during growth under conditions of starch-excess. Preparative isoelectric focusing of B. ovatus cytoplasmic extracts indicated the existence of three distinct starch-hydrolyzing enzymes. Incubation of active fractions from the isoelectric focusing cell with maltose and a variety of low-molecular-weight oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose) identified a single amylase activity, an enzyme with combined β-amylase and glucoamylase/α-glucosidase properties, and also a possible pullulanase. The ability of B. ovatus to synthesize several starch-hydrolyzing enzymes with different specificities and activities may confer a significant competitive advantage to this organism in the colonic ecosystem. Received: 14 August 1996 / Accepted: 29 October 1996     

Identification of an extracellular thermostable glycosyl hydrolase family 13 α-amylase from <Emphasis Type="Italic">Thermotoga neapolitana</Emphasis>

We cloned the gene for an extracellular α-amylase, AmyE, from the hyperthermophilic bacterium Thermotoga neapolitana and expressed it in Escherichia coli. The molecular mass of the enzyme was 92 kDa as a monomer. Maximum activity was observed at pH 6.5 and temperature 75°C and the enzyme was highly thermostable. AmyE hydrolyzed the typical substrates for α-amylase, including soluble starch, amylopectin, and maltooli-gosaccharides. The hydrolytic pattern of AmyE was similar to that of a typical α-amylase; however, unlike most of the calcium (Ca²⁺)-dependent α-amylases, the activity of AmyE was unaffected by Ca²⁺. The specific activities of AmyE towards various substrates indicated that the enzyme preferred maltooligosaccharides which have more than four glucose residues. AmyE could not hydrolyze maltose and maltotriose. When maltoheptaose was incubated with AmyE at the various time courses, the products consisting of maltose through maltopentaose was evenly formed indicating that the enzyme acts in an endo-fashion. The specific activity of AmyE (7.4 U/mg at 75° C, pH 6.5, with starch as the substrate) was extremely lower than that of other extracellular α-amylases, which indicates that AmyE may cooperate with other highly active extracellular α-amylases for the breakdown of the starch or α-glucans into maltose and maltotriose before transport into the cell in the members of Thermotoga sp.     

Extracellular amylase production bySaccharomycopsis capsularis and its evaluation for starch saccharification

A strain of starch-assimilating yeast,Saccharomycopsis capsularis, isolated from Indian cereal-based fermented foods, produced significant levels of extracellular α-amylase and glucoamylase. The enzymes reached their peak activities during the stationary phase at the end of the 5th and 4th day of cultivation, respectively. The amylase yields were maximized by a proper choice of carbon and nitrogen sources, starting pH of the culture medium and growth temperature. High activities of the enzymes were obtained through inexpensive agricultural commodities, such as wheat bran and corn meal as carbon sources, and defatted soybean meal and peanut meal as nitrogen sources. A temperature of 28–32°C and an initial pH of 4.5–5.0 were optimum. The crude amylase mixture could liquefy and saccharify a 1% starch solution completely in 24 h at 50°C.     

Molecular cloning and expression of an extracellular α-amylase gene from an Antarctic deep sea psychrotolerant <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain 7193

Psychrotolerant Pseudomonas stutzeri strain 7193 capable of producing an extracellular α-amylase was isolated from deep sea sediments of Prydz Bay, Antarctic. The 59678-Da protein (AmyP) was encoded by 1665-bp gene (amyP). The deduced amino acid sequence was identified with four regions, which are conserved in amylolytic enzymes and form a catalytic domain, and was predicted to be maltotetraose forming extracellular amylase by using the I-TASSER online server. Purification of AmyP amylases from both the recombinant of Escherichia coli Top 10 F′ and strain 7193 was conducted. Biochemical characterization revealed that the optimal amylase activity was observed at pH 9.0 and temperature 40°C. The enzymes were unstable at temperatures above 30°C, and only retain half of their highest activity after incubation at 60°C for 5 min. Thin-layer chromatography analysis of the products of the amylolytic reaction showed the presence of maltotetraose, maltotriose, maltose and glucose in the starch hydrolysate.     

Regulation of maltose metabolism in stationary phase cultures of an asporogenous mutant of Bacillus licheniformis

An asporogenous strain of Bacillus licheniformis accumulated maltose by an energy dependent transport mechanism during an extended stationary phase. Maltose transport was sensitive to the effects of the uncoupler tetrachlorosalicylanide (TCS), and was also inhibited by glucose. Maltose stimulated synthesis of a p -nitrophenyl-α- D -glucoside-hydrolysing enzyme ( p NPGase) in log phase and in stationary phase cells. In the presence of glucose this induction was inhibited. Glucose was used preferentially to maltose in stationary phase cells. The uptake of maltose from the medium, and the synthesis of p NPGase, were immediately and completely inhibited in the presence of glucose. These results are consistent with a mechanism of inducer exclusion mediating the repressive effect of glucose upon p NPGase synthesis in stationary phase cells. Catabolite repression of α-amylase synthesis by glucose was also demonstrated in late stationary phase mutant cells.     

Extracellular α-glucosidase of an alkalophilic microorganism, Bacillus sp. ATCC 21591

Abstract Bacillus sp. ATCC 21591, an alkalophilic bacterium, produces 3 enzymes associated with degradation of starch-α-amylase, pullulanase and α-glucosidase. The latter reached a maximum after 24 h growth. Highest activities of α-glucosidase and pullulanase were obtained when the initial pH of the medium was 9.7 and although at pH 10.4 highest biomass was attained after 48 h no α-glucosidase was present. The pH optimum for activity with maltose as substrate was 7.0, which is surprisingly low for an alkalophilic organism. The enzyme was substrate specific for p -nitrophenyl- α -D-glucoside, maltose and maltotriose in that order. Forty eight times the activity was located in the cell-free supernatant, relative to that found intracellulary. Transferase activity was detected - the major end-product formed from maltose was a compound with an R _f -value similar to isomaltose.     

Clostridium cellulolyticum Viability and Sporulation under Cellobiose Starvation Conditions

Depending on the moment of cellobiose starvation, Clostridium cellulolyticum cells behave in different ways. Cells starved during the exponential phase of growth sporulate at 30%, whereas exhaustion of the carbon substrate at the beginning of growth does not provoke cell sporulation. Growth in the presence of excess cellobiose generates 3% spores. The response of C. cellulolyticum to carbon starvation involves changes in proteolytic activities; higher activities (20% protein degradation) corresponded to a higher level of sporulation; lower proteolysis (5%) was observed in cells starved during the beginning of exponential growth, when sporulation was not observed; with an excess of cellobiose, an intermediate value (10%), accompanied by a low level of sporulation, was observed in cells taken at the end of the exponential growth phase. The basal percentage of the protein breakdown in nonstarved culture was 4%. Cells lacking proteolytic activities failed to induce sporulation. High concentrations of cellobiose repressed proteolytic activities and sporulation. The onset of carbon starvation during the growth phase affected the survival response of C. cellulolyticum via the sporulation process and also via cell-cellulose interaction. Cells from the exponential growth phase were more adhesive to filter paper than cells from the stationary growth phase but less than cells from the late stationary growth phase.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Purification, partial characterization, and covalent immobilization–stabilization of an extracellular α-amylase from Aspergillus niveus

An extracellular amylase secreted by Aspergillus niveus was purified using DEAE fractogel ion exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5 % polyacrylamide gel electrophoresis (PAGE) and 10 % sodium dodecyl sulfate (SDS-PAGE). The enzyme exhibited 4.5 % carbohydrate content, 6.6 isoelectric point, and 60 and 52 kDa molar mass estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The amylase efficiently hydrolyzed glycogen, amylose, and amylopectin. The end-products formed after 24 h of starch hydrolysis, analyzed by thin layer chromatography, were maltose, maltotriose, maltotetraose, and maltopentaose, which classified the studied amylase as an α-amylase. Thermal stability of the α-amylase was improved by covalent immobilization on glyoxyl agarose (half-life of 169 min, at 70 °C). On the other hand, the free α-amylase showed a half-life of 20 min at the same temperature. The optima of pH and temperature were 6.0 and 65 °C for both free and immobilized forms.     

Kinetics of α-amylase production in a continuous culture ofBacillus licheniformis

As found during continuous cultivation ofBacillus licheniformis on a semisynthetic medium (glucose or maltose as C source), the specific rate of α-amylase production is proportional to growth rate but is repressed by higher substrate concentrations. Besides glucose or maltose, peptone was also used as an alternative carbon source during cultivation. The specific rate of production of the enzyme on maltose is half that found with glucose.     

Purification of pullulanase from <Emphasis Type="Italic">Aureobasidium pullulan</Emphasis>s

Purification and characterization of pullulanase from Aureobasidium pullulans. Pullulanase was purified by using gel—filtration column then on ion exchange using Q-sepharose column yielding a single peak. Purification was further carried out on SP-sepharose column. Molecular weight of pullulanase from A. pullulans was found to be about 73 KDa on the SDS-PAGE 10%. Native-PAGE 10% showed the activity of pullulanase, using polyacrylamide gel containing pullulan. Hydrolysis products from pullulanase activity with soluble starch, glycogen and pullulan on thin layer chromatography appeared as one band which is maltotriose, while α-amylase with soluble starch and glycogen showed two bands which are maltose and maltotriose but α-amylase gave negative result with pullulan on TLC chromatography only. Pullulanase could degrade α-1,6 glycosidic linkage of the previous substrates, while amylase could degrade α-1,4 glycosidic linkage of glycogen, soluble starch and pullulan. MALDI-Ms was employed to deduce protein sequence of pullulanase.     

Cloning and expression of a Bacillus licheniformisα-amylase gene in Pseudomonas aeruginosa

Abstract The gene for B. licheniformis α-amylase has been cloned in P. aeruginosa . Synthesis of the enzyme occurs in late log phase and goes on during stationary phase. Although P. aeruginosa is a secretory bacterium, α-amylase is not efficiently secreted into the extracellular medium; 85% of the enzyme is retained in the periplasm.     

Fermentation of maltotriose by brewer's and baker's yeasts

Two brewer's yeasts and one baker's yeast grew with 95% (w/w) pure maltotriose as carbon source in the presence of antimycin A to block respiration. Biomass yields (0.15 and 0.24 g dry yeast g^–1 sugar, respectively, with and without antimycin A) were similar for growth on maltose and maltotriose, and yields of ethanol were 80% of stoichiometric. Yeasts harvested during growth on glucose and containing low maltose transport activity did not begin to use maltotriose in the presence of antimycin A until after a long lag phase (up to 50 h), but yeast harvested during growth on maltose, and containing high maltose transport activity, began to use maltotriose after about 25 h. Much shorter lags were observed before growth started in the absence of antimycin A.     

Effect of growth rate on α-amylase production by Streptomyces sp. IMD 2679

The α-amylase of Streptomyces sp. IMD 2679 was subject to catabolite repression. Four different growth rates were achieved when the organism was grown at 40 °C and 55 °C in the presence and absence of cobalt, with an inverse relationship between α-amylase production and growth rate. Highest α-amylase yields (520 units/ml) were obtained at the lowest growth rate (0.062 h⁻¹), at 40 °C in the absence of cobalt, while at the highest growth rate (0.35 h⁻¹), at 55 °C in the presence of cobalt, α-amylase production was decreased to 150 units/ml. As growth rate increased, the rate of specific utilisation of the carbon source maltose also increased, from 46 to 123 μg maltose (mg biomass)⁻¹ h⁻¹. The pattern and levels of α-glucosidase (the enzyme degrading maltose) detected intracellularly in each case, indicate that growth rate effectively controls the rate of feeding of glucose to the cell, and thus catabolite repression. Received: 17 February 1997 / Received revision: 29 April 1997 / Accepted: 11 May 1997     

Microarray karyotyping of maltose‐fermenting Saccharomyces yeasts with differing maltotriose utilization profiles reveals copy number variation in genes involved in maltose and maltotriose utilization

Aims: We performed an analysis of maltotriose utilization by 52 Saccharomyces yeast strains able to ferment maltose efficiently and correlated the observed phenotypes with differences in the copy number of genes possibly involved in maltotriose utilization by yeast cells. Methods and Results: The analysis of maltose and maltotriose utilization by laboratory and industrial strains of the species Saccharomyces cerevisiae and Saccharomyces pastorianus (a natural S. cerevisiae/Saccharomyces bayanus hybrid) was carried out using microscale liquid cultivation, as well as in aerobic batch cultures. All strains utilize maltose efficiently as a carbon source, but three different phenotypes were observed for maltotriose utilization: efficient growth, slow/delayed growth and no growth. Through microarray karyotyping and pulsed‐field gel electrophoresis blots, we analysed the copy number and localization of several maltose‐related genes in selected S. cerevisiae strains. While most strains lacked the MPH2 and MPH3 transporter genes, almost all strains analysed had the AGT1 gene and increased copy number of MALx1 permeases. Conclusions: Our results showed that S. pastorianus yeast strains utilized maltotriose more efficiently than S. cerevisiae strains and highlighted the importance of the AGT1 gene for efficient maltotriose utilization by S. cerevisiae yeasts. Significance and Impact of the Study: Our results revealed new maltotriose utilization phenotypes, contributing to a better understanding of the metabolism of this carbon source for improved fermentation by Saccharomyces yeasts.