Production and Characterization of Amylase from Calvatia gigantea

α-Amylase (EC 3.2.1.1) was excreted by Calvatia gigantea in liquid growth media containing different sources of starch. Among the factors affecting enzyme production in shake flasks were the type and the concentration of starch and the nitrogen source supplied. Optimum cultural conditions for maximum enzyme production were: soluble starch concentration, 5%; inoculum size, 3.75 × 10⁵ conidia per ml; 5-day cultivation time at 28 to 30°C. The observed maximum yield of 81.3 U of saccharifying enzyme activity per ml of growth medium was the highest ever reported in the literature for submerged cultures. Partially purified enzyme functioned optimally at pH 4.5 to 5.5 and 53 to 58°C. The activation energy of enzymic hydrolysis of starch in the range of 20 to 40°C was 8,125 cal/mol (ca. 3.41 × 10⁴ J). The apparent K_m value of the enzyme at 25°C was 7.68 × 10⁻⁴ g/ml. Some of the properties of the enzyme under investigation were similar to those of α-amylases excreted from molds producing large amounts of the enzyme.     

Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22

A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The K_m and k_cat values for the pure enzyme were 1.35 mg ml⁻¹ and 249 μM mg⁻¹ min⁻¹, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l⁻¹ gelatinized soluble starch or 125 g l⁻¹ raw wheat starch at an enzyme concentration of 10 U g⁻¹ of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.     

Expression of a fungal glucoamylase in transgenic rice seeds

Glucoamylase, which catalyses the hydrolysis of the α-1,4 glycosidic bonds of starch, is an important industrial enzyme used in starch enzymatic saccharification. In this study, a glucoamylase gene from Aspergillus awamori, under the control of the promoter of seed storage protein Gt1, was introduced into rice by Agrobacterium-mediated transformation. Significant glucoamylase activity was detected specifically in the seeds but not other tissues of the transgenic rice lines. The highest enzymatic activity was found in the transgenic line Bg17-2, which was estimated to have about 500 units per gram of seeds (one unit is defined as the amount of enzyme that produces 1 μmol of reducing sugar in 1 min at 60 °C using soluble starch as substrate). The optimum pH for the activity of the rice produced enzyme is 5.0–5.5, and the optimum temperature is around 60 °C. One part of this transgenic glucoamylase rice seed flour fully converted 25 parts of corn starch pre-liquefied by an α-amylase also produced by a transgenic rice into glucose in 16 h incubation. This study suggests that this hydrolysis enzyme may substitute commercial fermentation enzymes for industrial starch conversion.     

Purification and Characterization of an Extremely Thermostable Cyclomaltodextrin Glucanotransferase from a Newly Isolated Hyperthermophilic Archaeon, a Thermococcus sp.

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 μm in diameter. The new isolate grew at temperatures between 60 and 95°C (optimum, 85°C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120°C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100°C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110°C. The enzyme formed mainly α-cyclodextrin with small amounts of β- and γ-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.     

Factors Affecting Yield and Safety of Protein Production from Cassava by Cephalosporium eichhorniae

The properties of Cephalosporium eichhorniae 152 (ATCC 38255) affecting protein production from cassava carbohydrate, for use as an animal feed, were studied. This strain is a true thermophile, showing optimum growth at 45° to 47°C, maximum protein yield at 45°C, and no growth at 25°C. It has an optimum pH of about 3.8 and is obligately acidophilic, being unable to sustain growth at pH 6.0 and above in a liquid medium, or pH 7.0 and above on solid media. The optimum growth conditions of pH 3.8 and 45°C were strongly inhibitive to potential contaminants. It rapidly hydrolyzed cassava starch. It did not utilize sucrose, but some (around 16%) of the small sucrose component of cassava was chemically hydrolyzed during the process. Growth with cassava meal (50 g/liter [circa 45 g/liter, glucose equivalent]) was complete in around 20 h, yielding around 22.5 g/liter (dry biomass), containing 41% crude protein (48 to 50% crude protein in the mycelium) and 31% true protein (7.0 g/liter). Resting and germinating spores (10⁶ to 10⁸ per animal) injected by various routes into normal and γ-irradiated 6-week-old mice and 7-day-old chickens failed to initiate infections.     

Isolation and physiological study of an amylolytic strain of Lactobacillus plantarum

Summary An amylolytic lactic acid bacterium identified as Lactobacillus plantarum was isolated from cassava roots (Manihot esculenta var. Ngansa) during reting. The amylolytic enzyme synthesized was an extracellular -amylase with an optimum pH of 5.0 and an optimum temperature of 55° C. Cultured on starch, the strain displayed a growth rate of 0.43 h^–1, a biomass yield of 0.19 g·g^–1 and a lactate yield of 0.81 g·g^–1. The growth kinetics were similar on starch and glucose. Sufficient enzyme was synthesized and starch hydrolysis was not a limiting factor for growth. Biosynthesis of the enzyme was observed when the glucose concentration was less than 6.7 g·l^–1 and reached up to 4 IU·ml^–1 at the end of the fermentation. Offprint requests to: M. Raimbault     

Production of α-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

α-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55°C and 5.5, respectively, and the apparent K_m for starch was 0.8 mg ml⁻¹. The products of α-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of α-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial α-amylase. Activities of α-amylase up to 4.4 U ml⁻¹ (1 U represents 1 μmol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml⁻¹ in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml⁻¹), α-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed α-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of α-amylase (initial concentration, 2.5 mg ml⁻¹), they were found to be less effective than starch, but maltose was almost as effective. The fungal α-amylase was found to be stable at 60°C in the presence of low concentrations of starch (≤5%), suggesting that it may be suitable for industrial application.     

Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

Pullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase from Bacillus deramificans were selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that the K_m values for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and the K_cat/K_m values increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.     

Characterization of an endo-Acting Amylopullulanase from Thermoanaerobacter Strain B6A

A thermoanaerobe (Thermoanaerobacter sp.) grown in TYE-starch (0.5%) medium at 60°C produced both extra- and intracellular pullulanase (1.90 U/ml) and amylase (1.19 U/ml) activities. Both activities were produced at high levels on a variety of carbon sources. The temperature and pH optima for both pullulanase and amylase activities were 75°C and pH 5.0, respectively. Both the enzyme activities were stable up to 70°C (without substrate) and at pH 4.5 to 5.0. The half-lives of both enzyme activities were 5 h at 70°C and 45 min at 75°C. The enzyme activities did not show any metal ion activity, and both activities were inhibited by β- and γ-cyclodextrins but not by α-cyclodextrin. A single amylolytic pullulanase responsible for both activities was purified to homogeneity by DEAE-Sepharose CL-6B column chromatography, gel filtration using high-pressure liquid chromatography, and pullulan-Sepharose affinity chromatography. It was a 450,000-molecular-weight glycoprotein composed of two equivalent subunits. The pullulanase cleaved pullulan in α1,6 linkages and produced multiple saccharides from cleavage of α-1,4 linkages in starch. The K_ms for pullulan and soluble starch were 0.43 and 0.37 mg/ml, respectively.     

A Thermostable Glucoamylase from Bispora sp. MEY-1 with Stability over a Broad pH Range and Significant Starch Hydrolysis Capacity

Background

Glucoamylase is an exo-type enzyme that converts starch completely into glucose from the non-reducing ends. To meet the industrial requirements for starch processing, a glucoamylase with excellent thermostability, raw-starch degradation ability and high glucose yield is much needed. In the present study we selected the excellent Carbohydrate-Activity Enzyme (CAZyme) producer, Bispora sp. MEY-1, as the microbial source for glucoamylase gene exploitation.

Methodology/Principal Findings

A glucoamylase gene (gla15) was cloned from Bispora sp. MEY-1 and successfully expressed in Pichia pastoris with a high yield of 34.1 U/ml. Deduced GLA15 exhibits the highest identity of 64.2% to the glucoamylase from Talaromyces (Rasamsonia) emersonii. Purified recombinant GLA15 was thermophilic and showed the maximum activity at 70°C. The enzyme was stable over a broad pH range (2.2–11.0) and at high temperature up to 70°C. It hydrolyzed the breakages of both α-1,4- and α-1,6-glycosidic linkages in amylopectin, soluble starch, amylose, and maltooligosaccharides, and had capacity to degrade raw starch. TLC and H¹-NMR analysis showed that GLA15 is a typical glucoamylase of GH family 15 that releases glucose units from the non-reducing ends of α-glucans. The combination of Bacillus licheniformis amylase and GLA15 hydrolyzed 96.14% of gelatinized maize starch after 6 h incubation, which was about 9% higher than that of the combination with a commercial glucoamylase from Aspergillus niger.

Conclusion/Significance

GLA15 has a broad pH stability range, high-temperature thermostability, high starch hydrolysis capacity and high expression yield. In comparison with the commercial glucoamylase from A. niger, GLA15 represents a better candidate for application in the food industry including production of glucose, glucose syrups, and high-fructose corn syrups.     

Enzymatic Analysis of an Amylolytic Enzyme from the Hyperthermophilic Archaeon Pyrococcus furiosus Reveals Its Novel Catalytic Properties as both an α-Amylase and a Cyclodextrin-Hydrolyzing Enzyme

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90°C. The substrate specificity of PFTA suggests that it possesses characteristics of both α-amylase and cyclodextrin-hydrolyzing enzyme. Like typical α-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and β-cyclodextrin, which are resistant to α-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent α-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both α-amylase and cyclodextrin-hydrolyzing enzyme.     

Production and Characteristics of Raw-Potato-Starch-Digesting α-Amylase from Bacillus subtilis 65

A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting α-amylase. The electrophoretically homogeneous preparation of enzyme (molecular weight, 68,000) digested and solubilized raw corn starch to glucose and maltose with small amounts of maltooligosaccharides ranging from maltotriose to maltoheptaose. This enzyme was different from other amylases and could digest raw potato starch almost as fast as it could corn starch, but it showed no adsorbability onto any kind of raw starch at any pH. The mixed preparation with Endomycopsis glucoamylase synergistically digested raw potato starch to glucose at 30°C. The raw-potato-starch-digesting α-amylase showed strong digestibility to small substrates, which hydrolyzed maltotriose to maltose and glucose, and hydrolyzed p-nitrophenyl maltoside to p-nitrophenol and maltose, which is different from the capability of bacterial liquefying α-amylase.     

Purification and Characterization of Pea Epicotyl beta-Amylase

The most abundant β-amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this β-amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a β-amylase by end product analysis and by its inability to hydrolyze β-limit dextrin and to release dye from starch azure. Pea β-amylase is an approximate 55 to 57 kilodalton monomer with a pl of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a K_m of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromer-curiphenylsulfonic acid and N-ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea β-amylase is competitively inhibited by its end product, maltose, with a K_i of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with α-cyclohexaamylose being a stronger inhibitor than β-cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea β-amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea β-amylase is the multichain type. Possible roles of pea β-amylase in cellular glucan metabolism are discussed.     

Partial Purification and Characterization of a Thermostable Actinomycete β-Amylase

A thermostable amylase, possibly a β-amylase from Thermoactinomyces sp. no. 2 isolated from soil, is reported. The enzyme was purified 36-fold by acetone precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration, and the molecular weight was estimated at 31,600. The enzyme was characterized by demonstration of optimum activity at 60°C and pH 7 and by retention of 70% activity at 70°C (30 min). It was stimulated by Mn²⁺ and Fe²⁺ but strongly inhibited by Hg²⁺. Maltose was the only detectable product of hydrolysis of starches and was quantitatively highest in plantain starch hydrolysate.     

Nitrogen-induced changes in the growth and metabolism of developing maize kernels grown in vitro

Cereal kernel growth and grain yield are functions of endosperm starch accumulation. The objective of this study was to examine how various metabolic factors in developing maize (Zea mays L.) endosperm influence starch deposition. Kernels were grown in vitro on medium with: (a) zero N (−N), (b) optimum N (+N), or (c) −N from 3 to 20 days after pollination followed by +N until maturity (±N) to produce different degrees of endosperm growth and to promote an enhancement of starch synthesis midway through development. At intervals, kernels were harvested and levels of enzyme activities and carbohydrate and N constituents examined. Endosperm starch and protein accumulation were decreased in −N compared to +N kernels, but relief of N starvation increased both constituents. With greater movement of N into ±N kernels, endosperm sugar concentrations declined suggesting an inverse relationship between C and N transport. Unusually high concentrations of sugar in N stressed kernels did not appear to limit or enhance starch production. Rather, increased accumulation of starch in ±N endosperm was correlated with significant increases in the enzymatic activities of sucrose synthase and PPi-linked phosphofructokinase, and to a lessor extent hexokinase. In addition, the occurrence of specific proteins of the albumin/globulin fraction either increased, decreased, or remained unchanged in relation to starch synthesis. These data suggest that lack of N limits starch deposition in maize endosperm primarily through an influence on synthesis of key proteins.     

Utilization of Low Concentrations of Starch by a Flavobacterium Species Isolated from Tap Water

Experiments in well-cleaned glass flasks revealed that addition of starch in concentrations of 10 and 25 μg of substrate C per liter to the filtrate of slow sand filters stimulated the development of a yellow-pigmented bacterium which was identified as a Flavobacterium species. The isolate was able to multiply in tap water without substrates added, but addition of starch and glucose in amounts as low as 1 μg of substrate C per liter clearly enhanced growth. The substrate affinities of the Flavobacterium for these compounds were 3.9 μg of starch C and 3.3 μg of glucose C per liter. The results of this study indicate that microorganisms which rapidly utilize starch at a level of a few micrograms per liter commonly occur in water.     

Production and Characteristics of Raw-Starch-Digesting α-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher α-amylaseactivity than the parent strain under submerged culture at 30°C for 24 h. About 70% of the total α-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable α-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable α-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal α-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.     

Heterologous Expression of the Saccharomyces cerevisiae PGU1 Gene in Schizosaccharomyces pombe Yields an Enzyme with More Desirable Properties for the Food Industry

The Saccharomyces cerevisiae PGU1 gene was successfully expressed in Schizosaccharomyces pombe. The optimum pH and temperature for the recombinant enzyme were 5 and 40°C, respectively, these being around 0.5 U higher and 5°C lower than those shown by the native enzyme. The K_m value was about fourfold higher than that of the S. cerevisiae enzyme. The recombinant endopolygalacturonase was more efficient in reducing the viscosity of polygalacturonic acid and was also more stable at different pHs and temperatures than the native enzyme.     

Purification and Properties of a Maltotetraose- and Maltotriose-Producing Amylase from Chloroflexus aurantiacus

A maltotetraose- and maltotriose-producing amylase which is stable at alkaline pHs and high temperatures was detected in the culture filtrate of a strain of Chloroflexus aurantiacus J-10-F1, a thermophilic, green, photosynthetic bacterium. The enzyme was purified to homogeneity, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by means of ultrafiltration, ammonium sulfate fractionation, and DEAE-cellulose, hydroxyapatite, and high-performance liquid chromatographies. The molecular mass of the purified enzyme was estimated to be about 210,000 Da. The isoelectric point of the enzyme was estimated to be 6.24 by polyacrylamide gel electrofocusing. The amylase was stable up to 55°C and at alkaline pHs of up to 12.0. The optimum pH and temperature of the enzyme activity were 7.5 and 71°C, respectively. Metal ions such as Hg²⁺, Zn²⁺, Cu²⁺, Mn²⁺, and Ni²⁺ strongly inhibited the enzyme activity. The enzyme activity was reactivated specifically by Ca²⁺ after the enzyme was treated with 1 mM EDTA. This enzyme could digest various kinds of raw-starch granules from corn, cassava, and potato. Both maltotetraose and maltotriose were formed as the main enzymatic products from soluble starch.     

Purification and Properties of a Thermoactive Glucoamylase from Clostridium thermosaccharolyticum

A bacterial glucoamylase was purified from the anaerobic thermophilic bacterium Clostridium thermosaccharolyticum and characterized. The enzyme, which was purified 63-fold, with a yield of 36%, consisted of a single subunit with an apparent molecular mass of 75 kDa. The purified enzyme was able to attack α-1,4- and α-1,6-glycosidic linkages in various α-glucans, liberating glucose with a β-anomeric configuration. The purified glucoamylase, which was optimally active at 70°C and pH 5.0, attacked preferentially polysaccharides such as starch, glycogen, amylopectin, and maltodextrin. The velocity of oligosaccharide hydrolysis decreased with a decrease in the size of the substrate. The K_m values for starch and maltose were 18 mg/ml and 20 mM, respectively. Enzyme activity was not significantly influenced by Ca²⁺, EDTA, or α- or β-cyclodextrins.