Digestive α-amylase activity in Aelia acuminata L. (Hemiptera: Pentatomidae)

Activity of α-amylase was revealed in the midgut and salivary glands of the wheat and barley pentatomid pest, A. acuminata. The activity was determined in salivary gland more than those in midgut. Optimal activity of the enzyme occurred at 40°C. Optimal pH activity in salivary gland (pH = 6) was more than those in the midgut (pH = 4.5). pH stability analysis of the enzyme showed that the enzyme is more stable at slightly acidic pHs than those at acidic and alkaline pHs. However, α-amylase is more stable at acidic pH in long period of time. Temperature stability analysis determined the enzyme was remarkably active over a broad range of temperature (5–40°C). α-Amylase activity was decreased after addition of MgCl₂, Tris, Triton X-100, CuSO₄, SDS, urea and CaCl₂. The salts NaCl and KCl increased the enzyme activity from midgut and salivary glands. Zymogram analysis of midgut and salivary gland extract showed at least two bands of amylase activity in the midgut and salivary glands.     

Characteristic Decrease of Nuclear Protein Kinase Activity in G1-Arrested Cells of Saccharomyces cerevisiae cdc28

An alkalopsychrotrophic strain, Micrococcus sp. 207, inducibly and extracellularly produced amylase and pullulanase. The main hydrolysis product from amylose, with a crude enzyme preparation, was maltotetraose. The optimum temperature for activity of the amylase was 60°C and that for pullulanase 55°C. The activities at 0 to 30°C exhibited similar activation energy values. In an optimized production medium at pH 9.7, the highest yields of these enzymes were obtained after cell growth at 18°C for 4 days. At pH 8.5, the yields of amylase and pullulanase became maximum after 3 days cultivation. With more prolonged cultivation, the yield of amylase but not that of pullulanase activity decreased. These enzymes were not produced at temperatures above 30°C. Sucrose was not effective as an inducer, but it stimulated cell growth and enhanced the enzyme productivities with soluble starch.     

Characteristics of the amylase of Arthrobacter psychrolactophilus

Properties of the extracellular amylase produced by the psychrotrophic bacterium, Arthrobacter psychrolactophilus, were determined for crude preparations and purified enzyme. The hydrolysis of soluble starch by concentrated crude preparations was found to be a nonlinear function of time at 30 and 40 °C. Concentrates of supernatant fractions incubated without substrate exhibited poor stability at 30, 40, or 50 °C, with 87% inactivation after 21 h at 30 °C, 45% inactivation after 40 min at 40 °C and 90% inactivation after 10 min at 50 °C. Proteases known to be present in crude preparations had a temperature optimum of 50 °C, but accounted for a small fraction of thermal instability. Inactivation at 30, 40, or 50 °C was not slowed by adding 20 mg/ml bovine serum albumin or protease inhibitor cocktail to the preparations or the assays to protect against proteases. Purified amylase preparations were almost as thermally sensitive in the absence of substrate as crude preparations. The temperature optimum of the amylase in short incubations with Sigma Infinity Amylase Reagent was about 50 °C, and the amylase required Ca⁺² for activity. The optimal pH for activity was 5.0–9.0 on soluble starch (30 °C), and the amylase exhibited a K _m with 4-nitrophenyl-α-D-maltoheptaoside-4,6-O-ethylidene of 120 μM at 22 °C. The amylase in crude concentrates initially hydrolyzed raw starch at 30 °C at about the same rate as an equal number of units of barley α-amylase, but lost most of its activity after only a few hours.     

The production and activity of extracellular amylase by Rhizoctonia bataticola

Rhizoctonia bataticola produced the highest amounts of amylase in medium containing starch than that lacking starch within the 10 days of culture. Doubling the concentration of starch in the growth medium resulted in a near doubling of the amylase activity. Amylase production by the fungus is related to the type of carbon source in the medium with maximum amylase produced in medium containing starch. The maximum activity of the enzyme was detected in extracellular filtrates obtained from 4 days cultures. After this period, amylase activity decreased at first, and then increased through the 10 days incubation period. The fungus produced maximum levels of amylase prior to attainment of maximum mycelial biomass. Peak activity of the extracellular amylase was recorded at a temperature and pH range of 20–25°C and 4–5 respectively. The role of the exoenzyme in the deterioration of stored food products and its possible use in industrial fermentation processes are discussed.     

Digestive amylase and pectinase activity in the larvae of alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

The alfalfa weevil Hypera postica is a serious economic pest in most alfalfa grown in many countries worldwide. Digestive α-amylase and pectinase activities of larvae were investigated using general substrates. Midgut extracts from larvae showed an optimum activity for α-amylase against starch at acidic pH (pH 5.0). α-Amylase from larval midgut was more stable at mildly acidic pH (pH 5–6) than highly acidic and alkaline pH. The enzyme showed its maximum activity at 35°C. α-Amylase activity was significantly decreased in the presence of Ca²⁺, Mg²⁺ and sodium dodecylsulfate. On the contrary, K⁺ and Na⁺ did not significantly affect the enzyme activity. Zymogram analysis revealed the presence of one band of α-amylase activity in in-gel assays. Pectinase activity was assayed using agarose plate and colorimetric assays. Optimal pH for pectinase activity in the larval midgut was determined to be pH 5.0. Pectinase enzyme is more stable at pH 4.0–7.0 than highly acidic and alkaline pH. However, the enzyme was more stable at slightly acidic pH (pH 6.0) when incubation time increased. Maximum activity for the enzyme incubated at different temperatures was observed to be 40°C. Optimum pH activity for α-amylase and pectinase is not completely consistent with the pH prevailing in the larval midgut. This is the first report of the presence of pectinase activity in H. postica.     

Fermentative production of extracellular amylase from novel amylase producer,Tuber maculatum mycelium,and its characterization

Abstract

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22?±?2?°C) under static conditions which resulted in 1.41?U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50?°C. The enzyme showed good thermostability at 50?°C by retaining 98% of the maximal activity after 100?min of incubation. The amylase activity was marginally enhanced in presence of Cu²⁺ and Na⁺ and slightly reduced by K⁺, Ca²⁺, Fe²⁺, Mg²⁺, Co²⁺, Zn²⁺, and Mn²⁺ ions at 1?mM concentration.     

Structure‐based replacement of methionine residues at the catalytic domains with serine significantly improves the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica

The alkaline amylase requires high resistance towards chemical oxidation for use in the detergent and textile industries. This work aims to improve the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica by site‐directed mutagenesis based on the enzyme structure model. Five mutants were created by individually replacing methionine at positions 145, 214, 229, 247, and 317 in the amino acid sequence of alkaline amylase with oxidative‐resistant serine. The pH stability of the mutant enzymes was almost the same as that of the wild‐type (WT) enzyme (pH 7.0–11.0). The stable temperature range of the mutant enzymes M145S and M247S decreased from <50°C of the WT to <40°C, while the thermal stability of the other three mutant enzymes (M214S, M229S, and M317S) was almost the same as that of the WT enzyme. The catalytic efficiency (k_cat/K_m) of all the mutant enzymes decreased when compared to WT enzyme. The mutant enzymes showed increased activity in the presence of surfactants Tween‐60 and sodium dodecyl sulfate. When incubated with 500 mM H₂O₂ at 35°C for 5 h, the WT enzyme retained only 13.3% of its original activity, while the mutant enzymes M145S, M214S, M229S, M247S, and M317S retained 55.6, 70.2, 54.2, 62.5, and 46.4% of the original activities, respectively. The results indicated that the substitution of methionine residues at the catalytic domains with oxidative‐resistant serine can significantly improve the oxidative stability of alkaline amylase. This work provides an effective strategy to improve the oxidative stability of amylase, and the high oxidation resistance of the mutant enzymes shows their potential applications in the detergent and textile industries. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Cloning and Sequencing of an Original Gene Encoding a Maltogenic Amylase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. US149 Strain and Characterization of the Recombinant Activity

A gene encoding maltogenic amylase from acidic Bacillus sp. US149 (maUS149) was cloned, sequenced and over-expressed in Escherichia coli. The nucleotide sequence analysis revealed an open reading frame (ORF) of 1749 bp encoding a protein of 582 residues. The alignment of deduced amino acid sequence revealed a relatively low homology with the already reported maltogenic amylases. In fact, its highest identity, of only 60%, was found with the maltogenic amylase of Thermus sp. IM6501. The recombinant enzyme (MAUS149) was found to be intracellular and was purified to homogeneity from the cell crude extract with a yield of 23%. According to PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of 135 kDa and is composed of two identical subunits of 67.5 kDa each. The maximum activity was obtained at 40°C and pH 6.5. MAUS149 could be classified as a maltogenic amylase since it produces mainly maltose from starch, maltose and glucose from β-cyclodextrin, and panose from pullulan.     

Purification and characterization of the amylase of B. subtilis NRRL B3411

The amylase of Bacillus subtilis NRRL B3411 has been purified and partially characterized. The specific activity can be increased from 300,000 units/g to 6,000,000 units/g with a 60% recovery of total units. The purified material consists of one major and one trace anodic component as determined by disc gel electrophoresis. The molecular weight was 48,000 as determined by bio-gel filtration; the molecular weight was 44,900 ± 2400 as determined by sedimentation equilibrium methods. This purified enzyme is stable at, 70°C in the presence of 0.01 M Ca⁺⁺ and 0.1 M NaCl over a broad pH range from 5.5–9.5. The pH activity profile indicates optimum activity at pH 6.0. This amylase exhibits maximum activity at 60°C. The enzyme is a liquefying α-amylase as determined by analysis of hydrolysis products and immunological studies.     

Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25

A bacterium that secretes maltooligosaccharide-forming amylase in a medium containing 12.5% (vol/vol) dimethylsulfoxide (DMSO) was isolated and identified as Brachybacterium sp. strain LB25. The amylase of the strain was purified from the culture supernatant, and its molecular mass was 60 kDa. The enzyme was stable at pH 7.0–8.5 and active at pH 6.0–7.5. The optimum temperature at pH 7.0 was 35°C in the presence of 5 mM CaCl₂. The enzyme hydrolyzed starch to produce maltotriose primarily. The enzyme was active in the presence of various organic solvents. Its yield and product selectivity of maltooligosaccharides in the presence of DMSO or ethanol were compared with those of the industrial maltotriose-forming amylase from Microbacterium imperiale. Both enzymes improved the production selectivity of maltotriose by the addition of DMSO or ethanol. However, the total maltooligosaccharide yield in the presence of the solvents was higher for LB25 amylase than for M. imperiale amylase.     

Preparation of Triglyceride Hydroperoxides

Corticium rolfsii AHU 9627, which we isolated from a tomato stem, is one of the most promising producers of a raw starch saccharifying enzyme. The effects of the cultural conditions and medium components on the enzyme production were investigated. The enzyme production was improved by increasing both the concentrations of carbon sources and organic nutrients in the medium. Under the optimum cultural conditions, the enzyme activity of the culture supernatant against raw starch reached a maximum after 8-days incubation at 27°C and the activity reached 80 units per ml (when determined at 40°C and pH 4.0). The optimal pH and temperature for the enzyme reaction were 4.0 and 65°C, respectively. The saccharifying reaction was scarcely inhibited even with a high substrate concentration, and raw starch was rapidly hydrolyzed into glucose.     

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.     

Enzymatic Properties of Alkaline Proteinase from Aspergillus candidus

Some enzymatic properties were examined with the purified alkaline proteinase from Aspergillus candidus. The isoelectric point was determined to be 4.9 by polyacrylamide gel disc electrofocusing. The optimum pH for milk casein was around 11.0 to 11.5 at 30°C. The maximum activity was found at 47°C at pH 7.0 for 10 min. The enzyme was stable between pH 5.0 and 9.0 at 30°C and most stable at pH 6.0 at 50°C. The enzyme activity over 95% remained at 40°C, but was almost completely lost at 60°C for 10 min. Calcium ions protected the enzyme from heat denaturation to some extent. No metal ions examined showed stimulatory effect and Hg²⁺ ions inhibited the enzyme. The enzyme was also inhibited by potato inhibitor and diisopropylphosphorofluoridate, but not by metal chelating agent or sulfhydryl reagents. A. candidus alkaline proteinase exhibited immunological cross-reacting properties similar to those of alkaline proteinases of A. sojae and A. oryzae.     

Purification and Properties of an Amylase from Bacillus cereus NY-14

An extracellular amylase was obtained from a culture medium of Bacillus cereus NY-14. This enzyme was purified to show a single band on disc gel electrophoresis by ammonium sulfate fractionation and Sephadex G-100 gel filtration to 1101-fold of the activity of the original culture liquor. The purified enzyme had a molecular weight of 55,000, an isoelectric point of 6.13, an optimum pH of 6.0, and an optimum temperature of 55°C. The pH-stability range was wide; the enzyme retained more than 80% of its initial activity in the range of pH 5.5 to 12. It was stable below 35°C and required calcium ions for the stabilization. The action pattern of this enzyme on amylaceous polysaccharides was unique in that the predominant product was maltopentaose. The purified amylase could also digest starch granules of such plants as rice, barley, corn, and kuzu to produce maltopentaose as the main product.     

Isolation and characterization of a cold-active,alkaline, detergent stable α-amylase from a novel bacterium Bacillus subtilis N8

A cold-active alkaline amylase producer Bacillus subtilis N8 was isolated from soil samples. Amylase synthesis optimally occurred at 15°C and pH 10.0 on agar plates containing starch. The molecular weight of the enzyme was found to be 205?kDa by performing SDS-PAGE. While the enzyme exhibited the highest activity at 25°C and pH 8.0, it was highly stable in alkaline media (pH 8.0–12.0) and retained 96% of its original activity at low temperatures (10–40°C) for 24?hr. While the amylase activity increased in the presence of β-mercaptoethanol (103%); Ba²⁺, Ca²⁺, Na⁺, Zn²⁺, Mn²⁺, H₂O₂, and Triton X-100 slightly inhibited the activity. The enzyme showed resistance to some denaturants: such as SDS, EDTA, and urea (52, 65, and 42%, respectively). N8 α-amylase displayed the maximum remaining activity of 56% with 3% NaCl. The major final products of starch were glucose, maltose, and maltose-derived oligosaccharides. This novel cold-active α-amylase has the potential to be used in the industries of detergent and food, bioremediation process and production of prebiotics.     

Untersuchungen zur Bildung extrazellulärer Hemicellulasen durch Pseudocercosporella herpotrichoides in vitro

Investigations on the production of extracellular hemicellulases by Pseudocercosporella herpotrichoides in vitro For all 15 isolates of Pseudocercosporella herpotrichoides investigated, xylanase as well as arabanase activity could be demonstrated. After cultivation of 3 weeks, the activity of the enzymes reached a peak. The activity of xylanase was considerably increased by addition of xylan in comparison to Maltzin as the sole source of carbohydrate. Also the arabanase activity could be increased significantly by addition of araban or xylan as compared to the Maltzin variant. The optimum temperature with regard to activity and stability of xylanase ranged at 50°C. The pH-optimum for xylanase activity was found to be at pH 5.0, and the enzyme was stable in ° range between pH4.0 and 8.0 (9.0). In case of arabanase, the temperature optimum varied between 40 and 50°C; up to this temperature, the enzyme was also stable. At pH 5.0, the arabanase activity reached its optimum; stability was observed in - pH range between 4.0 and 9.0. In extracts prepared from autoclaved wheat coleoptiles which were inoculated with Pseudocercosporella herpotrichoides, the presence of the enzymes xylanase, arabanase, cellulase and polymethylgalacturonase could be demonstrated. The enzyme activities of the inoculated samples were considerably higher than those of non-inoculated controls. The differences, in most cases, were statistically significant. Der Deutschen Forchungsgemeinschaft danken wir für finanzielle Unterstützung.     

Trehalase,invertase, and amylase activities in the black scale,Saissetia oleae,and their relation to host adaptability

In young females of the black scale, Saissetia oleae, the optimum conditions for invertase activity involve a reaction mixture of pH 5.5 and 2% sucrose at 37°C for 60 min; for amylase, pH 6.0 and 0.5% starch at 37°C for 45 min; and for trehalase, pH 5.5 and 1.5% trehalose at 37°C for 60 min. At optimal conditions and using standard enzyme activity units, both invertase and trehalase activities were much higher (about 8-fold) than that of amylase, indicating the importance of these enzymes in food digestion and energy supply.The enzyme activities were strongly affected by various host plants. Trehalase activity in scales reared on potato sprouts was about 3.5- and 4-fold that obtained in scales reared on oleander and citrus plants, respectively. An increase of about 40% for invertase and 60% amylase activity was obtained in scales reared on potato sprouts as compared with those reared on oleander or citrus plants.A good correlation was observed between enzyme activity-especially of trehalase-and scale development. The duration of one generation of the black scale reared on potato sprouts was 2.5 to 3 months, on oleander 4 to 5 months, and on citrus above 6 months. These results suggest that trehalase and to some extent invertase could be used as parameters to assess the adaptability of the black scale to its host plant.     

Starch-degrading enzymes of two species ofFusarium

Starch supported production of maximum α-amylases (dextrinizing and saccharifying) byFusarium oxysporum andF. scirpi. Addition of gibberellic acid resulted in an increased production of α-amylase. Presence of glucose depressed the enzyme production. pH 4.5 and 4.0 was found to be optimum for the dextrinizing enzyme secreted by both species. The temperature of 25 and 40 °C was optimum for the dextrinizing enzyme secreted byF. oxysporum andF. scirpi, respectively. Saccharifying enzymes of both species showed their optimum at pH 6.9. The optimum temperature for the activity of the saccharifying enzyme was 30 and 40 °C, respectively.     

Purification and characterization of an alkaline xylanase from alkaliphilic Micrococcus sp AR-135

An xylanase producing alkaliphilic Micrococcus sp was isolated from an alkaline soda lake. Xylose and xylan induced enzyme production but no activity was detected when it was grown using other carbohydrate sources. The level of xylanase production was higher in the presence of xylose than in the presence of xylan. The enzyme was purified to homogeneity and its molecular weight was estimated to be 56 kD on SDS-PAGE. The optimum temperature and pH for xylanase activity were 55°C and 7.5–9.0, respectively. Sixty per cent of the maximum activity was displayed at pH 11. The enzyme was very stable in the pH range of 6.5–10 and up to a temperature of 40°C. Xylanase activity was inhibited by Cu²⁺ and Hg²⁺. Received 03 October 1997/ Accepted in revised form 03 February 1998     

A new thermolabile alkaline phospholipase D from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS628

A phospholipase D (PLD₆₂₈), constitutively secreted by Streptomyces sp. CS628, was purified by ion exchange with CM Trisacryl and gel filtration with Sepharose CL-6B. The enzyme production was highest with peptone and starch as nitrogen and carbon sources, and at 30°C with an initial medium pH of 7.5. Molecular weight, optimum pH, optimum temperature, pH stability, and thermostability of the enzyme were 50 kDa, pH 9.6, 30°C, pH 5.7 ∼ 10.6 and ≤30°C, respectively. Detergents and metal ions had varied effects on the enzyme activity. Importantly, PLD₆₂₈ could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol or ethanolamine, which are extensively used to assess the activity, suggesting that PLD₆₂₈ lacks the transphosphatidylation activity. PLD₆₂₈ could be a novel PLD based on its biochemical characteristics, which are significantly different from previously reported PLDs, such as thermolability, highest activity at alkaline pH, and lack of transphosphatidylation activity.