Production and partial characterization of high molecular weight extracellular alpha-amylase from Thermoactinomyces vulgaris isolated from Egyptian soil

Optimizing production of alpha-amylase production by Thermoactinomyces vulgaris isolated from Egyptian soil was studied. The optimum incubation period, temperature and initial pH of medium for organism growth and enzyme yield were around 24 h, 55 degrees C and 7.0, respectively. Maximum alpha-amylase activity was observed in a medium containing starch as carbon source. The other tested carbohydrates (cellulose, glucose, galactose, xylose, arabinose, lactose and maltose) inhibited the enzyme production. Adding tryptone as a nitrogen source exhibited a maximum activity of alpha-amylase. Bactopeptone and yeast extract gave also high activity comparing to the other nitrogen sources (NH4CI, NH4NO3, NaNO3, KNO3, CH3CO2NH4). Electrophoresis profile of the produced two alpha-amylase isozymes indicated that the same pattern at about 135-145 kDa under different conditions. The optimum pH and temperature of the enzyme activity were 8.0 and 60 degrees C, respectively and enzyme was stable at 50 degrees C over 6 hours. The enzyme was significantly inhibited by the addition of metal ions (Na+, Co2+ and Ca2+) whereas CI- seemed to act as activator. The enzyme was not affected by 0.1 mM EDTA while higher concentration (10 mM EDTA) totally inactivated the enzyme.     

Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3

A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.     

Purification and physico-chemical properties of collagenase synthesized by a bacterium of the type Acinetobacter sp.]

The Acinetobacter spec collagenase has been almost completely purified. This enzyme is a true collagenase the activity of which is high on collagen. The enzyme is active on insoluble collagen, gelatin and the synthetic Pz-peptide, but has no proteolytic activity on casein or bovine serum-albumin. The collagenase was obtained on a simple medium with gelatin and yeast extract. The enzyme was purified by (NH4)2SO4 precipitation. DEAE cellulose column chromatography, Sephadex G 200 gel-filtration. The molecular weight of the enzyme was found to be 102 000 daltons, and its isoelectric point was found to be 7,7 +/- 0,2. The optimum pH and temperature for insoluble collagen hydrolysis were 7.6 and 37 degrees C, respectively; so, this collagenase corresponds to true collagenase. Hydrolysis of Pz-peptide is activated by Ca2+ and inhibited by metal ions (Cu2+, Fe3+, Zn2+, Pb2+, Hg2+). EDTA and o-phenanthroline induced a very significant reduction in enzyme activity. Iodoacetate and p-CMB induced a slight reduction in enzyme activity only at high concentrations (10-2M). The collagenase is most stable for temperatures less than or equal to 50 degrees C.     

Purification and characterization of an alpha-glucosidase from Rhizobium sp. (Robinia pseudoacacia L.) strain USDA 4280.

A novel alpha-glucosidase with an apparent subunit mass of 59 +/- 0. 5 kDa was purified from protein extracts of Rhizobium sp. strain USDA 4280, a nodulating strain of black locust (Robinia pseudoacacia L), and characterized. After purification to homogeneity (475-fold; yield, 18%) by ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic chromatography, dye chromatography, and gel filtration, this enzyme had a pI of 4.75 +/- 0.05. The enzyme activity was optimal at pH 6.0 to 6.5 and 35 degrees C. The activity increased in the presence of NH4+ and K+ ions but was inhibited by Cu2+, Ag+, Hg+, and Fe2+ ions and by various phenyl, phenol, and flavonoid derivatives. Native enzyme activity was revealed by native gel electrophoresis and isoelectrofocusing-polyacrylamide gel electrophoresis with fluorescence detection in which 4-methylumbelliferyl alpha-glucoside was the fluorogenic substrate. The enzyme was more active with alpha-glucosides substituted with aromatic aglycones than with oligosaccharides. This alpha-glucosidase exhibited Michaelis-Menten kinetics with 4-methylumbelliferyl alpha-D-glucopyranoside (Km, 0.141 microM; Vmax, 6.79 micromol min-1 mg-1) and with p-nitrophenyl alpha-D-glucopyranoside (Km, 0.037 microM; Vmax, 2.92 micromol min-1 mg-1). Maltose, trehalose, and sucrose were also hydrolyzed by this enzyme.     

Purification and characterization of an alpha-glucosidase from a hyperthermophilic archaebacterium, Pyrococcus furiosus, exhibiting a temperature optimum of 105 to 115 degrees C.

Pyrococcus furiosus is a strictly anaerobic hyperthermophilic archaebacterium with an optimal growth temperature of about 100 degrees C. When this organism was grown in the presence of certain complex carbohydrates, the production of several amylolytic enzymes was noted. These enzymes included an alpha-glucosidase that was located in the cell cytoplasm. This alpha-glucosidase has been purified 310-fold and corresponded to a protein band of 125 kilodaltons as resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited optimum activity at pH 5.0 to 6.0 and over a temperature range of 105 to 115 degrees C. Kinetic analysis conducted at 108 degrees C revealed hydrolysis of the substrates p-nitrophenyl-alpha-D-glucopyranoside (PNPG), methyl-alpha-D-glucopyranoside, maltose, and isomaltose. Trace activity was detected towards p-nitrophenyl-beta-D-glucopyranoside, and no activity could be detected towards starch or sucrose. Inhibition studies conducted at 108 degrees C with PNPG as the substrate and maltose as the inhibitor yielded a Ki for maltose of 14.3 mM. Preincubation for 30 min at 98 degrees C in 100 mM dithiothreitol and 1.0 M urea had little effect on enzyme activity, whereas preincubation in 1.0% sodium dodecyl sulfate and 1.0 M guanidine hydrochloride resulted in significant loss of enzyme activity. Purified alpha-glucosidase from P. furiosus exhibited remarkable thermostability; incubation of the enzyme at 98 degrees C resulted in a half life of nearly 48 h.     

Extracellular Maltase of Bacillus brevis

Bacillus brevis NRRL B-4389 produced extracellular maltase (alpha-glucosidase; EC 3.2.1.20) only in the presence of short alpha-1,4-glucosidic polymers, such as maltose and maltotriose. An optimum medium was developed; it contained 2.5% maltose, 0.5% nonfat dry milk, 0.4% yeast extract, and 0.01% CaCl(2). The enzyme was produced extracellularly during the logarithmic phase of growth; no cell-bound activity was detected at any time. Partial purification of the maltase was accomplished by using diethylaminoethyl cellulose batch adsorption, ammonium sulfate precipitation, and Sephadex G-200 gel filtration. Maltase, isomaltase (oligo-1,6-glucosidase), and glucosyltransferase activities were purified 20.0-, 19.1-, and 11.5-fold, respectively. Some properties of the partially purified maltase were determined: optimum pH, 6.5; optimum temperature, 48 to 50 degrees C; pH stability range, 5.0 to 7.0; temperature stability range, 0 to 50 degrees C; isoelectric point, pH 5.2; and molecular weight, 52,000. The relative rates of hydrolysis of maltose (G(2)), maltotriose (G(3)), G(4), methyl-alpha-d-maltoside, G(40), dextrin, and isomaltose were 100, 22, 12, 10, 10, 8, and 5%, respectively; the K(m) on maltose was 5.8 mM; d-glucose, p-nitrophenyl-alpha-d-glucoside, and tris (hydroxymethyl) aminomethane were competitive inhibitors; transglucosylase activity of the enzyme on maltose resulted in the synthesis of isomaltose, isomaltotroise, and larger oligosaccharides.     

Purification and characterization of trehalose-6-phosphate synthase from Saccharomycopsis fibuligera A11

Mutant A11, a mutant of Saccharomycopsis fibuligera Sdu with low acid and neutral trehalase was found to accumulate over 18% (w/w) trehalose from starch in its cells. In this study, trehalose-6-phosphate synthase (Tps1) was purified to homogeneity from this mutant, with a 30-fold increase in the specific enzyme activity, as compared to the concentrated cell-free extract, from initial cells. The molecular mass of the purified enzyme as determined by SDS-PAGE was 66 kDa. The optimum pH and temperature of the purified enzyme were 6.6 and 37 degrees C, respectively. The enzyme was activated by Ca2+, K+ and Mg2+, with K+ showing the highest activation at 35 mM. On the other hand, Mn2+, Cu2+, Fe3+, Hg2+ and Co2+ inhibited the enzyme. The enzyme was also strongly inhibited by protease inhibitors such as iodoacetic acid, EDTA and PMSF.     

Production of an extracellular maltase by thermophilic Bacillus sp. KP 1035.

Production of extracellular maltase was studied with thermophilic Bacillus sp. KP 1035, which was selected as the organism producing the highest levels of maltase. The final enzyme yield was increased by maltose, peptone, and yeast extract but reduced by succinate and fumarate. Maximum enzyme production was achieved at 55 degrees C and at an initial pH of 6.2 to 7.0 on a medium containing 0.3% maltose, 1% peptone, 0.1% meat extract, 0.3% yeast extract, 0.3% KH2PO4, and 0.1% KH2PO4. Maltase was synthesized in cytoplasm and accumulated as a large pool during the logarithmic growth phase, which preceded sporulation. At the end of this phase, the enzyme appeared in the culture broth, and its accumulation increased in parallel with a rise in the extracellular protein level. Maltase was stable for 24 h at 60 degrees C over a pH range of 5.6 to 9.0 and retained 95% of the original activity after treatment for 20 min at 70 degrees C at pH 6.8.     

Optimization of culture conditions and properties of immobilized sulfide oxidase from Arthrobacter species

Arthrobacter species strain FR-3, isolated from sediments of a swamp, produced a novel serine-type sulfide oxidase. The production of sulfide oxidase was maximal at pH 7.5 and 30 degrees C. Among various carbon and nitrogen sources tested, glucose and yeast extract were found to be the most effective substrates for the secretion of sulfide oxidase. The sulfide oxidase was purified to homogeneity and the molecular weight of the purified enzyme was 43 kDa when estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified sulfide oxidase can be effectively immobilized in DEAE (diethylaminoethyl)-cellulose matrix with a yield of 66%. The purified free and immobilized enzyme had optimum activity at pH 7.5 and 6.0, respectively. Immobilization increases the stability of the enzyme with respect to temperature. The half-life of the immobilized enzyme was 30 min at 45 degrees C, longer than that of the free enzyme (10 min). The purified free sulfide oxidase activity was completely inhibited by 1 mM Co2+ and Zn2+ and sulfhydryl group reagents (para-chloromercuribenzoic acid and iodoacetic acid). Catalytic activity was not affected by 1 mM Ca2+, Mg2+, Na+ and metal-chelating agent (EDTA).     

里氏木霉GXC木聚糖酶的研究

研究了里氏木霉GXC产木聚糖酶的条件和酶学性质。结果表明,适宜产酶碳源为乳糖、甘露糖、棉子糖、木聚糖和麸皮,氮源为牛肉膏和酵母膏;产酶的最适初始pH为4.0,30℃培养60h。对以麸皮为碳源的培养液进行纯化的酶特性研究表明,木聚糖酶的最适反应温度为50℃,pH为5.5,该酶在pH5.0(7.0和40℃以下相对稳定。Fe3+和Mn2+对木聚糖酶有较大的促进作用,Cu～2+、Fe～2+和Ca～2+ 具有抑制作用。     

Partial purification and characterization of a soluble protein kinase from Leishmania donovani promastigotes

A soluble protein kinase from the promastigote form of the parasitic protozoon Leishmania donovani was partially purified using DEAE-cellulose, Sephadex G-200 and phosphocellulose columns. The enzyme preferentially utilized protamine as exogenous phosphate acceptor. The native molecular mass of the enzyme was about 85 kDa. Mg2+ ions were essential for enzyme activity; other metal ions, e.g. Ca2+, Co2+, Zn2+ and Mn2+, could not substitute for Mg2+. cAMP, cGMP, Ca2+/calmodulin and Ca2+/phospholipid did not stimulate enzyme activity. The pH optimum of the enzyme was 7.0-7.5, and the temperature optimum 37 degrees C. The apparent Km for ATP was 60 microM. Phosphoamino acid analysis revealed that the protein kinase transferred the gamma-phosphate of ATP to serine residues in protamine. The thiol reagents p-hydroxymercuribenzoic acid, 5-5'-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide inhibited enzyme activity; the inhibition by p-hydroxymercuribenzoic acid and 5-5'-dithio-bis(2-nitrobenzoic acid) was reversed by dithiothreitol.     

Isolation and properties of a phosphatidylcholine-specific phospholipase C from bull seminal plasma

A phospholipase C which hydrolyzes [14C]phosphatidylcholine has been purified 1782-fold from 70% ammonium sulfate extract of bull seminal plasma. Purification steps included acid precipitation, chromatography on DEAE-Sephacel, concanavalin A, octyl-Sepharose 4B and Ultrogel AcA 34. The final step provided homogeneous phospholipase C as determined by polyacrylamide gel electrophoresis. The enzyme comprised two subunits, Mr 69,000 and Mr 55,000, respectively. The enzyme had an optimum at pH 7.2 and pI 5.0. EDTA, Cd2+, Pb2+, Ni2+, Fe2+, and Zn2+ inhibited phospholipase C activity. Km and Vmax on p-nitrophenyl phosphorylcholine and phosphatidylcholine substrates were 20 mM and 17 mumol/min/mg of the purified enzyme and 100 microM and 18 mumol/min/mg of the purified enzyme, respectively. The enzyme appeared to be localized in the acrosome as judged by the binding of anti-phospholipase C to the acrosome. This phospholipase C, unlike other known phospholipases (C), did not hydrolyze [1-14C]phosphatidylinositol. The testicular extract of the guinea pig contained inactive phospholipase C which was activated on incubation with acrosin and trypsin but not chymotrypsin.     

里氏木霉GXC木聚糖酶的研究*

研究了里氏木霉GXC产木聚糖酶的条件和酶学性质。结果表明,适宜产酶碳源为乳糖、甘露糖、棉子糖、木聚糖和麸皮,氮源为牛肉膏和酵母膏;产酶的最适初始pH为4.0,30℃培养60h。对以麸皮为碳源的培养液进行纯化的酶特性研究表明,木聚糖酶的最适反应温度为50℃,pH为5.5,该酶在pH5.0(7.0和40℃以下相对稳定。Fe3+和Mn2+对木聚糖酶有较大的促进作用,Cu～2+、Fe～2+和Ca～2+ 具有抑制作用。     

A specific short dextrin-hydrolyzing extracellular glucosidase from the thermophilic fungus Thermoascus aurantiacus 179-5

The thermophilic fungus Thermoascus aurantiacus 179-5 produced large quantities of a glucosidase which preferentially hydrolyzed maltose over starch. Enzyme production was high in submerged fermentation, with a maximal activity of 30 U/ml after 336 h of fermentation. In solid-state fermentation, the activity of the enzyme was 22 U/ml at 144 h in medium containing wheat bran and 5.8 U/ml at 48 h when cassava pulp was used as the culture medium. The enzyme was specific for maltose, very slowly hydrolyzed starch, dextrins (2-7G) and the synthetic substrate (alpha-PNPG), and did not hydrolyze sucrose. These properties suggest that the enzyme is a type II alpha-glucosidase. The optimum temperature of the enzyme was 70 degrees . In addition, the enzyme was highly thermostable (100% stability for 10 h at 60 degrees and a half-life of 15 min at 80 degrees), and stable within a wide pH range.     

Isolation and purification of an extracellular protease from a new strain of Bacillus subtilis, viz.NCIM 2711

A new extracellular protease having a prospective application in the food industry was isolated from Bacillus sUbtilis NCIM 2711 by (NH4)2SO4 precipitation from the cell broth. It was purified using DEAE-Cellulose and CM-Sephadex C-50 ion-exchange chromatography. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 7.0 and temperature 55 degrees C with Km 1.06 mg/ml. The enzyme was stable over a pH range 6.5-8.0 at 30 degrees C for 1 hr in presence of CaCl2 x 2H2O. At 55 degrees C, the enzyme retained 60% activity up to 15 min in presence of CaCl2 x 2H2O. EDTA and o-phenanthroline (OP) completely inhibited the enzyme activity while DFP, PMSF and iodoacetamide were ineffective. The enzyme was completely inhibited by Hg2+ and partially by Cd2+, Cu2+, Ni2+, Pb2+ and Fe2+. The OP inhibited enzyme could be reactivated by Zn2+ and Co2+ up to 75% and 69% respectively. It is a neutral metalloprotease showing a single band of 43 kDa on SDS-PAGE.     

Purification and characterization of a maltase from the extremely thermophilic crenarchaeote Sulfolobus solfataricus.

A soluble maltase (alpha-glucosidase) with an apparent subunit mass of 80 kDa was purified to homogeneity from Sulfolobus solfataricus. The enzyme liberates glucose from maltose and malto-oligomers. Maximal activity was observed at 105 degrees C, with half-lives of 11 h (85 degrees C), 3.0 h (95 degrees C), and 2.75 h (100 degrees C). The enzyme was generally resistant to proteolysis and denaturants including aliphatic alcohols. n-Propanol treatment at 85 degrees C increased both Km and Vmax for maltose hydrolysis.     

Purification and characterization of cyclic maltosyl-(1-->6)-maltose hydrolase and alpha-glucosidase from an Arthrobacter globiformis strain

Cyclic maltosyl-maltose [CMM, cyclo-[-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate alpha-1,4 and alpha-1,6 linkages. CMM is synthesized from starch by the action of 6-alpha-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and alpha-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 degrees C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 degrees C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the alpha-1,6-linkage of CMM. The molecular mass of alpha-glucosidase was determined to be 60.1 kDa by SDS-PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for alpha-glucosidase activity were 7.0 and 35 degrees C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 degrees C. alpha-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and alpha-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and alpha-glucosidase.     

Kinetics of alpha-amylase synthesis from Bacillus amyloliquefaciens

The microbial production of alpha-amylase from Bacillus amyloliquefaciens was investigated. The microorganism was grown using media containing glucose or maltose at 37 degrees C and under aerobic conditions in a 16-L fermentor. The alpha-amylase synthesis from maltose was not found to be inducible but was found to be subject to catabolite repression. The maltose uptake rate was observed to be the rate-limiting step compared to the conversion rate of maltose to glucose by intracellular alpha-glucosidase. The alpha-amylase activity achieved with maltose as a substrate was higher than that achieved with glucose. A slower growth rate and a higher cell density were obtained with maltose. The enzyme production pattern depended upon the nutrient composition of the medium.     

Latency of some glycosidases of rat liver lysosomes.

The latency of the alpha-glucosidase activity of intact rat liver lysosomes was studied by using four substrates (glycogen, maltose, p-nitrophenyl, alpha-glucoside, alpha-fluoroglucoside) at a range of substrate concentrations. The results indicate that the entire lysosome population is impermeable to glycogen and maltose, but a proportion of lysosomes are permeable to alpha-fluoroglucoside and a still higher proportion permeable to p-nitrophenyl alpha-glucoside. Incubation at 37 degrees C in an osmotically protected buffer of of pH 5.0 caused lysosomes to become permeable to previously impermeant substrates and ultimately to release their alpha-glucosidase into the medium. The latencies of lysosomal beta-glucosidase and beta-galactosidase were examined by using p-nitrophenyl beta-glucoside and beta-galactoside as substrates. The results indicate permeability properties to these substrates similar to that to p-nitrophenyl alpha-glucoside. On incubation in an osmotically protected buffer of pH 5, lysosomes progressively released their beta-galactosidase in soluble form, but beta-glucosidase remained attached to sedimentable material. Lysosomal beta-glucosidase was inhibited by 0.1% Triton X-100; alpha-glucosidase and beta-galactosidase were not inhibited.