alpha-Galactosidase from Bacillus megaterium VHM1 and its application in removal of flatulence-causing factors from soymilk

A bacterial strain capable of producing extracellular alpha-galactosidase was isolated from sugar cane industrial waste soil sample. Microbiological, physiological, and biochemical studies revealed that isolate belonged to Bacillus sp,. Furthermore, 16S rDNA sequence analysis of new isolates was identified as Bacillus megaterium VHM1. The production of alpha-galactosidase was optimized by various physical culture conditions. Guar gum and yeast extract acted as the best carbon and nitrogen source, respectively for the production of alpha-galactosidase. The enzyme showed an optimum pH at 7.5 and was stable over a pH between 5 and 9. The enzyme was optimally active in 55degreesC and the enzyme was thermostable with half life of 120 minutes at 55 degrees C and lost their 90%, residual activity in 120 minutes at 60 degrees C. alpha-Galactosidase was strongly inhibited by Ag2, Cu2, and Hg2+ at 1mM concentration. The metal ions Fe2, Mn2+, and Mg2+ had no effect on alpha-galactosidase activity, Zn2+,Ni2+, and Ca2+ reduced the enzyme activity slightly. The B megaterium VHM1 enzyme treatment completely hydrolyzed flatulence-causing sugars of soymilk within one and half hour.     

Characterization and biotechnological application of an acid alpha-galactosidase from Tachigali multijuga Benth. seeds

Tachigali multijuga Benth. seeds were found to contain protein (364 mg g(-1)dwt), lipids (24 mg g(-1)dwt), ash (35 mg g(-1)dwt), and carbohydrates (577 mg g(-1)dwt). Sucrose, raffinose, and stachyose concentrations were 8.3, 3.0, and 11.6 mg g(-1)dwt, respectively. alpha-Galactosidase activity increased during seed germination and reached a maximum level at 108 h after seed imbibition. The alpha-galactosidase purified from germinating seeds had an M(r) of 38,000 and maximal activity at pH 5.0-5.5 and 50 degrees C. The enzyme was stable at 35 degrees C and 40 degrees C, but lost 79% of its activity after 30 min at 50 degrees C. The activation energy (E(a)) values for p-nitrophenyl-alpha-d-galactopyranoside (pNPGal) and raffinose were 13.86 and 4.75 kcal mol(-1), respectively. The K(m) values for pNPGal, melibiose, raffinose, and stachyose were 0.45, 5.37, 39.62 and 48.80 mM, respectively. The enzyme was sensitive to inhibition by HgCl(2), SDS, AgNO(3), CuSO(4), and melibiose. d-Galactose was a competitive inhibitor (K(i)=2.74 mM). In addition to its ability to hydrolyze raffinose and stachyose, the enzyme also hydrolyzed galactomannan.     

Conditions of formation, purification, and characterization of an alpha-galactosidase of Trichoderma reesei RUT C-30.

Trichoderma reesei RUT C-30 formed an extracellular alpha-galactosidase when it was grown in a batch culture containing lactose or locust bean gum as a carbon source. Short-chain alpha-galactosides (melibiose, raffinose, stachyose), as well as the monosaccharides galactose, dulcitol, arabinose, and arabitol, also induced alpha-galactosidase activity both when they were used as carbon sources (at a concentration of 1%) in batch cultures and in resting mycelia (at concentrations in the millimolar range). The addition of 50 mM glucose did not affect the induction of alpha-galactosidase formation by galactose. alpha-Galactosidase from T. reesei RUT C-30 was purified to homogeneity from culture fluids of galactose-induced mycelia. The active enzyme was a 50 +/- 3-kDa, nonglycosylated monomer which had an isoelectric point of 5.2. It was active against several alpha-galactosides (p-nitrophenyl-alpha-D-galactoside, melibiose, raffinose, and stachyose) and galactomannan (locust bean gum) and was inhibited by the product galactose. It released galactose from locust bean gum and exhibited synergism with T. reesei beta-mannanase. Its activity was optimal at pH 4, and it displayed broad pH stability (pH 4 to 8). Its temperature stability was moderate (60 min at 50 degrees C resulted in recovery of 70% of activity), and its highest level of activity occurred at 60 degrees C. Its action on galactomannan was increased by the presence of beta-mannanase.     

Purification and properties of alpha-galactosidase from Klebsiella Sp. No. PG-2

alpha-Galactosidase has been purified from Klebsiella Sp. No. PG-2, a bacterium isolated from rat small intestine, using calcium phosphate gel, DEAE-cellulose column chromatography and gel filtration technique. About 130-fold increase in specific activity was observed, the pH optimum of 6.5-7.0 characterizes the enzyme as neutral alpha-galactosidase. The optimum temperature was 37 degrees C and the energy of activation was 11,856 cal/mole. Km values obtained for raffinose, mellibose, stachyose and p-nitrophenyl-alpha-D-galactopyranoside were 20.0, 6.6 33.3 and 4.0 mM respectively. The activity was inhibited by p-CMB; iodoacetate, Ag2+, Hg2+, Cu2+, Pb2+ and galactose. Examination of the enzyme activity indicated that the enzyme is cytosolic and is inducible in nature.     

Production, Purification, and Characterization of alpha-Galactosidase from Monascus pilosus

A Monascus pilosus strain was selected for production of intracellular alpha-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Galactose was one of the best enzyme inducers. The enzyme was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography and was demonstrated to be homogeneous by slab gel electrophoresis. The molecular weight of this enzyme, estimated by gel filtration, was about 150,000. The optimum conditions for the enzyme reaction was pH 4.5 to 5.0 at 55 degrees C. The purified enzyme was stable at 55 degrees C or below and in buffer at pH 3 to 8. The activity was inhibited by mercury, silver, and copper ions. The kinetics of this enzyme, with p-nitrophenyl-alpha-d-galactoside as substrate, was determined: K(m) was about 0.8 mM, and V(max) was 39 mumol/min per mg of protein. Enzymatic hydrolysis of melibiose, raffinose, and stachyose was analyzed by thin-layer chromatography.     

一种来源于Leifsonia shinshuensis DICP 16菌体的b-D-木糖苷酶的分离纯化及其性质

采用盐析、DE 52、Q-Sepharose Fast Flow阴离子交换层析、Toyopearl Butyl 650C疏水层析以及Sephacryl S-300 HR凝胶过滤层析联用的方法, 从Leifsonia shinshuensis DICP 16菌体中纯化出一种b-木糖苷酶。分离后该酶在SDS-PAGE 上呈单一蛋白质条带, 通过SDS-PAGE和凝胶过滤层析法, 测得该酶是一个由两个分子量约为91 kD的相同亚基组成的同源二聚体。其水解对硝基苯酚木糖苷(pNPX)的最适反应温度为55°C, pH值为7.0。该木糖苷酶在45°C以下, pH 6.0~11.0之间具有很好的稳定性。在45°C, pH值为7.0的条件下, 水解pNPX的Km, Vmax分别为1.04 mmol/L, 0.095 mmol/(min·mg)。研究不同的金属离子对该酶的活性影响, 发现Fe2+和Cu2+是很强的抑制剂。通过对天然木糖苷化合物的水解测试, 发现该酶可以水解人参皂苷Rb3的木糖基, 产生人参皂苷Rd, 却不能水解紫杉烷木糖苷的木糖基。     

Properties and application of a partially purified alkaline xylanase from an alkalophilic fungus Aspergillus nidulans KK-99

An alkalophilic Aspergillus nidulans KK-99 produced an alkaline, thermostable xylanase (40 IU/ml) in a basal medium supplemented with wheat bran (2% w/v) and KNO3 (at 0.15% N) pH 10.0 and 37 degrees C. The partially purified xylanase was optimally active at pH 8.0 and 55 degrees C. The xylanase was stable in a broad pH range of 4.0-9.5 for 1 h at 55 degrees C, retaining more than 80% of its activity. The enzyme exhibited greater binding affinity for xylan from hardwood than from softwood. The xylanase activity was stimulated (+25%) by Na+ and Fe2+ and was strongly inhibited (maximum by 70%) by Tween-20, 40, 60, SDS, acetic anhydride, phenylmethane sulphonyl fluoride, Triton-X-100. The xylanase dose of 1.0 IU/g dry weight pulp gave optimum bleach boosting of Kraft pulp at pH 8.0 and temperature 55 degrees C for 3 h reaction time.     

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.     

毛壳霉内切菊粉酶的纯化与性质

毛壳霉 (Chaetomiumsp .)C34发酵液经硫酸铵分级沉淀、DEAE 纤维素 11离子交换层析、Q SepharoseFastFlow离子交换层析、SephacrylS 2 0 0凝胶过滤、PhenolSepharoseTM HP疏水层析 ,得到电泳纯的内切菊粉酶组分 ,纯化倍数为 30 8倍 ,活力回收率为 7 7%。用SDS PAGE测得该酶亚基的分子量为 6 6kD。菊粉酶的最适pH为 6 0 ,最适温度为 5 0～ 5 5℃。菊粉酶在 5 0℃以下 ,pH5 0～ 8 0时较稳定。Cu2 完全抑制酶的活性 ,Mn2 、Zn2 、Fe2 、EDTA以及NBS(N bromosuccinimide ,N 溴代丁二酰亚胺 )对该酶有很强的抑制作用。该酶对菊粉有较强底物专一性 ,产物主要为低聚果糖 ,也可作用于蔗糖 ,I S值为 2 0。以菊粉为底物时 ,Km 为 0 199mmol L ,Vmax为 115 μmol (mg·min)。     

极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37℃下在pH8～10的缓冲液中保温1h酶活几乎不改变。重组酶反应的最适温度为75℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的KmKG为7.559mmol/L,VmaxKG为0.086mmol/(L·min),KmAsp为2.031mmol/L,VmaxAsp为0·024mmol/(L·min)。Ca2 、Fe3 、Mn2 等金属离子对酶活性有微弱抑制作用。     

Extracellular alpha galactosidase (E.C. 3.2.1.22) from Aspergillus ficuum NRRL 3135 purification and characterization

Extracellular alpha-galactosidase, a glycoprotein from the extracellular culture fluid of Aspergillus ficuum grown on glucose and raffinose in a batch culture system, was purified to homogeneity in five steps by ion exchange and hydrophobic interaction chromatography. The molecular mass of the enzyme was 70.8 Kd by SDS polyacrylamide gel electrophoresis and 74.1 Kd by gel permeation HPLC. On the basis of a molecular mass of 70.7 Kd, the molar extinction coefficient of the enzyme at 279 nm was estimated to be 6.1 X10(4) M-1 cm-1. The purified enzyme was remarkably stable at 0 degrees C. It had a broad temperature optimum and maximum catalytic activity was at 60 degrees C. It retained 33% of its activity after 10 min. at 65 degrees C. It had a pH optimum of 6.0. It retained 62% of its activity after 12 hours at pH 2.3. The Kms for p-nitrophenyl-alpha-D-galactopyranoside, o-nitrophenyl-alpha-D-galactopyranoside and m-nitrophenyl-alpha-D-galactopyranoside are: 1462, 839 and 718 microM. The enzyme was competitively inhibited by mercury (19.8 microM), silver (21.5 microM), copper (0.48 mM), zinc (0.11 mM), galactose (64.0 mM) and fructose (60.3 mM). It was inhibited non-competitively by glucose (83.2 mM) and uncompetitively by mannose (6.7 mM).     

Purification and characterization of alkaline phosphatase from plasma membranes of rat ascites hepatoma.

Alkaline phosphatase was purified from plasma membranes of rat ascites hepatoma AH-130, the homogenate of which had 50-fold higher specific activity than that found in the liver homogenate. The presence of Triton X-100, 0.5%, was essential to avoid its aggregation and to stabilize its activity. The purified enzyme, a glycoprotien, was homogeneous in polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a protein molecular weight of 140,000. The addition of beta-mercaptoethanol caused the dissociation of the alkaline phosphatase into two subunits of identical molecular weight, 72,000. Isoelectric focusing revealed that the pI of this enzyme is 4.7. The pH optimum for the purified enzyme was 10.5 or higher with p-nitrophenylphosphate, and slightly lower pH values (pH 9.5--10.2) were obtained when other substrates were used. Of the substrates tested, p-nitrophenylphosphate (Km-0.3 mM) was most rapidly hydrolyzed. Vmax values of other substrates relative to that of p-nitrophenylphosphate were as follows; beta-glycerophosphate, 76%; 5'-TMP, 82%; 5'-AMP, 62%; 5'-IMP, 43%; glucose-6-phosphate, 39%; ADP, 36% and ATP, 15%. More than 90% of the activity of the purified enzyme was irreversibly lost when it was heated at 55 degrees C for 30 min, or exposed either to 10 mM beta-mercaptoethanol for 10 min to 3 M urea for 30 min, or to an acidic pH below pH 5.0 for 2 h. Of the effects by divalent cations, Mg2+ activated the enzyme by 20% whereas Zn2+ strongly inhibited it by 95% at 0.5 mM. EDTA at higher than 1 mM inactivated the enzyme irreversibly, although the effect of EDTA at lower than 0.1 mM was reversible by the addition of divalent cations, particularly by Mg2+. The enzyme was most strongly inhibited by L-histidine among the amino acids tested, and also strongly inhibited by imidazole. These results suggest that alkaline phosphatase of rat hepatoma AH-130 is very similar to that of rat liver in most of the properties reported so far.     

Purification and some important characters of extracellular inulinase of Alternaria alternata (Fr.) Keissler

Protein precipitate of cell-free dialysate of extracellular inulinase (2,1-beta-fructan fructanohydrolase, EC 3.2.1.7) of A. alternata was maximally obtained by methanol. Such protein was fractionated by using 2-step column chromatography on Sephadex G150 and DEAE-cellulose. The partially purified enzyme had activity of 81 x 10(3) U/mg protein, with a yield of 69% of the original activity and the fold of purification was 62. Optimum temperature and pH for the activity of the purified enzyme were found to be 55 degrees C and 4.5, respectively. The enzyme was found to be stable up to 55 degrees C and in pH range of 4 to 5. Ba2+ and Ca2+ were found to stimulate the enzyme activity while Cu2+, Fe3+, Hg2+, and iodoacetate were recorded as strong inhibitors. T(1/2) of the enzyme was estimated to be two weeks and its apparent Km was calculated to be 0.066 M. The enzyme recorded hydrolyzing activity against sucrose and raffinose recording I/S ratio of 0.50. Molecular mass of the enzyme preparation was estimated by gel filtration and found to be 115 +/- 5 kDa.     

Purification and characterization of a thermostable-cellulase free xylanase from Syncephalastrum racemosum Cohn

Syncephalastrum racemosum Cohn. produces an extracellular xylanase that was shown to potentially bleach pulp at pH 10 and 50 degrees C. The enzyme was found to be a dimer with an apparent molecular weight of 29 kDa as determined by SDS-PAGE. The optimum activity was found at two pH values 8.5 and 10.5; however the activity sharply decreased below pH 6 and above pH 10.5. The enzyme was stable for 72 h at pH 10.5 and at 50 degrees C. Kinetic experiments at 50 degrees C gave V(max) and K(m) of 1,400 U/ml min(-1) mg(-1) protein and 0.05 mg/ml respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by group II b metal ions like Zn2+, Hg2+, etc. Xylan completely protected the enzyme from being inactivated by N-bromosuccinimide.     

海枣曲霉β—葡萄糖苷酶的提纯与性质

A beta-glucosidase has been purified to electrophoretically homogeneity from the wheat bran culture of Aspergillus phoenicis by PEG 6000-phosphate biphasic separation, column chromatography on Sephadex G-100, DEAE-Sephadex A-50 and SE-Sephadex C-50. The enzyme showed optimal activity at pH 5.0 and 60 degrees C. It was stable in the pH range of 4.0-7.5 and up to 55 degrees C. The enzyme activity was strongly inhibited by Ag+ and Hg2+. The molecular weight of the enzyme was 118000 as determined by SDS-PAGE and 195000 by gradient-PAGE. The isoelectric point was pI 3.95 as determined by PAGIF.     

Molecular cloning, purification and characterization of thermostable beta-1,3-1,4 glucanase from Bacillus subtilis A8-8

A gene encoding a beta-1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant beta-1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60 degrees C, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60 degrees C and retained 30% of its original activity at 70 degrees C for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.     

Purification and properties of extracellular alpha-glucosidase of a thermophile, Bacillus thermoglucosidus KP 1006.

An extracecular alpha-glucosidase (alpha-D-glucoside glycohydrolase, EC 3.2.1.20) of a thermophile, Bacillus thermoglucosidius KP 1006, was purified about 350-fold. The purified enzyme had a specific activity of 164 mumol of p-nitrophenyl-alpha-D-glucopyranoside hydrolyzed per min at 60 degrees C and pH 6.8 per mg of protein. The molecular weight was estimated at 55 000. The pH and temperature optima for activity were 5.0--6.0 and 75 degrees C, respectively. Below 40 degrees C, the activity was less than 4.5% of the optimym. The enzyme showed a high specificity for alpha-D-glucopyranoside. The maximal hydrolyzing velocity per substrate diminished in the order: phenyl-alpha-D-glucopyranoside, p-nitrophenyl-alpha-D-glucopyranoside, isomaltose, methyl-alpha-glycopyranoside. The respective Km values were 3.0, 0.23, 3.2 and 27 mM. The activity was trace for turanose, and not detectable for sucrose, trehalose, raffinose, melezitose, maltose, maltotriose, phenyl-alpha-D-maltoside, dextran, dextrin and starch. Tris, p-nitrophenyl-alpha-D-xylopyranoside, glucose and glucono-delta-lactone blocked competitively the enzyme with respect to p-nitrophenyl-alpha-D-glucopyranoside. The Ki values were 0.12, 0.14, 2.2 and 2.4 mM, respectively. The activity was affected by heavy metal ions, but insensitive to EDTA, p-chloromercuribenzoate and iodoacetate. The enzyme was stable up to 60 degrees C, and inactivated rapidly at temperatures beyond 72 degrees C. The pH range for stability was 4.0--11.0 at 31 degrees C, and 6.0--8.5 at 55.5 degrees C. At 25 degrees C, the enzyme failed to be inactivated in 45% ethanol, in 7.2 M urea, and in 0.06% sodium dodecyl sulfate, but the tolerance was extremely reduced at 60 degrees C.     

A study of extracellular alkaline protease from Bacillus subtilis NCIM 2713

An alkaline protease was isolated from culture filtrate of B. subtilis NCIM 2713 by ammonium sulphate precipitation and was purified by gel filtration. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 8.0 and temperature 70 degrees C. The purified protease had molecular weight 20 kDa, Isoelectric point 5.2 and km 2.5 mg ml(-1). The enzyme was stable over the pH range 6.5-9.0 at 37 degrees C for 3 hr. During chromatographic separation this protease was found to be susceptible to autolytic degradation in the absence of Ca2+. Ca2+ was not only required for the enzyme activity but also for the stability of the enzyme above 50 degrees C. About 62% activity was retained after 60 min at pH 8.0 and 55 degrees C. DFP and PMSF completely inhibited the activity of this enzyme, while in the presence of EDTA only 33% activity remained. However, it was not affected either by sulfhydryl reagent, or by divalent metal cations, except SDS and Hg2+. The results indicated that this is a serine protease.     

根霉α-半乳糖苷酶的三相分离及其酶学性质

根霉Rhizopus sp. A01发酵豆渣产α-半乳糖苷酶,粗酶液依次经过三相分离、Sephadex G-100凝胶过滤获得了电泳纯的α-半乳糖苷酶,纯化了6.7倍,总酶活回收率达到46%;凝胶过滤和SDS-PAGE显示该酶为相对分子质量为87.6kDa的单体蛋白。该酶水解对硝基苯-α-D-吡喃半乳糖苷的最适pH值为5.0,最适温度为55℃,表观Km、kcat/Km分别为2.56mmol/L、47,400L/mol·s;能微弱水解蜜二糖和棉子糖,水解蜜二糖的速率是水解棉子糖速率的3.4倍;水解活性受多种     

Purification, crystallization and properties of triacylglycerol lipase from Pseudomonas fluorescens.

Triacylglycerol lipase of Pseudomonas fluorescens was purified from the crude enzyme by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-cellulose. The crystallization of the lipase was successfully carried out. The purified lipase was demonstrated to be homogenous on disc electrophoresis and its molecular weight was calculated to be 32 000 by gel filtration. The optimum pH for hydrolysis of sesame oil was 7.0. The enzyme was stable up to 40 degrees C under the condition of pH 7.0 for 30 min and had more than 80% of the remaining activity between pH 5.0--11.0 at 37 degrees C for 60 min. The lipase was strongly inhibited by iodine and partially inhibited by FeCl3 and N-bromosuccinimide, and showed the most activity on tricaproyglycerol, among the triacylglycerols used.