Characterization of a thermostable family 10 endo-xylanase (XynB) from Thermotoga maritima that cleaves p-nitrophenyl-beta-D-xyloside

Thermotoga maritima MSB8 possesses two xylanase genes, xynA and xynB. The xynB gene was isolated from the genomic DNA of T. maritima, cloned, and expressed in Escherichia coli. XynB was purified to homogeneity by heat treatment, affinity chromatography and ion-exchange column chromatography. The purified enzyme produced a single band upon SDS-PAGE corresponding to a molecular mass of 42 kDa. At 70 degrees C, the enzyme was stable between pH 5.0 and pH 11.4, and it was stable at temperatures of up to 100 degrees C from pH 7.0 to pH 8.5. At 50 degrees C, XynB displayed an optimum pH of 6.14 and at this pH the temperature for optimal enzyme activity was 90 degrees C. XynB exhibited broad substrate specificity and was highly active towards p-nitrophenyl-beta-D-xylobioside with K(m) and k(cat) values of 0.0077 mM and 5.5 s(-1), respectively, at 30 degrees C. It was also active towards p-nitrophenyl-beta-D-xyloside. The initial product of the cleavage of p-nitrophenyl-beta-D-xyloside was xylobiose, indicating that the major reaction in the cleavage was transglycosylation, not hydrolysis.     

Purification and characterization of the Clostridium josui porphobilinogen deaminase encoded by the hemC gene from a recombinant Escherichia coli

The porphobilinogen deaminase encoded by the Clostridium josui hemC gene was purified from a recombinant Escherichia coli strain and its properties were characterized. The optimal temperature and pH of the purified enzyme were 65 degrees C and 7.0, respectively. This enzyme was quite thermostable: it retained 86% of the original activity after incubation at 70 degrees C for 1 h. The Km and Vmax values of the enzyme were 65 microM and 3.3 micromol/h/mg for porphobilinogen, respectively.     

Purification and characterization of an alkaline manganese peroxidase from Aspergillus terreus LD-1

We report that Aspergillus terreus LD-1 produces an extracellular ligninolytic enzyme, manganese peroxidase (MnP), that reacts under alkaline conditions. This MnP was purified 13.1-fold from the culture supernatant to elicit a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of this MnP was estimated as either 43 kDa by SDS-PAGE or 44 kDa by gel permeation chromatography, suggesting a monomeric structure. The optimum pH and temperature of this MnP are 12.5 and 37 degrees C, respectively. This MnP is stable in the pH range 11.0 to 12.5 and also up to 40 degrees C. The K(m) values of this MnP for hydrogen peroxide, 2,6-dimethoxyphenol (2,6-DMP) and Mn2+ were 320 microM, 20 microM and 33 microM at pH 12.5, respectively. The activity of the MnP is completely inhibited by Hg2+, Pb2+, Ag+ and lactate. On the other hand, the MnP is activated by oxalate, maleate and fumarate. Maleate at 5 mM increased the MnP activity 5-fold. EDTA at 1 mM inhibited the MnP activity completely, but this inhibition was not observed in the presence of 1 mM Fe2+.     

Peptide hydrolases of Lactobacillus casei: isolation and general properties of various peptidase activities.

Discovery of an endopeptidase by gel chromatography and separation of 3 exopeptidases (a dipeptidase, an aminopeptidase and a specific carboxypeptidase) from Lactobacillus casei NCDO 151 by affinity chromatography is described. The 3 exopeptidases were strongly inhibited by the metal chelators EDTA and 1,10-phenanthroline but were reactivated with Co2+ and Mn2+. The pH optima for aminopeptidase, dipeptidase and carboxypeptidase activities were 6.5, 7.6 and 7.2, respectively. Maximum activity was obtained at 45 degrees C for the aminopeptidase, at 30 degrees C for the dipeptidase and at 40 degrees C for the carboxypeptidase. The substrate specificities of the 3 enzymes were also studied. The properties of these 3 enzymes are compared with those of other bacteria.     

Purification and characterization of cis-aconitic acid decarboxylase from Aspergillus terreus TN484-M1

cis-Aconitic acid decarboxylase (CAD) was assumed to be a key enzyme in the production of itaconic acid by comparing the activity of CAD from Aspergillus terreus TN484-M1 with that of CAD from the low-itaconate yielding strain Aspergillus terreus CM85J. The constitutive CAD was purified to homogeneity from A. terreus TN484-M1 by ammonium sulfate fractionation, and column chromatography on DEAE-toyopearl, Butyl-toyopearl, and Sephacryl S200HR, and then characterized. A molecular mass of 55 kDa for the native enzyme was determined by SDS-PAGE. The enzymic activity was optimal at a pH of 6.2 and temperature of 45 degrees C. The K(m) value for cis-aconitic acid was determined as 2.45 mM (pH 6.2, 37 degrees C). The enzyme was completely inactivated by Hg+, Cu2+, Zn2+, p-chloromercuribenzoate, and 5,5'-dithio-bis(2-nitrobenzoate).     

Purification and characterization of a beta-glucosidase with beta-xylosidase activity from Aspergillus sojae

A beta-glucosidase (EC 3.2.1.21) was purified as an electrophoretically homogeneous protein from a solid culture of Aspergillus sojae. The molecular mass of the purified enzyme was estimated to be 250 kDa by gel filtration chromatography and 118 kDa by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point of the enzyme was 3.80. The maximum velocity of rho-nitrophenyl beta-d-glucopyranoside degradation by the beta-glucosidase was attained at 60 degrees C and at pH 5.0. The purified enzyme was stable from pH 6.0 to 8.0, and up to 50 degrees C. The activity of the enzyme was significantly inhibited by Hg2+ and Cu2+, and stimulated by Mn2+ and Fe3+. The purified enzyme hydrolyzed beta-D-xylopyranosides as well as beta-D-glucopyranosides; the Km and Vmax values on rho-nitrophenyl beta-D-glucopyranoside were 0.14 mM and 16.7 micromol/min/mg protein, and on rho-nitrophenyl beta-D-xylopyranoside 0.51 mM and 12.2 micromol/min/mg protein, respectively.     

产β-D-半乳糖苷酶马克斯克鲁维酵母的分离鉴定及其产酶特性

从藏灵菇中分离筛选到的一株高产β-D-半乳糖苷酶菌株ZX-5,经ITS DNA序列分析,鉴定为马克斯克鲁维酵母。对产酶培养基的最佳碳源和氮源优化结果为半乳糖2.0%,胰蛋白胨1.0%;产酶优化条件:温度30℃,培养基初始pH 6.5,装液量30%,转速100 r/min,接种量2.0%,发酵36 h。粗酶液酶活力为2.60 U/mL;经硫酸铵分级沉淀和DEAE离子交换层析,获得纯化酶的比活力为157.35 U/mg。酶最适反应温度35℃,最适pH 6.0,在20~40℃和pH 5.0~7.0的范围内酶的稳定性较好;Mn2+对酶的活性有促进作用。利用菌株ZX-5β-D-半乳糖苷酶分解乳糖并合成低聚半乳糖(galacto-oligosaccharide,GOS),在35℃、乳糖质量浓度60 g/100 mL、酶浓度1.0 U/mL条件下,乳糖水解率达68.34%(50 h),GOS产率达34.70%(40 h),具有潜在的应用前景。     

Molecular cloning and characterization of thermostable beta-lactam acylase with broad substrate specificity from Bacillus badius

The gene (pac) encoding beta-lactam acylase from Bacillus badius was cloned and expressed in Escherichia coli. The pac gene was identified by polymerase chain reaction (PCR) using degenerated primers, on the basis of conserved amino acid residues. By using single specific primer PCR (SSP-PCR) and direct genome sequencing, a complete pac gene with its promoter region was obtained. The ORF consisted of 2415 bp and the deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a signal sequence, an alpha-subunit, a spacer peptide and a beta-subunit. The pac gene was expressed with its own promoter in different E. coli host strains and a maximum recombinant PAC (1820 U l(-1)) was obtained in E. coli DH5alpha. The recombinant PAC was purified by Ni-NTA chromatography and the purified PAC had two subunits with apparent molecular masses of 25 and 62 kDa. This enzyme exhibited a high thermostability with a maximum activity at 50 degrees C. This enzyme showed stability over a wide pH range (pH 6.0-8.5) with a maximum activity at pH 7.0 and activity on a wide beta-lactam substrate range. The K(m) values obtained for the hydrolysis of penicillin G and a chromogenic substrate, 6-nitro-3-phenylacetylamidobenzoic acid, from B. badius PAC were 39 and 41 microM, respectively. The PAC activity was competitively inhibited by PAA (K(i), 108 microM) and noncompetitively by 6-APA (K(i), 17 mM). The constitutive production of B. badius PAC in E. coli and its easier purification together with the advantageous properties, such as thermostability, pH stability and broad substrate specificity, make this as a novel enzyme suitable for beta-lactam industry.     

AN APPROACH TO THE IDENTIFICATION OF A ß-GLUCAN SOLUBILASE FROM BARLEY1

A ß-glucan solubilase activity was demonstrated in barley extract. This enzyme catalyzed the dissolution of barley ß-glucan by releasing a product having a narrow molecular weight distribution and a molecular weight of about 20,000. The enzyme was partially purified by ion exchange chromatography on DEAE-cellulose and gel permeation chromatography on Bio-Gel P-100. Although carboxypeptidase activity was present in the crude extract of barley flour the partially purified ß-glucan solubilase did not hydrolyse N-CBZ-Phe-ala. Examination of extracts from different barley tissues indicated that the ß-glucan solubilase activity was associated with the husks only; a large portion of the activity was extractable from whole barley kernels. About 85% of the enzyme activity in crude extracts from barley flour was retained after 40 min at 62°C. However, the enzyme was much more heat-labile in extracts of whole barley kernels. The pH of maximal activity was found to be about pH 5.7 and results from column chromatography suggested that the enzyme had a low pl value and a MW between 5 × 10⁴ and 6 × 10⁴.     

Human N-acetylglucosaminyltransferase I. Expression in Escherichia coli as a soluble enzyme, and application as an immobilized enzyme for the chemoenzymatic synthesis of N-linked oligosaccharides

N-Acetylglucosaminyltransferase I (GnT-I), which catalyzes the transfer of an N-acetylglucosamine residue from UDP-N-acetylglucosamine to the alpha1,3-linked mannose on Man5GlcNAc2 (M5), is a critical enzyme for the synthesis of high-mannose-type to complex-type glycan structures in N-linked glycan processing. We developed a large-scale preparation system for recombinant human GnT-I (hGnT-I) using the maltose binding protein (MBP) fusion system to facilitate the chemoenzymatic route for complex-type N-linked glycan synthesis. MBP-fused GnT-I was purified by affinity chromatography on an amylose resin column. The relative activity of MBP-fused GnT-I toward high-mannose-type N-linked oligosaccharides was 100% for Man5GlcNAc2, 52% for Man3GlcNAc2, 17% for Man6GlcNAc2. MBP-fused GnT-I exhibited optimal activity at pH 6.5-9.5 and was more active between pH 6.5-9.0. The optimum temperature for MBP-fused GnT-I activity was 40 degrees C, but the enzyme was active between 0-70 degrees C. Mn2+ and Co2+ were critical for the enzyme activity, while Zn2+ and Ca2+ inhibited the activity. Kinetic analysis of the purified enzyme showed an apparent K(m) value of 0.483 mM and a V(max) of 101 nmol/mg/min for M5. Immobilization of MBP-fused GnT-I on the amylose resin led to an 80% yield of the high mannose-type-of oligosaccharide.     

Purification and characterization of a beta-galactosidase from Fusarium oxysporum var. lini

Beta-D-Galactosidase was purified from a cellular extract of Fusarium oxysporum var. lini by heat shock and successive chromatography on DEAE-cellulose DE-52 and Sephadex G-100. The purified enzyme was homogeneous on SDS gel electrophoresis. It was inhibited by divalent cations such as Zn++, Mg++, and Ca++. The Michaelis constant and maximum velocity values for o-nitrophenyl beta-D-galacto-pyranoside were 6.76 mM and 816.7 mumol X mg protein-1 X min-1. The isoelectric point was 3.83, and the optimal pH and temperature were 5.0 and 55 degrees C. The estimated molecular weight of the enzyme was 224,000 by gel filtration and 36,300 by SDS-PAGE. The enzyme was considered a hexamer. o-Nitrophenyl-beta-D-galacto-pyranoside hydrolysis was activated by lactose, suggesting an allosteric nature of the enzyme.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

Purification and characterization of dibenzothiophene sulfone monooxygenase and FMN-dependent NADH oxidoreductase from the thermophilic bacterium Paenibacillus sp. strain A11-2

A dibenzothiophene (DBT) sulfone monooxygenase (TdsA), which catalyses the oxidative CS bond cleavage of DBT sulfone to produce 2-(2-hydroxyphenyl)benzenesulfinate (HPBS) was purified from the thermophilic DBT desulfurizing bacterium Paenibacillus sp. strain A11-2 by multistep chromatography. The molecular mass of the purified enzyme was determined to be 120 kDa by gel filtration and the subunit molecular mass was calculated to be 48 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicating a dimeric structure. The N-terminal amino acid sequence of the purified TdsA was determined to be MRQMHLAGFFAAGNTHH, which revealed no significant similarity to any other known amino acid sequences. The purified TdsA absolutely required an oxidoreductase for its activity. This oxidoreductase (TdsD) was also purified to homogeneity, and its molecular size was calculated to be 50 kDa and 25 kDa by gel filtration and SDS-PAGE, respectively. TdsD was completely FMN-dependent, and FAD could not act as a cofactor. The N-terminal amino acid sequence of the purified TdsD was determined to be TSQTAEQSIAPIVAQYRHPEQPISALFVNR, which showed significant similarity to kinesin-like protein (44% identity). The optimal temperatures for the activity of TdsA and TdsD were 45 degrees C and 55 degrees C, respectively. Both enzymes showed optimal activity at pH 5.5. TdsA was slightly inhibited by sulfate, but not by 2-hydroxybiphenyl (2-HBP), which is another end product of DBT. TdsA showed higher activity toward bulkier substrates than its mesophilic counterpart, DszA. These properties suggest the applicability of biodesulfurization to the processing of actual petroleum fractions.     

Purification, characterization, and steady-state kinetics of a meta-cleavage compound hydrolase from Pseudomonas fluorescens IPO1

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IP01, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0-9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20 degrees C. The enzyme has a K(m) of 7.3 microM and a k(cat) of 21 s(-1) for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25 degrees C), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodR Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10(5)-fold lower activity than that of the wild-type enzyme.     

Purification and characterization of a monoacylglycerol lipase from Pseudomonas sp. LP7315

A monoacylglycerol lipase (MGL) was purified from Pseudomonas sp. LP7315 by ammonium sulfate precipitation, anion-exchange chromatography, and preparative electrophoresis. The purified enzyme was homogeneous on SDS-PAGE with a molecular mass of 59 kDa. Its hydrolytic activity was confirmed to be specific for monoglycerides: the enzyme did not hydrolyze di- and triglycerides. MGL was found to be stable even after 1-h incubation at 65 degrees C. The optimum pH for monopalmitin hydrolysis was approximately 8. The hydrolytic activity depended not only on temperature and pH but also on the type of monoglyceride used. MGL also catalyzed monoglyceride synthesis at 65 degrees C in a solvent-free two-phase system, in which fatty acid droplets were dispersed in the glycerol phase with a low water content. The synthetic reaction proceeded at a constant rate for approximately 24 h and approximately reached an equilibrium after 48 h of reaction. The initial rate and equilibrium yield of the synthetic reaction depended on the type of fatty acid used as the substrate.     

PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM OYSTER (CRASSOTREA GIGAS)

Proteases in oyster (Crassotrea gigas) were extracted with 10 mM Tris-HCl buffer solution and purified by ammonium sulfate fractionation, Sephadex G-150 gel filtration, repeated DEAE-Sephadex A-50 and CM-Sepharose CL-6B chromatography. Three fractions with caseinolytic activity, named I, II and III, were obtained from CM-Sepharose CL-6B and DEAE-Sephadex A-50 chromatography. The three proteases were purified to electrophoretic homogeneity. Substrate specificity studies indicated that protease I was a carboxypeptidase A-like enzyme; II and III were trypsin-like enzymes. The optimal pH of protease I for hydrolysis of hippuryl-L-phenylalanine was 9.0, II and III for hydrolysis of p-toluenesulfonyl-L-arginine methyl ester (TAME) was 8.0. The temperatures which inactivated 50% of enzymes were 78°C for protease I in 30 min; 50 and 52°C for protease II and III, respectively, in 5 min. The molecular weights of proteases I, II and III were 23,000, 34, 400 and 31, 000, respectively.     

Characterization of Clostridium paraputrificum chitinase A from a recombinant Escherichia coli

Clostridium paraputrificum chitinase A (ChiA) was purified from a recombinant Escherichia coli. ChiA was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. ChiA showed maximum activity at pH 6.0 and its optimum temperature was 45 degrees C. ChiA was stable between pH 6.0 and 9.0 and at temperatures up to 40 degrees C. The K(m) and V(max) values of ChiA for 4-MU-(GlcNAc)2 were estimated to be 6.9 microM and 43 micromol/min/mg, respectively. Thin-layer chromatography indicated that ChiA hydrolyzes chitooligosaccharides to mainly chitobiose. ChiA was found to adsorb not only chitinous polymers but also cellulosic polymers.     

Proteolysis by toxigenic Aspergillus nidulans from Nigerian palm produce

The submerged cultures of Aspergillus nidulans had optimal growth and protease production at 37 degrees C and within 6 days of incubation. A rapid drop in pH of the growth medium from 6.9 to 4.8 and a subsequent gradual rise was recorded with the period of incubation. The acid-protease produced was purified by a combination of ethanolic precipitation, ultrafiltration and fractionation on DEAE-cellulose and Sephadex G-200. A single peak showing protease activity was subsequently obtained with a 16-fold increase in specific activity and a recovery value of 36%. The purified enzyme had optimal activity on casein and gelatin at pH 5.4 and a temperature of 40 degrees C.     

Purification and characterization of a novel serine protease compositain from compositae (Scorzonera hispanica L.)

In this research, a protease from the compositae (Scorzonera hispanica L.) was extracted and purified through (NH₄)₂SO₄ precipitation, CM-Sephadex and Sephacryl S200. At the end of purification by gel filtration on a Sephacryl S-200 column, 87.11-fold purification was achieved. It was shown that purified enzyme was homogeneous in terms of SDS-PAGE with molecular mass estimate of 30?kDa. The enzyme named compositain depicted an optimal pH of 8.0 and was stable at pH 7.0–9.0, and its optimal temperature was at 50?°C. While Tween 80 (0.2?%) was activated to the purified protease enzyme, it was partially inhibited by 5?mM concentration of some metal salts and EDTA, PMSF, dithiothreitol, H₂O₂ and β-mercaptoethanol. The enzyme activity was stable even in the presence of detergents and organic solvents. In addition, it was investigated whether the purified and characterized protease enzyme would cause to congeal milk. As a result, it was determined that the compositain could congeal milk and it would be used for cheese production. The compositain had potential application in food processing.     

Synthesis of raffinose by fungal α-galacotosidase from Absidia corymbifera

In order to investigate the optimal conditions for raffinose synthesis, α-galactosidase was purified from Absidia corymbifera IFO8084 with a recovery yield of approximately 8.1% (8.36 mg). The molecular weight of the wild-type α-galactosidase was about 83 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The native molecular mass of the enzyme was approximately 330 kDa by gel filtration chromatography, indicating that α-galactosidase from A. corymbifera IFO8084 is a homotetrameric enzyme. The purified enzyme displayed optimal enzyme activity at pH 4.5 and 60°C. When the purified α-galactosidase was incubated in a substrate solution of sucrose and D-galactose for 48 hr at 37°C, raffinose was synthesized and was confirmed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and ¹³C-nuclear magnetic resonance (¹³C-NMR) spectrometer analysis. Maximum rates of conversion were observed with 1.67 M galactose, 2.04 M sucrose, and 100 U α-galactosidase at pH 6.0 and 70°C. Under the optimized conditions, the overall conversion ratio was 10%(w/v), representing 2.5 times the synthesis yield that would be possible without the optimized conditions.