日本根霉IFO5318胞外β-葡萄糖苷酶的纯化及部分特性

采用硫酸铵沉淀及柱层析等步骤纯化了日本根霉IFO5318的β—葡萄糖苷酶,回收率为22％。该酶分子量约为4.0×10~5,由四个相同大小的亚基组成;最适反应温度55℃,最适反应pH5.5;对热较敏感,但能在较大的pH范围内保持稳定。用对硝基苯基—β-D-吡喃葡糖苷为底物,测得的K_m和V_(max)值分别为0.825mg·ml~(-1)和135.4μmol·min~(-1)·mg~(-1)。该酶对纤维二糖的水解能力最强,SDS、Fe³⁺、Hg²⁺等对酶活力有抑制作用。     

一株β-葡萄糖苷酶产生菌株的分离鉴定及酶学性质研究

【目的】分离获得β-葡萄糖苷酶高产菌株,确定该菌分类地位,并对其所产β-葡萄糖苷酶的酶学性质进行初步研究。【方法】采用七叶灵显色法从土壤样品中筛选β-葡萄糖苷酶产生菌,再用对硝基苯基-β-D-吡喃葡萄糖苷(PNPG)显色法进行复筛;通过形态特征、生理生化特征及16S rDNA序列相似性分析等方法确定其分类学地位;利用超滤、疏水层析、阴离子层析、分子筛层析法对β-葡萄糖苷酶进行分离纯化;以PNPG为底物,测定β-葡萄糖苷酶的最适反应pH及最适反应温度,通过双倒数作图法确定β-葡萄糖苷酶催化不同底物水解的米氏常数Km值。【结果】从土壤样品中筛选得到一株β-葡萄糖苷酶高产菌株ZF-6C,初步鉴定为Bacillus korlensis;芽胞杆菌ZF-6C所产β-葡萄糖苷酶的分子量约为90 kD,最适反应pH和温度分别为7.0和40°C,该酶具有水解β(1,4)糖苷键的活性,最适底物为邻硝基苯-β-D-吡喃葡萄糖苷,Km值为0.73 mmol/L。金属离子Ca2+、Pb2+增强酶活,而Cu2+、Fe2+抑制酶活。【结论】首次报道从Bacillus korlensis中分离得到β-葡萄糖苷酶,Bacillus korlensis ZF-6C所产β-葡萄糖苷酶在分子量、最适反应条件及底物特异性等方面均不同于已知酶,可能为一结构新颖且催化效率较高的β-葡萄糖苷酶。     

新型辅酶依赖型立体选择性氧化还原酶性质的初步研究

为了进一步认识立体选择性转化用菌株近平滑假丝酵母(Candida parapsilosis)SYB-1的转化机理及其转化酶系的酶学特性,考察了该菌种来源的粗酶的辅酶依赖型及立体选择性,发现依赖于辅酶NADP ,该酶将(R)-苯基乙二醇氧化为β-羟基苯乙酮;而依赖于辅酶NADH,该酶不对称还原β-羟基苯乙酮为(S)-苯基乙二醇。在研究该粗酶酶学特性后发现该粗酶对5C的伯醇专一性较强;在包括仲醇和二元醇的手性醇中,对于二元醇的专一性较强;而在还原反应中对2-丁酮专一性较强。金属螯合剂及重金属离子会对该粗酶的活力产生抑制作用。该粗酶催化氧化的最适pH为8.0,最适温度为50℃;催化还原的最适pH为6．0,最适温度为40℃。     

3-氰基吡啶水合酶的反应条件及影响因子

研究了芳腈水合酶催化水合3-氰基吡啶生成尼克酰胺的反应条件及影响因子.酶反应的最适pH为8.0,最适温度为25℃.酶在pH8.5于25℃保温4小时或在25—30℃于pH8.0保温3小时是稳定的.反应液中加入Fe~(3 )(1.5 mmol/L)可使酶活力增加 50%,而加入NH_4~ (300 mmol/L)则使酶活降低了67%.Ag~ 和 Hg(2 )”强烈地抑制酶反应活性,在浓度均为 5mmol/L时,抑制率分别为99.7%和100%.NaCN(50 mmol/L)和苯甲腈(100 mmol/L)对酶活性的抑制率分别为78%和85%.该酶作用于 3-氰基吡啶的Km为62.5 mmol/L,V_(max)为85.8 μmol·min~(-1)·mg~(-1).     

嗜碱菌碱性淀粉酶的研究

分离自内蒙古自治区察汗淖碱湖的嗜碱菌株No．1 0-1,好气,运动,细胞杆状,革兰氏染色阴性。该菌生长pH范围为8．0—13．0,最适生长pH1 0.0-ll.0,为专性嗜碱菌。在含淀粉培养基中产生胞外碱性淀粉酶,最适产酶条件是： 碳源为土豆淀粉,氮源为复合蛋白胨,Nacl浓度为2．O％,Na2CO3浓度为1．0—1．5％(pH9．9-10．5)。 酶的最适反应pH为10．0,稳定pH8．0,最适反应温度为50℃。作用于直链淀粉其水解产物为β-构型,主要产物是麦芽糖,其次为麦芽三糖、葡萄糖和麦芽四糖。嗜碱菌No.10-1产生的酶为碱性β-淀粉酶。     

黑曲霉β-葡萄糖苷酶的酶学特性研究

研究黑曲霉β-葡萄糖苷酶的酶学特性,采用酶学研究方法,通过硫酸铵沉淀、Sephadex G-25脱盐和Sephadex G-100纯化了β-葡萄糖苷酶,并进行了黑曲霉β-葡萄糖苷酶的最适反应温度、最适pH、热稳定性、pH稳定性及米氏常数等特性研究,采用SDS-PAGE凝胶电泳测定了分子量。研究表明,β-葡萄糖苷酶的最适反应温度为70℃、最适反应pH为4.5;在40、50和60℃下较稳定,80℃以上稳定性差;β-葡萄糖苷酶在pH为3、7、8、9的缓冲液中的稳定性很差,在pH为4、5、6的缓冲液中稳定性较好,其中在pH为5时,稳定性最好;酶的Km=41.67 mmol/L,Vmax=23.81 U/L;其分子量为65.2 ku。β-葡萄糖苷酶在饲料工业具有良好的应用前景。     

绿色木霉MJ1产内切β-葡聚糖苷酶的分离纯化及酶学性质

利用KTAUPC-900快速蛋白液相色谱系统(FPLC)从绿色木霉MJ1固体发酵产物中分离纯化出内切β-葡聚糖苷酶。分离纯化后酶的比活力提高了28·6倍,回收率为19·7%。SDS-PAGE后经BIO-RAD凝胶成像系统分析该内切酶的分子量为64·7kD。酶学试验研究表明:该酶的最适反应温度53℃,最适pH为4·2,Lineweaver-Burk法求得动力学参数,Km和Vmax分别为1·230×10-2g/mL、2·396×10-2mg/(mL·min)。并确定了FPLC层析缓冲液的离子强度为2·2mmol/L时分离效果达到最佳。     

半胱氨酸合成酶与β-氰基丙氨酸合成酶活性检测

植物半胱氨酸合成酶(Cysteine synthase,CSase)和β-氰基丙氨酸合成酶(β-cyanoalanine synthase,β-CAS)分别催化合成半胱氨酸(Cysteine,Cys)和β-氰基丙氨酸(β-Cyanoalanine,β-CA),它们在功能上冗余。本研究以山黧豆、苜蓿和玉米为主要材料,结合电泳对8种常见植物CSase和β-CAS粗酶活性进行了分析。结果表明,检测CSase活性时,8种植物两类粗酶的最适反应时间均为10min,最适pH均为8.0,底物O-乙酰-丝氨酸和Na2S最适浓度分别是10和5 mmol·L-1。检测β-CAS活性时,8种植物两类粗酶的最适反应时间均为30min,最适pH均在9~10范围内,底物Cys最适浓度均为3mmol·L-1,而底物KCN最适浓度前者为80mmol·L-1,后者为3mmol·L-1。8种植物中,CSase活性在种、种内组织间差别不是很大,但β-CAS活性则相反,尤其在茎叶和根中差别较大。     

木瓜凝乳蛋白酶的酶学性质研究

以酪蛋白为底物,木瓜凝乳蛋白酶的最适反应温度为80℃(pH7.0),最适pH为3-5(37℃)。在pH为7.0、反应温度为37℃的条件下,木瓜凝乳蛋白酶的Km值为1.25g·L-1,Vmax为0.1 g·L-1min-1。低浓度的NaCl和Ca2+对木瓜凝乳蛋白酶有激活作用,盐酸胍对其有抑制作用。     

海枣曲霉β-半乳糖苷酶的提纯与性质

 通过过聚乙二醇6000-磷酸钾缓冲液双相分离、Sephadex G-100凝胶过滤、DEAE-Sephadex A-50离子交换层析、羟基磷灰石层析及SephadexG-100凝胶过滤等提纯步骤,从海枣曲霉(Aspergillus phoenicis)麦麸培养物抽提液中提纯得到凝胶电泳均一的β-半乳糖苷酶。该酶的最适pH为3.5—4.0,最适温度为60℃(反应15分钟),在pH5.0—8.5之间及60℃以下稳定。在65℃和70℃保温时失活50％的时间分别为27和2分钟。用SDS凝胶电泳法和梯度凝胶电泳法分别测得该酶的分子量为115,000和118,000。薄层凝胶等电聚焦法测得其等电点为pH4.6。     

Purification and characterization of a beta-glucosidase from Trichoderma reesei

A beta-glucosidase has been purified from culture filtrates of the fungus Trichoderma reesei QM9414 grown on microcrystalline cellulose. The beta-glucosidase was purified using two successive DEAE-Sephadex anion-exchange chromatography steps, followed by SP-Sephadex cation-exchange chromatography and concanavalin-A--agarose chromatography. Evidence for homogeneity is provided by polyacrylamide disc gel electrophoretic patterns, which show a single protein band. Sedimentation equilibrium analysis yielded a molecular mass of 74.6 +/- 2.4 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a single protein band with a molecular mass of 81.6 kDa. Thus, the enzyme appears to be a single, monomeric polypeptide. The beta-glucosidase is isoelectric at pH 8.5. The enzyme is rich in basic amino acids and contains few half-cystine and methionine residues. The purified beta-glucosidase contains less than 1% by weight of neutral carbohydrate. The beta-glucosidase catalyzes the hydrolysis of cellobiose, p-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-glucopyranoside; the values of V/Km for each substrate were determined to be 2.3 X 10(4), 6.9 X 10(5) and 2.9 X 10(6) M-1 S-1 respectively. The enzyme is optimally active from pH 4.5 to 5.0 and is labile at higher hydrogen ion concentrations. The beta-glucosidase has an unusually high affinity for D-glucose (Ki = 700 microM). Comparison of inhibition constants for cello-oligosaccharides suggests that the substrate-binding region of the beta-glucosidase comprises multiple subsites.     

Characterization of beta-glucosidase as a peripheral enzyme of lysosomal membranes from mouse liver and purification

Lysosomal membrane fractions were prepared from lysosomes of mouse liver by freeze-thawing in a hypotonic buffer: 54% of beta-glucosidase [EC 3.2.1.45] in lysosomes was associated with the membrane fractions, whereas 96% of beta-glucuronidase [EC 3.2.1.31] was recovered in the soluble fractions of lysosomes. beta-glucosidase was solubilized by pH 9.5 treatment or by Triton treatment of membranes. The enzyme solubilized with alkali and concentrated with (NH4)2SO4 was rapidly inactivated in a solution of pH 9.5, but could be protected against inactivation by acidic detergent. Gel filtration analysis indicated that beta-glucosidase was in an aggregated form at neutral pH and could be disaggregated by alkali and detergents. The enzyme dissociated with detergents also showed a higher activity than the alkali-treated enzyme. These results suggested that beta-glucosidase is a peripheral enzyme bound to acidic lipids in membranes. beta-Glucosidase was purified to apparent homogeneity by (NH4)2SO4 fractionation and chromatographies with Sephacryl S-300, hydroxylapatite and cation exchangers in the presence of detergents. The catalytic activity of the purified enzyme was maximally stimulated by phosphatidylserine and heat-stable protein in the presence of a low concentration of Triton X-100. The stimulation was mainly due to an increase in Vmax.     

beta-Glucosidase of Penicillium funiculosum. II. Properties and mycelial binding

The properties of two isozymes of beta-glucosidase of Penicillium funiculosum (part I of this series) are described. The molecular weights of isozyme 1 was 2.3 x 10(5) by gel filtration and 1.2 x 10(5) by SDS gel electrophoresis, indicating two subunits. The molecular weight of isozyme 2 was unusually low for a fungal beta-glucosidase: 1.6 x 10(4) by gel filtration and 3.7 x 10(4) in the presence of isopropanol. The two enzymes differed from other fungal beta-glucosidases in their substrate specificities. They showed high activity with pNPG, cellobiose, cellotriose, cellotetraose, cellopentaose, gentiobiose, and laminarin, but were inactive with filter paper, CM cellulose, or derivatives or stabilized by bovine serum albumin and several alcohols such as butanol and propanol. It was inhibited by glucono-delta-lactone (K(i) = 0.67muM) and glucose (K(i) = 0.92mM).The enzyme was quantitatively adsorbed by P. funiculosum mycelium at pH 4 and the immobilized enzyme was as enzymically active as the free enzyme, but more heat stable. The binding efficiency was very high (5000 IU enzyme/g mycelium). It could be quantitatively eluted with buffers at pH 7 or by 0.02M Ca, Mg, or Al chlorides. The binding was selective, since mycelium grown on lactose could produce and also bind only beta-glucosidase isozyme 1, whereas mycelium grown on cellulose could produce as well as bind both beta-glucosidase isozymes as well as cellulases. Mycelial binding was unaffected by washing with EDTA or trypsinization, but was totally lost by washing with dilute KOH, HCl, or ethylenediamine.     

Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.     

Affinity chromatography of beta-glucosidase and endo-beta-glucanase from Aspergillus niger on concanavalin A-Sepharose: implications for cellulase component purification and immobilization

Affinity chromatography of a commercial preparation of beta-glucosidase from Aspergillus niger using concanavalin A-Sepharose (CAS) was employed as a means of purifying this glycoprotein. However, mannose (up to 1.08 M) was ineffective as an eluent of this enzyme from CAS, as were several other sugars and their derivatives, including 0.5 M glucose. Also, washing the CAS: beta-glucosidase complex with buffer at pH 3.5 in the absence of MnCl2 and CaCl2 (required to preserve the binding activity of concanavalin A below pH 5.0) did not result in elution of this enzyme. On the contrary, endoglucanase activity present in a crude cellulase complex (A. niger) which bound to CAS could be eluted by mannose (0.5-0.7 M) and was fractionated into at least two components. The CAS: beta-glucosidase complex hydrolyzed cellobiose to glucose and possessed an activity of 2,158 units/g dry CAS. It could be used, therefore, for continuous cellobiose hydrolysis without leakage of enzyme from the support.     

Characterization of beta-glucosidase isoenzymes possibly involved in lignification from chick pea (Cicer arietinum L.) cell suspension cultures.

Crude cell wall preparations from Cicer arietinum L. cell suspension cultures show high activity for the hydrolysis of coniferyl alcohol beta-D-glucoside (coniferin). Various beta-glucosidase activities could be solubilized from these preparations by 0.5 M NaCl treatment and one of these could be shown to possess a high activity for the hydrolysis of coniferin. The enzyme activities were purified to near homogeneity by Sephadex G-200 and CM-Sephadex chromatography. Isoelectric focussing indicated the occurrence of beta-glucosidase isoenzymes with identical catalytic activity (pI 8.5-10). Molecular weights were determined as 110 000, with two subunits of 63 000 and 43 000. Maximum hydrolytic activity is at pH 5. The beta-glucosidase isoenzymes catalyze the hydrolysis of various beta-glucosides with aromatic aglycone structure and different sugar moieties. However, coniferin has been found to be one of the best substrates (km = 0.8 mM; V = 6 mumol.min-1.mg protein-1) for these beta-glucosidase isoenzymes. The data suggest that beta-glucosidase-catalyzed reaction might be involved in lignification of these plant cell cultures.     

Purification and properties of a novel beta-glucosidase, hydrolyzing ginsenoside Rb1 to CK, from Paecilomyces Bainier

A novel ginsenoside-hydrolyzing beta-glucosidase was purified from Paecilomyces Bainier sp. 229 by a combination of QSepharose FF, phenyl-Sepharose CL-4B, and CHT ceramic hydroxyapatite column chromatographies. The purified enzyme was a monomeric protein with a molecular mass estimated to be 115 kDa. The optimal enzyme activity was observed at pH 3.5 and 60oC. It was highly stable within pH 3-9 and at temperatures lower than 55oC. The enzyme was specific to beta-glucoside. The order of enzyme activities against different types of beta-glucosidic linkages was beta-(1- 6)>beta-(1-2)>beta-(1-4). The enzyme converted ginsenoside Rb1 to CK specifically and efficiently. An 84.3% amount of ginsenoside Rb1, with an initial concentration of 2 mM, was converted into CK in 24 h by the enzyme at 45 degrees and pH 3.5. The hydrolysis pathway of ginsenoside Rb1 by the enzyme was Rb1-->Rd-->F2-->CK. Five tryptic peptide fragments of the enzyme were identified by a newly developed de novo sequencing method of post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. By comparing the five identified peptide sequences with the NCBI database, this purified beta-glucosidase proves to be a new protein that has not been reported before.     

Purification and Properties of beta-Glucosidase from Aspergillus terreus

A beta-glucosidase (EC 3.2.1.21) from the fungus Aspergillus terreus was purified to homogeneity as indicated by disc acrylamide gel electrophoresis. Optimal activity was observed at pH 4.8 and 50 degrees C. The beta-glucosidase had K(m) values of 0.78 and 0.40 mM for p-nitrophenyl-beta-d-glucopyranoside and cellobiose, respectively. Glucose was a competitive inhibitor, with a K(i) of 3.5 mM when p-nitrophenyl-beta-d-glucopyranoside was used as the substrate. The specific activity of the enzyme was found to be 210 IU and 215 U per mg of protein on p-nitrophenyl-beta-d-glucopyranoside and cellobiose substrates, respectively. Cations, proteases, and enzyme inhibitors had little or no effect on the enzyme activity. The beta-glucosidase was found to be a glycoprotein containing 65% carbohydrate by weight. It had a Stokes radius of 5.9 nm and an approximate molecular weight of 275,000. The affinity and specific activity that the isolated beta-glucosidase exhibited for cellobiose compared favorably with the values obtained for beta-glucosidases from other organisms being studied for use in industrial cellulose saccharification.     

One-step purification and characterization of an intracellular β-glucosidase from Metschnikowia pulcherrima

A collection of 60 non-Saccharomyces yeasts isolated from grape musts in Uruguayan vineyards was screened for beta-glucosidase activity and Metschnikowia pulcherrima was the best source of this enzyme activity. Its major beta-glucosidase was successfully purified to homogeneity by ion-exchange chromatography on amino-agarose gel. The enzyme exhibited an optimum catalytic activity at 50 degrees C and pH 4.5 and was active against (1 --> 4)-beta and (1 --> 2)-beta glycosidic linkages. In spite of preserving 100% of its activity and stability in the presence of 12% (v/v) ethanol and 5 g glucose/l, the enzyme was unstable below pH 4. We characterized the beta-glucosidase from M. pulcherrima with a view to its potential applications in wine-making.     

Characterization of beta-glucosidase from corn stover and its application in simultaneous saccharification and fermentation

Purification and characterization of beta-glucosidase from corn stover was performed and the enzyme was tried in SSF to evaluate the suitability of plant glycosyl hydrolases in lignocellulose conversion. A beta-glucosidase with M(w) of 62.4 kDa was purified to homogeneity from post-harvest corn stover. The following physicochemical and kinetic parameters of the beta-glucosidase were studied respectively: optimum temperature, thermal stability, optimum pH, pH stability, K(m), V(max), V(i), cellobiose inhibition, tryptic peptide mass spectrometry and effect of metal ions and other reagents on the activity. The beta-glucosidase activity on salicin was optimal at pH 4.8 and 37 degrees C. The unique property of optimum temperature makes the beta-glucosidase potentially useful in SSF. In SSF of steam explosion pretreated corn stover, the supplementation of the purified beta-glucosidase was more effective than Aspergillus niger beta-glucosidase.