木霉GXC产β-葡聚糖酶条件和酶学性质

研究了木霉GXC产β-葡聚糖酶的条件.结果表明,最适产酶碳源为麸皮,氮源为硫酸铵;产酶的最适条件为初始pH为4.0～5.0,30℃培养44h.粗酶液经硫酸铵沉淀、Sephadex G-25、Sephadex G-100和DEAE-Sehadex A-50柱层析得到纯β-葡聚糖酶,SDS-PAGE凝胶电泳显示一条带,测得分子量为35kD.该酶最适反应pH5.0,最适反应温度为60℃,在40℃以下、pH4.0～5.0酶活力相对稳定.5.0mmol/L以下的Ca2+、Zn2+和Fe2+,以及10.0mmol/L以下的Co2+对酶活力有激活作用;而Cu2+和Fe3+具有抑制作用.     

一株耐热脂肪酶产生菌的筛选及酶学性质研究

从云南省富油地采取了60份土样中,利用透明圈法筛选出一株耐热脂肪酶产生菌。对其酶学性质和发酵条件进行了研究,酶学性质表明,该酶最适作用温度为50℃,最适pH6.0,在pH3.0-8.0范围内稳定,在60℃保温60 min酶活还保留70%;70℃保温60 min残余50%;具有良好的热稳定性;不同金属离子有不同的作用,Ca+,K+对酶有激活作用,Fe3+、Pb2+、Mn2、Cu2+、Al3+、Zn2+对酶活有抑制作用。EDTA对酶影响不大。产酶最佳条件为:MgSO4.7H2O 0.05 g,K2HPO40.1 g,CaCO30.25 g,可溶性淀粉2.5 g,大豆粉2.5 g,装液量50 mL。这株细菌通过培养基优化酶活达到20.3 U/mL。     

白灵侧耳纤溶酶的纯化及酶学性质分析

白灵侧耳子实体浸提液经过硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换层析、凝胶过滤层析和羟基磷灰石色谱柱层析后,纯化得到一种纤溶酶。该酶在SDS-PAGE中显单条带,其分子量约为30kDa。该酶在45℃以下,pH6.5-10.0的范围内稳定,最适pH为8.0,最适温度为25℃。金属离子K+对该酶有明显的激活作用,Zn2+、Mg2+、Cu2+对酶有部分抑制作用。金属离子鳌合剂EDTA和丝氨酸蛋白酶抑制剂PMSF不抑制该酶活性,初步说明此酶既不是金属酶,也不是丝氨酸类蛋白酶。该酶既具有纤溶酶作用,又具有激活纤溶酶原的作用。     

球孢白僵菌胞外壳聚糖酶的纯化和性质*

球孢白僵菌Beauveria bassiana 1316-V1的培养上清液经硫酸铵分级沉淀,Sephadex G-75凝胶过滤,Chitosan-bead亲和层析,第二次Sephadex G-75凝胶过滤, 得到电泳纯的一种胞外壳聚糖酶,比活力达到45u/mg 。此酶的分子量为36 kD; 最适酶反应温度为60℃;最适pH为4.0;最适离子强度为 0.25mol/L NaCl; 37℃以下,pH 2.0~5.0之间稳定性好; Cu2+、Hg2+、Pb2+、Ni2+ 对该酶有强烈抑制作用;Ag+、Mn2+也有较强抑制作用;Fe2+有轻微激活作用。该壳聚糖酶是一种糖蛋白,含糖约为12.6%。酶的最适底物为脱乙酰度为90%的胶体壳聚糖;也能轻微水解CMC、DEAE-Cellulose和胶体几丁质;但不能水解片状的壳聚糖和几丁质。     

黄鳝碱性磷酸酶的分离纯化及其部分性质研究

经Tris-HCl缓冲液(pH8.6)抽提,正丁醇处理,30%-75%硫酸铵分级沉淀分离,DEAE-Sepharose离子交换柱层析,Sephacryl S-200凝胶过滤纯化,从黄鳝内脏组织中分离纯化出电泳纯的碱性磷酸酶。该酶提纯倍数为564倍,比活力达到3015U/mg。酶学性质和动力学性质研究表明,该酶催化磷酸苯二钠的水解反应,最适pH值为10.2,pH小于7和大于12均不稳定;最适温度为40℃,温度高于50℃不稳定;米氏常数Km值为1.17mmo1/L。金属离子对该酶的催化活力有不同的影响,K+对该酶活力无影响,Mg2+对该酶有激活作用,Zn2+对该酶有抑制作用。       

酸性α-淀粉酶的分离纯化与酶学性质研究

纯化了枯草芽胞杆菌xm-1菌株酸性α-淀粉酶,并对其酶学性质进行了研究。通过硫酸铵沉淀和Sephadex G-75凝胶层析将酸性α-淀粉酶粗酶液纯化了32.5倍,活力回收率为10.0%。酶性质测定结果表明,该酸性α-淀粉酶分子量约为60kD,最适反应温度为45℃、最适作用pH5.0,该酶在pH3.4-6.0下稳定,高温耐受性差。Cu2+、Zn2+、EDTA对酶有不同程度的抑制作用,Ca2+和Mn2+对酶具有较强的激活作用。     

一组厌氧真菌菌系产阿魏酸酯酶的初步研究

利用玉米秸秆粉为唯一碳源的筛选培养基,从肉牛瘤胃液中筛选构建了一组厌氧真菌菌系,研究了该菌系产阿魏酸酯酶的特征.阿魏酸酯酶的最适pH为8.0,最适温度为40℃,最高酶活力为19.1 mU/mL,在pH 6.0 ～8.0及35～45℃之间,酶活性保持相对稳定.Mg2+对酶活力具有激活作用,Fe2、Cu2+、Fe3+等均抑制酶活力.     

天然巴曲酶的酶学性质研究

目的 研究巴西矛头蝮蛇蛇毒中纯化的天然巴曲酶的酶学性质.方法 测定不同的温度、pH值和金属离子等条件对重组定点突变巴曲酶活性的影响.结果 实验表明,该酶在温度30～40℃,pH 6.5～9.0活性较为稳定;其最适反应温度为37℃,pH为7.5;Mg2+、K+和Ca2+离子对酶活有激活作用,而Cu2+、Fe2+和Zn2+离子对该酶有抑制作用.结论 从蛇毒提取的天然巴曲酶酶学性质比较稳定.     

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。     

嗜热脂肪芽孢杆菌高温蛋白酶的产生条件及酶学性质

对嗜热脂肪芽孢杆菌(Bacillusstearothermophilis)WF146的产蛋白酶的条件进行了研究,在58℃条件下,WF146在pH值为75的Fd培养基中振荡发酵培养48h后,发酵液中高温蛋白酶产量可达600u/mL以上。对该酶性质的研究表明,酶分子量为34kD,最适作用pH为80,最适作用温度为80℃,具有良好的pH稳定性及热稳定性。Ca2+对该酶的稳定性具有重要影响,PMSF、DFP及IAA能强烈抑制酶活力,而DTT对该蛋白酶活力无影响     

枯草芽胞杆菌突变株ZC-7中性蛋白酶催化区域氨基酸突变导致其活力提高

目的：探讨枯草芽胞杆菌突变株ZC-7高产中性蛋白酶的原因。方法：用PCR方法分别扩增突变株ZC-7与出发菌株枯草芽胞杆菌AS1．398产中性蛋白酶的编码基因,测序比较二者基因的不同;在CPHmodels Server网站进行氨基酸序列分析,模拟突变前后中性蛋自酶的二级结构。结果：对比结果显示成熟肽中有5个氨基酸位点发生突变,其中3个位于酶的催化区域内;从预测的二级结构模型上可以看到突变位点所处区域的折叠结构发生细微变化。结论：先前研究中发现枯草芽胞杆菌AS1．398和突变株ZC-7发酵液中的酶蛋白含量基本相同,因此推测高产的原因不是酶量的增加,而是突变的氨基酸使酶与底物结合的部位更加适合催化水解反应,从而提高其比活力。     

Regulation of Neutral Protease Productivity in Bacillus subtilis: Transformation of High Protease Productivity

A transformable strain of Bacillus subtilis 6160, a derivative of B. subtilis 168, produces three kinds of casein hydrolytic enzymes (alkaline protease, neutral protease, and esterase) in a culture medium. B. natto IAM 1212 produces 15 to 20 times as much total proteolytic activity as does B. subtilis. Extracellular proteases produced by the two strains were separated into each enzyme fraction by diethylaminoethyl-Sephadex A-50 column chromatography. The difference in the total protease activities of extracellular proteases between the two strains was due to the amount of neutral protease. The ratios of neutral protease activity to alkaline protease activity (N/A) were 1.1 in B. subtilis 6160 and 13.0 in B. natto IAM 1212. Enzymological and immunological properties of alkaline protease and neutral protease obtained from the two strains were quite similar or identical, respectively. Specific activities measured by an immunological analysis of the two neutral proteases against casein were also equal. A genetic character of high protease productivity in B. natto IAM 1212 was transferred to B. subtilis 6160 by the deoxyribonucleic acid-mediated transformation. Among 73 transformants that acquired high protease productivity, 69 produced a higher amount of neutral protease and the ratios of N/A were changed to 15 to 60. Three other strains were transformed in the productivity of neutral protease and alpha-amylase simultaneously, and one showed considerable change in the production of alkaline protease and neutral protease. The specific activities (casein hydrolytic activities/enzyme molecules) of neutral proteases from the representative four transformants were equal to those of the two parental strains. These results suggested the presence of a specific gene(s) that participated in the productivity of neutral protease in B. subtilis.     

Studies on Bacterial Protease

A neutral protease of Bacillus subtilis var. amylosacchariticus was purified and crystallized by sequential chromatography on columns of Duolite A-2 anion-exchange resin, CM-cellulose and DEAE-sephadex A-50. The crystalline preparation was chromatographically homogeneous and confirmed to be monodispersive by physicochemical criteria. The enzyme was most active at near pH 7 against casein and stabilized by calcium salts. Some metalchelating agents and metal ions such as Hg?, Pb?, Cu? and Fe? markedly inactivated the enzyme, whereas diisopropyl phosphorofluoridate, sulfhydryl reagents and protease inhibitor of potato did not affect the activity. The neutral protease obtained here was rather stable as compared with the neutral protease ever reported and was able to be freeze-dried without any appreciable lose in enzyme activity.     

猪血蛋白的酶水解及氨基酸含量

以AS1.398中性蛋白酶、胰蛋白酶、木瓜蛋白酶和菠萝蛋白酶对猪血蛋白进行水解,AS1.398中性蛋白酶对猪血蛋白的水解能力最强。采用活性炭对酶水解液进行脱色。脱色后的酶水解液中含有18种氨基酸,必需氨基酸占总氨基酸的39.18%。     

Overexpression and characterization of a lipase from Bacillus subtilis

A novel plasmid, pBSR2, was constructed by incorporating a strong lipase promoter and a terminator into the original pBD64. A mature lipase gene from Bacillus subtilis strain IFFI10210, an existing strain for lipase expression, was cloned into the plasmid pBSR2 and transformed into B. subtilis A.S.1.1655. Thus, an overexpression strain, BSL2, was obtained. The yield of lipase is about 8.6 mg protein/g of wet weight of cell mass and 100-fold higher than that in B. subtilis strain IFFI10210. The recombinant lipase was purified in a three-step procedure involving ammonium sulfate fractionation, ion exchange, and gel filtration chromatography. Characterizations of the purified enzyme revealed a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, maximum activity at 43 degrees C and pH 8.5 for hydrolysis of p-nitrophenyl caprylate. The values of Km and Vm were found to be 0.37 mM and 303 micromol mg-1 min-1, respectively. The substrate specificity study showed that p-nitrophenyl caprylate is a preference of the enzyme. The metal ions Ca2+, K+, and Mg2+ can activate the lipase, whereas Fe2+, Cu2+, and Co2+ inhibited it. The activity of the lipase can be increased about 48% by sodium taurocholate at the concentration of 7 mM and inhibited at concentrations over 10 mM.     

A new method for the preparation of a calcium activated neutral protease highly sensitive to calcium ions

A Ca²⁺--activated neutral protease has been purified from chicken skeletal muscle to homogeneity by a new method which employs affinity chromatography on casein CH-Sepharose 4B. SDS polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single polypeptide chain with a molecular weight of 76,000. For half-maximum activity this protease requires 50 μM Ca²⁺ ions and its optimum pH is 7.6. The protease is inhibited by leupeptin, antipain, E-64 and endogenous inhibitor. The purified protease is very labile upon storage; after 3 days at 4°C no detectable activity remained.     

Purification and Properties of Mucor pusillus Acid Protease

The protease produced by Mucor pusillus was recovered from a wheat bran medium by treatment with ammonium sulfate, ethyl alcohol, gel filtration and ion-exchange chromatography. The yield of the enzyme was 55%. The overall increase in the specific activity of the protease was 34-fold. The purified protease was most active at pH 3.8 and 5.6 against hemoglobin and casein, respectively. Optimal hydrolysis of casein was observed at 55 C. The enzyme was stable from pH 3.0 to 6.0. Enzyme inactivated by metal ions was reactivated by ethylenediaminetetraacetate and o-phenanthroline. Reducing agents and thiol poisons had no effect on the protease, suggesting that free sulfhydryl groups were not required for enzyme activity. Diisopropyl fluorophosphate did not inhibit the protease, indicating the probable absence of serine in the active center. The Michaelis-Menten constant for casein was 0.357%. Electrophoretic analysis of active protein recovered by ion-exchange chromatography showed that the protease preparation was homogeneous.     

InhA-like protease secreted by Bacillus sp. S17110 inhabited in turban shell

A strain producing a potent protease was isolated from turban shell. The strain was identified as Bacillus sp. S17110 based on phylogenetic analysis. The enzyme was purified from culture supernatant of Bacillus sp. S17110 to homogeneity by ammonium sulfate precipitation, SP-Sepharose, and DEAE-Sepharose anion exchange chromatography. Protease activity of the purified protein against casein was found to be stable at pH 7 to pH 10 and around 50 degrees . Approximately 70% of proteolytic activity of the enzyme was detected either in the presence of 100 mM SDS or Tween 20. The enzyme activity was enhanced in the presence of Ca2+, Zn2+, Mg2+, but was inhibited by EDTA, indicating that it requires metal for its activity. The purified enzyme was found to be a monomeric protein with a molecular mass of 75 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The purified enzyme was analyzed through peptide fingerprint mass spectra generated from matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and a BLAST search, and identified as immune inhibitor A (inhA) deduced from nucleotide sequence of B. cereus G9241. Since InhA was identified as protease that cleave antibacterial proteins found in insect, inhA-like protease purified from Bacillus sp. S17110 might be pathogenic to sea invertebrates.     

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.