金属离子对蜡状芽孢杆菌合成多聚γ-谷氨酸的影响

研究金属离子对蜡状芽孢杆菌合成多聚γ-谷氨酸(γ-PGA)的影响。通过在培养基中加入定量的Mn2+、K+、Fe3+、Mg2+、Ca2+、Mo6+、Zn2+、Co2+、Cd2+、Cu2+,定时取发酵液用原子吸收分光光度法测定蜡状芽孢杆菌合成γ-PGA过程中金属离子的消耗情况、菌体生物量及γ-PGA产量;在发酵培养基中,分别加入不同质量浓度的Mn2+、Ca2+、Zn2+、Co2+、Mo6+,通过组合不同金属离子以及控制各组合中离子质量浓度测定菌体生物量和γ-PGA含量的变化,结合金属离子的消耗量探讨金属离子对蜡状芽孢杆菌合成γ-PGA的影响。结果表明：在蜡状芽孢杆菌合成γ-PGA的过程中均消耗一定量的Mn2+、K+、Fe3+、Mg2+、Ca2+、Mo6+、Zn2+、Co2+,其中Mn2+、K+、Fe3+、Mg2+、Ca2+在生长代谢过程中利用较多,而Cd2+、Cu2+的质量浓度不变化;Mn2+是γ-PGA合成的必需元素,在其质量浓度为0.08g/L时,γ-PGA合成达最高峰(1.25g/L)。当Mn2+质量浓度为0.08g/L时,适当增加Ca2+质量浓度就能较大幅度提高γ-PGA合成量;同时固定Mn2+、Ca2+质量浓度,适量加入不同质量浓度的Zn2+、使γ-PGA的合成量增加明显,最高可达2.94g/L,而Mo6+对γ-PGA的合成影响不明显,但对菌体生物量的影响较大。     

Isolation and characterization of heat stable proteinases from Pseudomonas isolate AFT 21

Pseudomonas strain AFT 21 produced three heat stable extracellular proteinases in milk and nutrient broth at 7 or 21 degrees C, but the proportions depended on medium and cultivation temperature. The three proteinases were EDTA- and o-phenanthroline-sensitive metalloenzymes and were not inhibited by N-ethylmaleimide or phosphoramidon. Proteinases I and II showed maximum activity at pH 7-7.5 and proteinase III at pH 8.5. All three enzymes showed maximum activity at 45-47.5 degrees C, but had relatively high (19-27% of maximum) activity at 4 degrees C. They were unstable at 55 degrees C in phosphate buffer, pH 6.6, or synthetic milk ultrafiltrate (SMUF) containing 12 mmol Ca2+, but were stabilized by short preheating at 100 degrees C. They were extremely heat stable in both phosphate buffer and SMUF, pH 6.6, at 70-150 degrees C. Their D-values at 140 degrees C were 69, 54 and 80 s respectively. The Z-values for Pseudomonas AFT 21 proteinase III in phosphate buffer and SMUF were 29.7 and 30.3 degrees C respectively; the corresponding activation energies for inactivation were 8.7 x 10(4) J mol-1 and 9.2 X 10(4) J mol-1.     

中国对虾蛋白酶性质的研究

采用酶学分析方法研究了中国对虾蛋白酶的性质。结果表明,该酶最适温度为60℃,最适pH为9.0,是一种碱性蛋白酶。此酶在50℃以下及pH 7.0~10.0稳定性较好。以酪蛋白为底物,蛋白酶的最适底物浓度为2%。60℃时,该酶的半衰期为22 min,70℃时则为7 min。低浓度的EDTA对酶活有抑制作用,Mn2+、Ca2+、Mg2+对蛋白酶有一定的激活作用,而Cu2+、Zn2+、Fe2+、Pb2+对酶活有不同程度的抑制作用,可推断该酶是一种金属蛋白酶。     

不同金属离子对多酚氧化酶的影响

采用匀浆法从茄子中提取多酚氧化酶(PPO),以绿茶粉为底物,研究Cu2+、Mn2+、Mg2+、Ca2+、Na+、Al3+和Fe3+7种金属离子对PPO活性的影响。结果表明:Cu2+、Mn2+和Mg2+3种金属离子对PPO的活性有不同程度的促进作用,而Ca2+、Na+、Al3+和Fe3+对PPO的活性有抑制作用。其中,Cu2+浓度为3mmol/L时对PPO有激活作用,此时茶黄素总量达到7.45mg;Fe3+浓度为5mmol/L对PPO有显著抑制作用,此时茶黄素总量降低至0.26mg。     

Purification and characterization of intracellular proteinase from Lactobacillus casei ssp. casei LLG

The intracellular proteinase of Lactobacillus casei ssp. casei LLG was isolated in the cytoplasmic fraction with 0.05 M Tris-HCl buffer (pH 7.5). The enzyme was purified by the fast protein liquid chromatography system equipped with ion-exchange and gel filtration chromatographies. This proteinase comprised a single monomeric form and had a molecular weight of about 55 kDa and an isoelectric point near pH 4.9. The optimum pH and temperature for the enzyme activity were determined to be pH 6.5 and 37 degrees C, respectively. The enzyme was inactivated by metal-chelating compounds (EDTA, 1,10-phenanthroline) and less affected by serine proteinase inhibitors (diisopropylfluorophosphate, phenylmethylsulfonyl fluoride). Proteinase activity was increased by Ca++, Mn++, and Co++, and inhibited by Cu++, Mg++, and Zn++. The activity of this enzyme to hydrolyze casein appeared to be more active on beta-casein than alphas1-casein and kappa-casein as monitored by polyacrylamide gel electrophoresis.     

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

An extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatography on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43,000 and contained 2 g atom Ca/mol. It was active over a pH range 4.8-9.5 and had optimum activity on casein at pH 7.0 with Km = 0.17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 degrees C; above 50 degrees C activity declined rapidly, but significant activity persisted at 4 degrees C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.     

鸭卵清溶菌酶的分离纯化及性质

通过硫酸铵的分级沉淀、CM-Sepharose阳离子交换层析、Superdex-200凝胶过滤层析等步骤,从鸭卵清中获得电泳纯的溶菌酶,该酶的比活力达到33687.26U/mg,纯化倍数为109.44,回收率为28.00%。测得该酶分子质量约为14.82kD,对溶壁微球菌的最适反应温度为50℃,最适pH值为7,且在50℃以下及pH5～9有较好的稳定性,同时在最适条件下测得其Km值为0.0864mg/mL。Fe2+、Mg2+、Mn2+等金属离子对该酶有较强的抑制作用,而Zn2+、Cu2+、Co2+对该酶有一定的激活作用。     

大豆胰蛋白酶抑制剂的异源表达与生化特性解析

本文对一疑似大豆胰蛋白酶抑制剂(STI;sti)的开放阅读框在毕赤酵母中进行了克隆表达与生化特性分析。结果表明,在摇瓶水平上,重组菌GS115(pPIC-STI)分泌表达了30 mg/L STI;重组STI在(40~80)℃或pH 2.0~11.0孵育1 h后,仍能保持85%以上的抑制活性;K+、Zn2+和Mg2+对其胰酶抑制活性有明显的激活作用,而Cu2+、Mn2+、Ca2+、Fe2+和Fe3+则有明显的抑制作用;重组STI对胰蛋白酶具有较强的、专一性的和非竞争性的抑制作用,是一种典型的多肽类胰酶抑制剂。良好的酶学性质使STI在食品、医药等行业具有潜在的应用价值。     

Purification and some properties of sodom-apple latex proteinases

Two thiol-activated proteinases with isoelectric points (pIs approximately 9–9·5) were purified from sodom-apple latex by chromatography on Q-Sepharose and Superdex 200. Proteinase I had an estimated molecular weight of approximately 25 000 and proteinase II one of about 30 000. The proteinases degraded casein and azocoll, proteinase I having a lower specific activity than proteinase II. Proteinase I was most active at pH 8–10, with the optimum at about pH 8. Proteinase II was most active at pH 6–8, with the optimum at about pH 7.     

低浓度金属离子体外抗病毒研究

采用细胞病变效应(CPE)抑制实验观察和分析低浓度Zn2+、Cu2+和Mn2+金属离子对病毒的抑制作用。低浓度Zn2+在体外有较好的抑制单纯疱疹病毒、呼吸道合胞病毒和副流感病毒的致病作用;低浓度Cu2+和Mn2+在体外有较好的抑制副流感的致病作用。研究表明,将金属离子进行复合不但可以提高抑制作用,而且可以降低单一离子的浓度。     

Substrate specificity of a tripeptidase as a metalloenzyme purified from Lactococcus lactis subsp. lactis biovar. diacetylactis ATCC 13675

The peptidase purified to homogeneity from Lactococcus lactis subsp. lactis biovar. diacetylactis ATCC 13675 was considered to be an aminotripeptidase (EC 3.4.11.4) from the results of substrate specificity. The K(m) value showed a tendency to decrease with the number of alanine residues, but to increase with the number of glycine residues in the substrate tripeptide. The effects of divalent metal ions on enzyme activity were considerably different depending on the tripepride used as a substrate. In the case of Mn2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+, there was apparent correlation between enzyme activities observed in the presence of metal ions and following metal ion replacement. The Zn2+-replaced enzyme showed almost the same K(m) and k(cat) values as the native enzyme, suggesting the enzyme to be a zinc metallopeptidase. The K(m) of the divalent metal-replaced enzyme increased in the order of Co2+, Zn2+, and Mn2+. As a result of replacement with Co2+ an enzyme having 2.3-fold higher activity compared to the native enzyme for GGF as a substrate was obtained. Thus, the change in substrate specificity observed following metal replacement may suggest a highly specific interaction between the enzyme, metal and substrate, leading to the activity expression and stability of the tripeptidase.     

产气肠杆菌B19中β-甘露聚糖酶基因的克隆表达及酶学性质研究

将产气肠杆菌B19中的β-甘露聚糖酶基因(ManE)进行克隆,获得全长ManE基因序列,将ManE基因与pET28a(+)表达载体连接,转入大肠杆菌BL21(DE3)中,构建重组菌株BL21(DE3)-pET-28a(+)-ManE,并成功表达了重组β-甘露聚糖酶。结果表明:克隆得到ManE基因序列全长为2196 bp,编码731个氨基酸。通过Ni柱亲和层析法分离纯化得到纯度较高的重组β-甘露聚糖酶,其分子量大小约为82.5 ku。重组β-甘露聚糖酶的酶学性质研究结果显示:最适反应温度和最适pH分别为55℃和6.5,在温度50~55℃,pH4.0~7.0的范围内都能保持较好的稳定性。1 mmol/L浓度的Co2+、Mn2+、Zn2+、Ba2+和Ca2+对重组β-甘露聚糖酶的酶活有不同程度的激活作用。而K+、Mg2+和Cu2+对该重组酶有不同程度的阻碍作用,其中Cu2+对该重组酶催化活性的抑制作用最强,酶活降低到最大酶活力的65.3%。本研究实现了β-甘露聚糖酶的异源表达,这些结果为该酶的进一步工业化应用提供了重要的理论依据。     

Peptidases from two strains of Pseudomonas fluorescens: partial purification, properties and action on milk

Pseudomonas fluorescens strains 240 and 32A expressed cell-associated peptidase activity which was shown by subcellular fractionation to be primarily intracellular. Two peptidases were partly purified from strain 32A. One specifically hydrolysed N-alpha-benzoyl-DL-arginine-4-nitroanilide and was termed endopeptidase and the other hydrolysed L-lysine- and L-leucine-4-nitroanilide and was termed aminopeptidase. The endopeptidase had very low activity on bovine serum albumin compared with that of trypsin and probably was not a proteinase. The endopeptidase had a mol. wt of 33,000 and a pH optimum of 8.0. The enzyme was stimulated by Ca2+ and Mg2+ and inhibited by Co2+, Mn2+, Hg2+, Zn2+ and leupeptin. Soya bean trypsin inhibitor and phenylmethane sulphonyl fluoride (PMSF) had no effect on its activity. The aminopeptidase had a mol. wt of 44,000 and a pH optimum of 8.0. It was inhibited by all the metal ions mentioned above and by PMSF. Little proteolysis was found when ultra high temperature (UHT) sterilized milk was treated with cell-free extract from strain 32A. It was concluded that the cell-associated peptidases from Pseudomonas strains normally present in raw milk may not contribute significantly to the deterioration of UHT sterilized milk.     

高产β-环糊精葡萄糖基转移酶菌株的筛选、产酶条件优化及酶学性质研究

豉香型白酒肥猪肉浸制和酝浸阶段会析出脂肪油。该研究通过定量分析脂肪油样本中的长链、中链脂肪酸和二元酸含量，推论其氧化产物的产生机理。结果表明，脂肪油的主要成分为饱和脂肪酸（SFA）和单不饱和脂肪酸（MUFA），而多不饱和脂肪酸（PUFA）含量均少于70 g/kg。脂肪油氧化程度越深，PUFA含量越少，中链脂肪酸和二元酸等氧化产物的含量越多，其最高含量分别达4.52 g/kg和3.02 g/kg。随着脂肪油氧化程度的加深，油酸氧化产物如辛酸和癸酸逐渐成为主要的中链脂肪酸，而壬二酸则始终是含量最多的二元酸。该成果可为后续肥肉浸制工艺创新提供理论基础。     

托柄菝葜果色素提取及其性质研究

以民族药托柄菝葜的成熟果实为材料,对其红色素的提取条件及理化性质进行研究.结果表明,托柄菝葜果色素提取的最佳条件为:40％vol乙醇水溶液、浸提温度60℃、浸提时间lh.托柄菝葜果色素在自然光照下稳定,但添加金属离子的色素溶液对光不稳定;色素溶液在酸性环境中呈现鲜艳的橙红色,在中性及碱性环境中变为褐绿色.VC对托柄菝葜色素具有增色作用;柠檬酸、EDTA对色素无明显影响;亚硫酸钠、高锰酸钾及苯甲酸钠对色素具有减色作用.金属离子Ca2+、Mn2+、Zn2+、K+、Na+、Fe2+、Mg2+、Cu2+、Co2+、Al3+、Pb2+等离子能减弱托柄菝葜果色素对光的稳定性;Sn2+、Fe3+影响色素稳定性.     

芽枝霉菌Lcxs9产两种耐热β-葡萄糖苷酶的快速纯化及酶学特性研究

以高产β-葡萄糖苷酶的芽枝霉菌（Cladosporium cladosporioides）Lcxs9为研究对象,对其进行固态发酵,采用硫酸铵沉淀及强阴离子交换柱对其所产β-葡萄糖苷酶进行分离纯化,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测其分子质量,并研究其酶学性质。结果表明,快速纯化获得两种β-葡萄糖苷酶,分别命名为BG CC1和BG CC2,分子质量分别为41 kDa、53 kDa,比酶活力分别为8.6 U/mg、12.2 U/mg。两种酶都具有水解活性,可以水解纤维二糖得到葡萄糖;同时具有转苷活性,可以利用低分子质量的单糖合成纤维三糖和纤维四糖。两种酶的最适作用pH及温度均分别为6.0、60 ℃,均在pH 4～10和20～80 ℃条件下较稳定,Ag2+、Co2+、Cu2+、Hg2+对两种酶均有抑制作用,而Mn2+、Ca2+和Mg2+均有明显的激活作用,Zn2+和Ni+无明显影响。利用4-硝基苯基-β-D-吡喃葡萄糖苷（pNPG）为底物,两种酶的动力学常数Km和最大反应速率（Vmax）均分别为2.76 mg/mL、20.6 U/mg。     

Interaction of inorganic arsenic with biogenic manganese oxide produced by a Mn-oxidizing fungus, strain KR21-2

In batch culture experiments we examined oxidation of As(III) and adsorption of As(III/V) by biogenic manganese oxide formed by a manganese oxide-depositing fungus, strain KR21-2. We expected to gain insight into the applicability of Mn-depositing microorganisms for biological treatment of As-contaminated waters. In cultures containing Mn2+ and As(V), the solid Mn phase was rich in bound Mn2+ (molar ratio, approximately 30%) and showed a transiently high accumulation of As(V) during the early stage of manganese oxide formation. As manganese oxide formation progressed, a large proportion of adsorbed As(V) was subsequently released. The high proportion of bound Mn2+ may suppress a charge repulsion between As(V) and the manganese oxide surface, which has structural negative charges, promoting complex formation. In cultures containing Mn2+ and As(III), As(III) started to be oxidized to As(V) after manganese oxide formation was mostly completed. In suspensions of the biogenic manganese oxides with dissolved Mn2+, As(III) oxidation rates decreased with increasing dissolved Mn2+. These results indicate that biogenic manganese oxide with a high proportion of bound Mn2+ oxidizes As(III) less effectively than with a low proportion of bound Mn2+. Coexisting Zn2+, Ni2+, and Co2+ also showed similar effects to different extents. The present study demonstrates characteristic features of oxidation and adsorption of As by biogenic manganese oxides and suggests possibilities of developing a microbial treatment system for water contaminated with As that is suited to the actual situation of contamination.     

一株产高活性多酚氧化酶菌株筛选鉴定及其酶学性质

采用变色圈法从土壤样品中分离筛选产多酚氧化酶的菌株,对其进行形态学观察、分子生物学鉴定,并对其所产多酚氧化酶的酶学性质进行研究。结果表明,筛选得到1株产多酚氧化酶活性较高的菌株,命名为ZL-2,其被鉴定为竹黄菌属（Shiraia sp.）。菌株ZL-2发酵8 d产多酚氧化酶酶活最高,达到521 U/mL。菌株ZL-2产多酚氧化酶最适底物为邻苯二酚,其最适pH值为6、最适温度为30 ℃;酶活在温度20～40 ℃及pH 3～6范围内稳定。金属离子Cu2+、Mg2+、Mn2+、Fe3+、Zn2+对多酚氧化酶都有激活作用,其中Cu2+激活作用最强;Ca2+、Al3+对酶活有一定抑制作用;L-抗坏血酸和亚硫酸氢钠对该酶抑制作用较强。     

紫色红曲霉中新型酯酶ESM1的纯化及其酶学特性研究

该研究以白酒大曲中筛选出的一株高产酯酶菌株HQ为研究对象,采用分子生物学对其进行菌种鉴定。通过硫酸铵二级沉淀、丁基琼脂糖凝胶柱和Superdex G-200柱对其所产的酯酶进行纯化,并进行N-端序列、质谱和酶学性质分析。结果表明,菌株HQ被鉴定为紫红曲霉（Monascus purpureus）,经纯化获得一个分子质量为60 kDa的酯酶,命名为ESM1。酯酶ESM1具有一段酸性蛋白酶的肽段序列,最适温度为60 ℃、最适pH值为7.0,在温度为50 ℃的环境和中性环境中表现出良好的稳定性。Zn2+、Ca2+和Na+对ESM1有促进作用,其中Zn2+促进作用最高,Cu2+、Mg2+、Fe2+和Mn2+有抑制作用,其中Cu2+的抑制作用最强。     

杂优-2平菇漆酶的分离纯化及酶学性质

将杂优-2平菇菌丝进行液体培养,发酵液经硫酸铵分级沉淀、DEAE（diethylaminoethyl）-Sepharose fastflow层析和Superdex-200 prep grade层析等方法纯化,获得了电泳纯的杂优-2平菇漆酶,并对纯化的漆酶进行了部分酶学性质研究。结果显示,杂优-2平菇漆酶比活力为115 U/mg,分子质量约为244.0 kD,亚基分子质量约为85.6 kD。最适反应pH值和最适反应温度分别为5.0和55 ℃,在pH 6.0～8.0及40～55 ℃范围内稳定性较好;最适条件下,以2,2’-联氮-二（3-乙基苯并噻唑-6-磺酸）为底物的Km值为2.1 mmol/L,最大反应速率（vmax）为0.117 μmol/（min·L）。Fe2＋、抗坏血酸对该酶活性具有完全抑制作用,乙二胺四乙酸、Ag＋、Mg2＋、Li＋对该酶活性影响较小;草酸、甲醇、正丁醇、K＋、Ca2＋、Ba2＋、Zn2＋、Cd2＋、Pb2＋、Mn2＋、Co2＋对该酶活性有不同程度的抑制作用;Cu2＋激活作用不明显;尿素、乙醇、异丙醇对该酶活性具有激活作用。