一种碱性γ-谷氨酰转肽酶在Bacillus subtilis 168中的分泌表达与酶学性质分析

将γ-谷氨酰转肽酶(GGT)在Bacillus subtilis 168同源过量表达,发现该酶能够自动分泌到细胞外,且重组菌酶活比原始菌提高了3 568倍。通过SDS-PAGE分析发酵液上清液发现,该γ-谷氨酰转肽酶是由一个相对分子质量为42 000的大亚基和一个22 000的小亚基组成。通过酶学性质分析发现,该γ-谷氨酰转肽酶是一个耐碱性的酶,最适反应pH为10;同时该酶具有良好的耐热性,最适反应温度达到50℃;另外,加入5.0 mmol/L的Mg^(2+)、Ca^(2+)、K^+、La^(3+)、Li^+和NH_4^+对GGT转肽活力具有明显的促进作用,其中添加的NH_4^+对GGT活力促进作用最为显著,GGT活力提高45%以上,而Cu^(2+)和Zn^(2+)则对其具有抑制作用。

Postharvest Biological Control of Apple Anthracnose by Bacillus subtilis BS80-6,A Mutant Strain through Ion Implantation

The original Bacillus subtilis was isolated from the soil of an apple orchard,and B. subtilis strain BS80-6,selected from Bacillus subtilis by ion implantation,was used as an antagonist for postharvest biological control against apple anthracnose（ colletotrichum gloeosporioides）. The mechanisms of action and efficacy of Bacillus subtilis strain BS80-6 against apple anthracnose caused by colletotrichum gloeosporioides were studied in vitro and on apples in controlled and semi-commercial conditions. An application of cell suspension（ 108 cells per mL） of the antagonist in artificial wounds of apples reduced growth of C. gloeosporioides after storage at different temperatures. The inhibitory actions of cell culture to hyphal growth and to spores germination were 86. 6% and 98. 65%,respectively,and the control efficacy of cell culture against the disease was 60. 34% at room temperature. The results showed that BS80-6 was more effective against apple anthracnose.The cell culture of BS80-6 received better control efficacy against apple anthracnose than culture filtrates and autoclaved cell. All treatments significantly inhibited the disease both in vitro and in vivo compared with control. Fruits treated with cell culture and stored at 10 ℃ had lower infection rate,more delayed formation of acervwlus and smaller lesion diameter than those stored at 20 ℃. There was better control efficacy in fruits inoculated with spores after application of BS80-6. The mechanisms of BS80-6 activity appeared to involve production of an antifungal substance,deformation of hyphal cell and disintegration of cell wall. Besides,BS80-6 could improve the activity of major defense enzymes of apple,such as peroxidase（ POD） and polyphenol oxidase（ PPO）.

枯草芽孢杆菌YT168-6产抗菌肽sublancin的发酵工艺优化及培养基筛选的研究

研究旨在探索枯草芽孢杆菌YT168-6产抗菌肽sublancin的最适发酵条件和最适培养基组成,以期提高发酵液中sublancin含量。试验先通过单因素试验筛选出最适发酵条件,确定发酵培养基需要的无机盐、碳源、氮源,再通过正交试验、最陡爬坡试验、Box-Behnken设计的响应面分析方法确定最适发酵培养基组成。结果表明:枯草芽孢杆菌YT168-6产抗菌肽sublancin的最适发酵条件为菌种接种量1.0%、发酵温度37℃、发酵液初始pH 7.0;最适发酵培养基为K_(2)HPO_(4)5 g/L、KH_(2)PO_(4)5 g/L、MgSO_(4)10 g/L、可溶性淀粉30.2 g/L、蛋白胨23.6 g/L、玉米浆18.2 g/L;优化后sublancin产量由756μg/mL提到1581μg/mL,是优化前的2.09倍。综上所述,本研究所筛选的最适发酵条件和最适培养基可为枯草芽孢杆菌YT168-6菌株工业化生产sublancin提供技术参考。

不同来源寡聚-1,6-葡糖苷酶异源表达及酶学特性

寡聚-1,6-葡糖苷酶(EC 3.2.1.10)对于乳酸发酵液残糖中含量较高的异麦芽糖有较高的利用活性。基于前期研究工作基础并经BRENDA数据库搜索挑选了5个可能耐高温、耐酸的寡聚-1,6-葡糖苷酶基因(Bacillus coagulans ATCC 7050的malL BF292011(BC1)和malL BF292004(BC2)、Bacillus subtilis 168的malL yvdL BSU34560(BS1)和yugT BSU31290(BS2)以及Bacillus thermoglucosidasius KP 1006的malL(KP)),首次利用pET表达系统在大肠杆菌中进行重组表达、纯化及酶学性质的系统研究。结果显示:BC1、BC2、KP的最适pH为5.0,BS1、BS2的最适pH为6.0。最适温度分别为:KP(60℃),BC1(56℃),BC2(56℃),BS1(43℃),BS2(43℃)。Mg^(2+)、NH_(4)^(+)对于BC1的酶催化活性具有激活作用,Co^(2+)、K+、NH_(4)^(+)对于KP有激活作用,但这些离子对于BC2、BS1和BS2都没有明显的激活作用。Zn^(2+)、Cu^(2+)、Ni^(+)对这5种寡聚-1,6-葡糖苷酶都有明显的抑制作用。以对硝基苯-α-D-葡萄糖苷(pNPG)为底物时,酶的转换数和催化效率从高到低依次为:KP>BC1>BS1>BS2>BC2。以乳酸发酵液中的残糖成分作为参考,在以麦芽糖、异麦芽糖、蔗糖、乳糖、海藻糖以及可溶性直链淀粉作为底物测定酶活时,BC1、BC2、KP、BS1的最适底物都是异麦芽糖,BS2的最适底物为麦芽糖。