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耐盐性谷氨酰胺酶在Bacillus subtilis 168中的整合表达 被引量:1
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作者 徐朝阳 张显 +2 位作者 徐美娟 杨套伟 饶志明 《食品与生物技术学报》 CAS CSCD 北大核心 2020年第6期22-29,共8页
微生物质粒过表达外源基因,会由于质粒分离稳定性低、种子培养基中需添加抗生素等原因,限制其在工业生产中的应用。为了得到一株可用于工业生产谷氨酰胺酶的菌株,选择食品安全级Bacillus subtilis 168作为宿主,将来源于Micrococcus lute... 微生物质粒过表达外源基因,会由于质粒分离稳定性低、种子培养基中需添加抗生素等原因,限制其在工业生产中的应用。为了得到一株可用于工业生产谷氨酰胺酶的菌株,选择食品安全级Bacillus subtilis 168作为宿主,将来源于Micrococcus luteus K-3的编码耐高浓度盐的谷氨酰胺酶基因(Mglu)插入到其染色体上进行整合表达。选择了2个内源性蛋白酶基因作为整合表达位点,通过lox序列的重组消除抗性基因,得到无抗性基因的重组谷氨酰胺酶表达菌株。遗传稳定性研究表明,在不添加抗生素的条件下,通过连续传代重组菌株并测定发酵酶活,发现重组菌株连续传42代时,酶活基本不变。随后,采用5 L发酵罐对重组菌株进行分批补料发酵,最高酶活达到了41.5 U/mL。本研究对提高谷氨酰胺酶重组菌株遗传稳定性提供了借鉴。 展开更多
关键词 谷氨酰胺酶 bacillus subtilis 168 整合表达 蛋白酶 遗传稳定性
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Expression of a Lysine-rich Gene in Bacillus subtilis 168 被引量:1
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作者 Li Xue-lin Liu Xiao-fei +3 位作者 Gao Xue-jun Qiao Bin Pan Hong-bao Ao Jin-xia 《Journal of Northeast Agricultural University(English Edition)》 CAS 2013年第1期43-48,共6页
Lysine-rich protein gene (lys) was cloned from winged bean (Psophocarpus tetragonolobus (L.) DC), and cloned into prokaryotie expression vector pHT43, the recombinant plasmid pHT43/lys were constructed and then ... Lysine-rich protein gene (lys) was cloned from winged bean (Psophocarpus tetragonolobus (L.) DC), and cloned into prokaryotie expression vector pHT43, the recombinant plasmid pHT43/lys were constructed and then transferred into Bacillus subtilis168, upon IPTG induction, the recombinant protein was expressed, and the content of lysine was detected by HPLC. The result showed that lysine content increased by 9.85%. It was suggested that introducing lys gene into Bacillus subtilis 168 was an effective way to improve its nutrition quality. 展开更多
关键词 lysine-rich protein gene bacillus subtilis 168 lysine content HPLC
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不同来源寡聚-1,6-葡糖苷酶异源表达及酶学特性 被引量:1
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作者 华烨 刘鹏 王永红 《华东理工大学学报(自然科学版)》 CAS CSCD 北大核心 2021年第4期445-454,共10页
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 BF29201... 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。 展开更多
关键词 寡聚-1 6-葡糖苷酶 bacillus coagulans ATCC 7050 bacillus subtilis 168 bacillus thermoglucosidasius KP 1006
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Application of Protein Feed Processed by Microbial Fermentation to Dairy Cow 被引量:9
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作者 Sun Zhe Liu Ying +1 位作者 Pan Hong-bao Gao Xue-jun 《Journal of Northeast Agricultural University(English Edition)》 CAS 2014年第1期39-44,共6页
Methionine (Met) and lysine (Lys) have been reported as the first two limiting amino acids (AA) for maximum milk yield and milk protein production. Supplying these AA may improve microbial protein synthesis and ... Methionine (Met) and lysine (Lys) have been reported as the first two limiting amino acids (AA) for maximum milk yield and milk protein production. Supplying these AA may improve microbial protein synthesis and therefore improve milk production without adding excess N to the environment. This observation utilized fermented soybean meal (SBM), cottonseed meal (CSM), rapeseed meal (RSM) and corn by Bacillus subtilis 168 and Leuconostoc mesenteroides as core feedstuffs to produce special biological protein feed for dairy cow. The results showed that the milk production, milk protein percentage, milk fat percentage and milk DM percentage of test groups in trial period were significantly more than those of the control group (P〈0.01), the results showed that adding fermenting protein feed in dairy cow diets could significantly improve milk yield, milk protein and milk fat content. The economic benefits of actual application were analyzed, the group of 0.5% was the best compared with the other groups. 展开更多
关键词 soybean meal cottonseed meal rapeseed meal corn fermentation bacillus subtilis 168 Leuconostoc mesenteroides methionine lysine
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N端截短CBM41对枯草芽孢杆菌来源普鲁兰酶酶学性质的影响 被引量:1
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作者 付巧 林啟兰 +2 位作者 薛强 熊海容 王亚伟 《生物技术通报》 CAS CSCD 北大核心 2022年第6期245-251,共7页
采用N端截短方式对Bacillus subtilis 168来源普鲁兰酶进行蛋白结构改造,构建不同形式的截短突变体,考察N端结构对酶学特性的影响。利用基因工程的手段分别删去CBM41结构域N端前2、4和6个氨基酸,获得突变体M1(ΔN2)、M2(ΔN4)和M3(ΔN6)... 采用N端截短方式对Bacillus subtilis 168来源普鲁兰酶进行蛋白结构改造,构建不同形式的截短突变体,考察N端结构对酶学特性的影响。利用基因工程的手段分别删去CBM41结构域N端前2、4和6个氨基酸,获得突变体M1(ΔN2)、M2(ΔN4)和M3(ΔN6)。3种突变体最适反应温度(40-45℃)和最适pH(6.0)均与WT一致,WT、M1、M2和M3的T_(m)值分别为48.57℃、50.03℃、48.43℃和49.50℃,M1、M2和M3的比活力均高于野生型,是WT的1.18、1.60和2.44倍,WT、M1、M2和M3的K_(m)值分别为23.89、29.01、17.29和19.08 mg/mL。上述结果表明,通过截短普鲁兰酶N端氨基酸可获得性质得到改良的突变体,为提高该酶的热稳定性、比活力和底物结合能力提供新的方法和思路。 展开更多
关键词 N端截短 普鲁兰酶 bacillus subtilis168 酶学特性
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