不同信号肽及分子伴侣对中性蛋白酶在枯草芽孢杆菌中分泌表达的影响

doi:10.13560/j.cnki.biotech.bull.1985.2017-0989

摘要/Abstract

摘要： 通过筛选5个不同的信号肽基因（AmyE、AprE、NprE、SacB、YncM）代替对照质粒pHT43-npr上原始的信号肽基因，整合2个不同的分子伴侣（PrsA、DnaK）到pHT43-npr质粒上，构建重组质粒并转化到枯草芽孢杆菌WB800N中进行诱导表达。结果表明，构建了5株信号肽重组菌株，通过牛奶平板验证无明显水解圈，通过酶活性比较，5株重组菌株中性蛋白酶活性均显著低于对照菌株；成功构建了2株整合分子伴侣的重组菌株，通过牛奶平板验证有水解圈，通过酶活性比较，两株菌株均表达中性蛋白酶，其中重组菌株WB800N/pHT43-npr-PrsA产蛋白酶活性比对照菌株WB800N/pHT43-npr提高了23%，重组菌株WB800N/pHT43-npr-DnaK产蛋白酶活性比对照菌株提高了33%。

关键词: 中性蛋白酶, 信号肽, 分子伴侣, 枯草芽孢杆菌

Abstract: By screening five different signal peptides to replace the original signal peptide of the plasmid pHT43-npr and integrating two different molecular chaperones（PrsA and DnaK）to the plasmid pHT43-npr，we constructed recombinant plasmids and then transformed them to Bacillus subtilis WB800N to have induced expressions. As a result，five recombinant strains with new signal peptide were constructed，no hydrolyzed circles emerged via milk plate testing，and all the five strains’ enzyme activities were obviously lower than that by control strain. We also successfully constructed two recombinant strains integrated the molecular chaperones，both of the two strains emerged hydrolyzed circles in milk plate testing. Both strains had neutral protease expressed，and the enzyme activity of recombinant strain WB800N/pHT43-npr-PrsA increased 23%，and the enzyme activity of recombinant strain WB800N/pHT43-npr-DnaK increased 33%，compared with control strain WB800N/pHT43-npr.

Key words: neutral protease, signal peptide, molecular chaperone, Bacillus subtilis

杨何宝 ,胡美荣 ,郑翔 ,牟庆璇 ,高沛汝. 不同信号肽及分子伴侣对中性蛋白酶在枯草芽孢杆菌中分泌表达的影响[J]. 生物技术通报, 2018, 34(6): 134-140.

YANG He-bao ,HU Mei-rong ,ZHENG Xiang ,MOU Qing-xuan ,GAO Pei-ru. Effects of Different Signal Peptides and Their Molecular Chaperones on the Secretion of Neutral Protease in Bacillus subtilis[J]. Biotechnology Bulletin, 2018, 34(6): 134-140.

参考文献

[1] Saran S, Mahajan RV, Kaushik R, et al. Enzyme mediated beam house operations of leather industry：a needed step towards greener technology[J] . Journal of Cleaner Production, 2013, 54（9）：315-322.
[2] Kumar MM, Saranraj P. Commercial production and application of bacterial alkaline protease：an overview[J] . Quaternary Science Reviews, 2015, 3（34）：1-23.
[3] 张玉红, 刘萌, 但卫华, 等. 4 种典型脱毛工艺的对比（续）[J] . 中国皮革, 2015, 44（14）：20-23.
[4] Pandeeti EV, Pitchika GK, Jotshi J, et al. Enzymatic depilation of animal hide：Identification of elastase（LasB）from Pseudomonas Aeruginosa MCM B-327 as a depilating protease[J] . PLoS One, 2011, 6（2）：e16742.
[5] 于士强, 桂俊鸿, 王海燕. 短小芽孢杆菌胞外蛋白质组双向电泳分析及碱性胁迫下胞外差异蛋白鉴定[J] . 应用与环境生物学报, 2014, 20（2）：217-222.
[6] Ohmura K, Shiroza T, Nakamura K, et al. A Bacillus subtilis secretion vector system derived from the Bacillus subtilis α-amylase promoter and signal sequence region, and secretion of Escherichia coli β-lactamase by the secretion vector system[J] . J Bio chem, 1984, 95：87.
[7] He XS, Bruckner R, Doi RH. The protease genes of Bacillus subtilis[J] . Res Microbiol, 1991, 142：797.
[8] Palva I, Sarvas M, Lehtovaara P, et al. Secretion of Escherichia coli β-lactamase from Bacillus subtilis by the aid of α-amylase signal sequence[J] . Proc Natl Acad Sci USA, 1982, 79：5582.
[9] Breitling R, Gerlach D, Hartmann M, et al. Secretory expression in Escherichia coli and Bacillus subtilis of human interferon αgenes directed by staphylokinase signals[J] . Mol Gen Genet, 1989, 217：384.
[10] Palva I, Sarvas M, Lehtouaara P, et al. Production and secretion of pertussis toxin subunits in Bacillus subtilis[J] . FEMS Microbiol Lett, 1990, 68：143-148.
[11] Zhang JH, Wu D, Chen J, et al. Enhancing functional expression of β-glucosidase in Pichia pastoris by co-expressing protein disulfide isomerase[J] . Biotechnol Bioproc E, 2011, 16（6）：1196-1200.
[12] Wu M, Liu W, et al. Engineering of a Pichia pastoris expression system for high-Level secretion of HSA/GH fusion protein[J] . Appl Biochem Biotechnol, 2014, 172（5）：2400-2411.
[13] 郭永华, 陈济琛, 贾宪波等. 分子伴侣共表达对嗜热环糊精葡萄糖基转移酶异源可溶性表达的影响[J] . 微生物学通报, 2016, 43（3）：518-526.
[14] 张网罗. 铜绿假单胞菌甲酸脱氨酶的分子伴侣共表达、纯化及酶活检测[D] . 上海：复旦大学, 2013. 
[15] Nagarajan N, Ramaley R, Albertson H, et al. Secretion of streptavi-din from Bacillus subtilis[J] . Applied and Environmental Microbiology, 1993, 59（11）：3894-3898.
[16] Brockmeier U, Caspers M, Freudl R, et al. Systematic screening of all signal peptides from Bacillus subtilis：A powerful strategy in optimizing heterologous protein secretion in gram-positive bacteria[J] . J Mol Biol, 2006, 362：393-402.
[17] Pfeffer J, Rusnak M, Hansen CE, et al. Functional expression of lipase A from Candida antarctica in Escherichia coli-A pre-requisite for high-throughput screening and directed evolution[J] . Journal of Molecular Catalysis B：Enzymatic, 2007, 45：62-67.
[18] 陈飞, 胡美荣, 江贤章, 等. 共表达分子伴侣PDI和Ero1对灰盖鬼伞过氧化物酶在毕赤酵母中表达的影响[J] . 生物工程学报, 2015, 31（12）：1682-1689.
[19] 郑翔, 杨何宝, 胡美荣, 等. 一种新型蛋白酶的酶学特性及脱毛应用[J] . 生物技术通报, 2017, 33（5）：183-189.
[20] Sambrook J, Russell DW. Molecular Cloning：A Laboratory Manual[M] . 3rd ed. Beijing：Cold Spring Harbor Laboratory Press, 2001.
[21] Soerensen NH, Hoff T, Oestergaard PR, et al. Dehairing of skins and hides：US, 9267182B2[P] . 2016-2-23.
[22] Dettmer A, Cavalli é, Ayub MAZ, et al. Environmentally friendly hide unhairing：enzymatic hide processing for the replacement of sodium sulfide and delimig[J] . Journal of Cleaner Production, 2013, 47（5）：11-18.
[23] 刘颖, 张彬彬, 孙冰玉, 等. 枯草芽孢杆菌高产中性蛋白酶发酵条件的优化[J] . 食品科学, 2014, 35（13）：166-170.
[24] 马宏颖. 中性蛋白酶高产菌株的诱变选育及发酵条件研究[D] . 保定：河北农业大学, 2008.
[25] 陈鸿图. 高产中性蛋白酶菌株选育及产酶特性研究[D] . 广州：华南理工大学, 2012.
[26] 李艳丽, 许少春, 许尧兴. 中性蛋白酶高产菌株的筛选及产酶酶系分析[J] . 生物技术, 2007, 17（1）：20-23.
[27] 许波. 中性蛋白酶基因的克隆及其在毕赤酵母中的高效表达[D] . 昆明：云南师范大学, 2004.
[28] 杜珊珊, 王颖, 张东杰. 解淀粉芽孢杆菌中性蛋白酶基因的克隆与表达[J] . 黑龙江八一农垦大学学报, 2015, 27（1）：51-54.
[29] 段春燕. 枯草芽孢杆菌信号肽筛选载体的构建木聚糖基因（xynA）的表达[D] . 杨凌：西北农林科技大学, 2010.
[30] 祝发明. 枯草芽孢杆菌Tat分泌表达青霉素G酰化酶初步研究[D] . 杨凌：西北农林科技大学, 2006.
[31] EllisJ. Proteins as molecular chaperones[J] . Nature, 1991, 328（6129）：378-379.
[32] 张海军, 杨君, 等. 应用分子伴侣共表达系统表达pfu 基因及酶活性测定[J] . 云南植物研究, 2009, 31（6）：499-503.
[33] 万娟, 邓会群, 张碧乾, 等. 分子伴侣协助下抗肿瘤抗生素美达霉素生物合成中的糖基转移酶Med-ORF8的原核表达[J] . 微生物学通报, 2011, 38（2）：221-227.
[34] 曾伶俐. 不同信号肽对脂肪酶A在枯草芽孢杆菌中分泌表达的影响[D] . 无锡：江南大学, 2009.
[35] 崔硕硕, 张镱, 林学政, 等. 分子伴侣共表达对低温脂肪酶Lip-837异源可溶性表达的影响[J] . 海洋科学进展, 2011, 29（1）：105-112.
[36] 蒋红亮, 张虹, 赵辅昆, 等. 分子伴侣CsaA过表达及wprA蛋白酶的缺失对枯草芽孢杆菌分泌表达外源蛋白的影响[J] . 浙江理工大学学报, 2011, 28（2）：260-266.