枯草杆菌蛋白酶与蛋白质工程

在以蛋白质三维结构及其与功能关系为基础的蛋白质工程的兴起中,枯草杆菌蛋白酶的定位诱变起了重要作用。以该酶为代表的蛋白质的工程研究,反映了这种生物技术所取得的进展和存在的主要问题。虽然,为了达到工程改造蛋白质的目标仍需要做大量基础性研究,但目前取得的成果足以说明蛋白质工程发展的光明前景。     

枯草杆菌蛋白酶E的156和165位突变

应用定点突变方法,在M222A突变的枯草杆菌蛋白酶E基因上进行E156S和V165I定点突变. 将突变基因插入大肠杆菌-枯草杆菌穿梭质粒pBE-2中,在碱性和中性蛋白酶缺陷型的枯草杆菌DB104中进行表达,得到突变种(M222A,E156S)和(M222A,E156S,V165I)蛋白酶E. 性质测定表明,E156S突变使蛋白酶比活力增加90%,并不影响酶的热稳定性和抗氧化性. 而V165I突变使蛋白酶比活力降低.     

枯草杆菌碱性蛋白酶蛋白质工程进展

枯草杆菌蛋白酶(Subtilisin,EC 3·4·21·14)一般是指枯草杆菌及其它芽孢杆菌产生的胞外碱性蛋白酶Subtilisin具有巨大的商业价值,被广泛应用于蚕丝工业、制革工业及洗涤剂工业。     

枯草蛋白酶E的突变体

Ser236位于横贯枯草蛋白酶E的α螺旋末端,远离催化活性中心,Ser236的突变不会对酶的活性产生大的影响。用定点突变的方法对枯草蛋白酶E的基因进行改造引入Ser236Cys,可能会形成分子间二硫键,有利于提高酶的稳定性。Ser236Cys变体酶(BP1)活性是野生型蛋白酶E的15倍,热稳定性提高3倍;进一步在其他位点引入突变的变体酶BU1(A1a15Asp/Gly20His/Ser236Cys)和BW1(Ser24His/Lys27Asp/Ser236Cys)活性都比野生型蛋白酶E低,但BW1的稳定性稍高于野生型蛋白酶E。     

枯草杆菌蛋白酶E的结晶及初步衍射研究

对来源于枯草芽孢杆菌的枯草杆菌蛋白酶E进行了初步晶体学研究。应用悬滴汽相扩散法生长出了该酶的单晶体,其空间群为P2_12_12_1,晶胞边长a=74.35A,b=80.98A,c=88.59A。已用面探测器衍射仪收集了一套中等分辨率的X射线衍射数据。     

枯草杆菌蛋白酶基因工程的研究进展

本文介绍了枯草杆菌蛋白酶（Ｓｕｂｔｉｌｉｓｉｎ）的研究现状,即利用定位诱变和体外重组等技术改变酶的性质,包括催化活性、底物特异性、稳定性、低温适应性以及酶在有机相中的性能等。对枯草杆菌蛋白酶的成功改造不仅有可观的商业价值,而且为蛋白质工程的发展作出了重要的贡献。     

枯草杆菌蛋白酶的基因工程改性

枯草杆菌蛋白酶（Ｓｕｂｔｉｌｉｓｉｎ）是一种工业上应用很广的酶,在洗涤剂、制革、丝绸等多种行业上有着广泛的用途。特别是用于生产加酶洗涤剂,帮助去除血渍、奶渍、汗渍及可可等各种蛋白污垢。１９６０年,丹麦人首先利用地衣芽孢杆菌生产了被称为Ｓｕｂｔｉｌｉｓｉｎ Ｃａｒｌｓｂｅｒｇ的碱性蛋白酶,随后该酶被用于生产加酶洗涤剂,目前有资料称国外市场上９０％是加酶洗涤剂。国内１９９０年枯草杆菌蛋白酶产量约为１．３万吨,加酶洗衣粉占洗涤剂总量约１０％。枯草杆菌蛋白酶的生产菌和研究对象主要是地衣芽孢杆菌、解淀粉芽…     

用遗传工程的方法构建一个分泌型高表达的枯草杆菌碱性蛋白酶E(Subtilisin E)的枯草杆菌质粒-宿主系统

将克隆了枯草杆菌蛋白酶E基因aprE的枯草杆菌质粒pPZW101和一个组成型強启动子Psk连接构建成质粒pPZW102,转入碱性和中性蛋白酶缺失枯草杆菌DB104并进行表达。此质粒-宿主系统具有Subtilisin E高表达和外分泌的特点。     

枯草杆菌蛋白酶突变体的纯化和晶体生长

应用基因工程手段,获得了枯草杆菌蛋白酶Ｅ的双突变体基因（Ｍ２２２Ａ,Ｎ１１８Ｓ）,此基因在枯草芽孢杆菌中表达得到了既抗氧又耐高温的碱性蛋白酶,含Ｍ２２２,Ｎ１１８Ｓ碱性蛋白酶基因的枯草杆菌发酵液经过硫酸铵分级沉淀和ＤＥＡＥＳｅｐｈａｄｅｘＡ－２５阴离子交换层析柱,再在ＦＰＬＣ层析系统上用Ｈｉｌｏａｄ２６／１０ＳＳｅｐｈａｒｏｓｅＨＰ阳离子交换柱分离得到ＳＤＳ－ＰＡＧＥ电泳纯的蛋白酶样品。突变体酶的等电点为ｐＨ８．９,分子量为２７４００,用四肽底物测得的动力学参数也有较大的变化。对该突变体酶进行了晶体生长研究,获得了较大的单晶体。     

枯草蛋白酶E的定点突变及其对酶性质的影响

用定点突变的方法研究S221C/P225A,N118S/S221C/P225A,D60N/S221C/P225A和Q103R/S221C/P225A突变对蛋白酶活性,酯酶活性与蛋白酶活性之比的影响。结果表明：S221C/P225A突变使蛋白酶活性比枯草蛋白酶E低73000多倍,酯酶活性与蛋白酶活性之比是Subtiligase的3倍;N118S/S221C/P225A突变使蛋白酶活性和酯酶活性分别比S221C/P225A突变下降3.6倍和15倍,酯酶与蛋白酶活性之比下降4倍,同时增加变体酶的热稳定性;D60N/N118S/S221C/P225A突变使蛋白酶活性比N118S/S221C/P225A突变体下降15倍,但对酯酶活性几乎没有影响,酯酶与蛋白酶活性之比增加14倍,分别是S221C/P225A突变体和Subtiligase的3.3倍和10.3倍;但是,Q103R/N118S/S221C/P225A突变使蛋白酶活性比N118S/S221C/P225A突变体增加5倍,酯酶活性下降55倍,酯酶与蛋白酶活性之比下降1000倍。     

用蛋白质工程的方法改良枯草杆菌蛋白酶E(Subtilisin E)的抗氧化性

以含有蛋白酶E基因(aprE)的单链M13mp18-aprE DNA为模板,合成的寡核苷酸5′-3′为诱变引物,用缺口双链法对aprE进行Met-222-Ala点突变。经菌落印迹杂交筛选,选出阳性噬斑。用SaⅡ酶解M13mp18-aprE得到aprE,将它和pPZW103重组,转化中性、碱性蛋白酶缺失宿主菌DB104。经含卡那霉素和脱脂奶粉板筛选和比较aprE限制性内切酶NcoⅠ和SacⅡ水解电泳图谱分析,完成构建一个分泌抗氧化的枯草杆菌蛋白酶E的工程菌PW8888。     

枯草杆菌蛋白酶基因工程的研究进展

本文介绍了枯草杆菌蛋白酶(Subtilisin)的研究现状,即利用定位诱变和体外重组等技术改变酶的性质,包括催化活性、底物特异性、稳定性、低温适应性以及酶在有机相中的性能等。对枯草杆菌蛋白酶的成功改造不仅有可观的商业价值,而且为蛋白质工程的发展作出了重要的贡献 。     

Chimeric Protein Engineering

Protein stability can be enhanced by the incorporation of non-natural amino acids and semi-rigid peptidomimetics to lower the entropic penalty upon protein folding through preorganization. An example is the incorporation of aminoisobutyric acid (Aib, α-methylalanine) into proteins to restrict the Φ and Ψ backbone angles adjacent to Aib to those associated with helix formation. Reverse-turn analogs were introduced into the sequences of HIV protease and ribonuclease A that enhanced their stability and retained their native enzymatic activity. In this work, a chimeric protein, design_4, was engineered, in silico, by replacing the C-terminal helix of full sequence design protein (FSD-1) with a semi-rigid helix mimetic. Residues 1–16 of FSD-1 was ligated in silico with the N-terminus of a phenylbipyridyl-based helix mimetic to form design_4. The designed chimeric protein was stable and maintained the designed fold in a 100-nanosecond molecular dynamics simulation at 280 K. Its β-hairpin adopted conformations that formed three additional hydrogen bonds. Compared to FSD-1, design_4 contained fewer peptide bonds and internal degrees of freedom; it should, therefore, be more resistant to proteolytic degradation and denaturation.     

Microneme Protein 5 Regulates the Activity of Toxoplasma Subtilisin 1 by Mimicking a Subtilisin Prodomain

Toxoplasma gondii is the model parasite of the phylum Apicomplexa, which contains obligate intracellular parasites of medical and veterinary importance. Apicomplexans invade host cells by a multistep process involving the secretion of adhesive microneme protein (MIC) complexes. The subtilisin protease TgSUB1 trims several MICs on the parasite surface to activate gliding motility and host invasion. Although a previous study showed that expression of the secretory protein TgMIC5 suppresses TgSUB1 activity, the mechanism was unknown. Here, we solve the three-dimensional structure of TgMIC5 by nuclear magnetic resonance (NMR), revealing that it mimics a subtilisin prodomain including a flexible C-terminal peptide that may insert into the subtilisin active site. We show that TgMIC5 is an almost 50-fold more potent inhibitor of TgSUB1 activity than the small molecule inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN). Moreover, we demonstrate that TgMIC5 is retained on the parasite plasma membrane via its physical interaction with the membrane-anchored TgSUB1.     

A mutant subtilisin E with enhanced thermostability

A mutant subtilisin E with remarkably thermostability is reported. It is more active against the typical substrate s-AAPF-pna than the wild-type subtilisin E. The time required for getting 50% residual activity of Ser236Cys subtilisin E at 60 °C in aqueous solution was approximately 80 min which is 4 times longer than that of wild-type subtilisin E. Similar to the wild-type subtilisin E, the amidase activity of Ser236Cys subtilisin E is dramatically reduced in the presence of dimethylformamide (DMF).