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。     

Subtilisin modification of monodeamidated ribonuclease-A

Limited proteolysis of RNAase-Aa₁ (monodeamidated ribonuclease-A) by subtilisin results in the formation of an active RNAase-S type of derivative, namely RNAase-Aa₁S. RNAase-Aa₁S was chromatographically distinct from RNAase-S, but exhibited very nearly the same enzymic activity, antigenic conformation and susceptibility to trypsin as did RNAase-S. Fractionation of RNAase-Aa₁S by trichloroacetic acid yielded RNAase-Aa₁S-protein and RNAase-Aa₁S-peptide, both of which are inactive by themselves, but regenerate active RNAase-Aa₁S′ when mixed together. RNAase-Aa₁S-peptide was identical with RNAase-S-peptide, whereas the protein part was distinct from that of RNAase-S-protein. Titration of RNAase-Aa₁S-protein with S-peptide exhibited slight but noticeably weaker binding of the peptide to the deamidated S-protein as compared with that of native protein. Unlike the subtilisin digestion of RNAase-A, which gives nearly 100% conversion into RNAase-S, the digestion of RNAase-Aa₁ gives only a 50% conversion. The resistance of RNAase-Aa₁ to further subtilisin modification after 50% conversion is apparently due to the interaction of RNAase-Aa₁ with its subtilisin-modified product. RNAase-S was also found to undergo activity and structural changes in acidic solutions, similar to those of RNAase-A. The initial reaction product (RNAase-Sa₁) isolated by chromatography was not homogeneous. Unlike the acid treatment of RNAase-A, which affected only the S-protein part, the acid treatment of RNAase-S affected both the S-protein and the S-peptide region of the molecule.     

枯草杆菌蛋白酶E的蛋白质工程

用定点突变和随机突变的方法,对枯草杆菌碱性蛋白酶E基因进行改造。突变后的基因插入大肠杆菌-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DBl04中进行表达,得到突变种的碱性蛋白酶．它们的突变位点分别是(M222A)、(M222A、N118S)、(M222A、N118S、Q103R)、(M222A、N118S、Q103R、D60N)。各突变种酶的性质测定 结果表明．M222A突变使酶抗氧化,N118S突变使酶增加热稳定性,Q103R和D60N突变虽然能增加酶的比活,但使酶的热稳定性大大下降,尤其是D60N突变使酶变得极不稳定。野生型碱性蛋白酶与(M222A)突变种的等电点均为8．92．而M222A,N118S)。(M222A,N118S ,Ql03R)和(M222A,118S．Q103R,D60N)突变酶分别为8.88．9.10和9．17。用Nsuc-AAPF-pNA作为底物时酶反应景适pH值为7.5～9.5,而用酪蛋白底物时最适pH值为10～12。     

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.     

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…     

Subtilisin Protein Inhibitor from Potato Tubers

A protein with molecular weight of 21 kD denoted as PKSI has been isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii). The isolation procedure includes precipitation with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion-exchange chromatography on CM-Sepharose CL-6B. The protein effectively inhibits the activity of subtilisin Carlsberg (Ki = 1.67 +/- 0.2 nM) by stoichiometric complexing with the enzyme at the molar ratio of 1 : 1. The inhibitor has no effect on trypsin, chymotrypsin, and the cysteine proteinase papain. The N-terminal sequence of the protein consists of 19 amino acid residues and is highly homologous to sequences of the known inhibitors from group C of the subfamily of potato Kunitz-type proteinase inhibitors (PKPIs-C). By cloning PCR products from the genomic DNA of potato, a gene denoted as PKPI-C2 was isolated and sequenced. The N-terminal sequence (residues from 15 to 33) of the protein encoded by the PKPI-C2 gene is identical to the N-terminal sequence (residues from 1 to 19) of the isolated protein PKSI. Thus, the inhibitor PKSI is very likely encoded by this gene.     

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 。     

Limited Proteolysis of Disulfide-reduced Ovalbumin by Subtilisin

Our previous study [Takahashiet al., J. Biochem., 109, 846–851 (1991)] has shown that the disulfide-reduced form of ovalbumin was proteolyzed by subtilisin into three major fragments. It was investigated whether or not these three fragments would be folded into one molecule. Gel permeation and ion-exchange chromatography indicated that the three fragments were eluted in a single peak. The proteolyzed protein had a CD spectrum that was almost indistinguishable from the disulfide-reduced, non-proteolyzed, form of ovalbumin. Differential scanning calorimetry, however, revealed, that the proteolyzed ovalbumin was denatured at a lower temperature than that of the disulfide-reduced, non-proteolyzed. protein. Thus, it is concluded that the three fragments were folded into a native-like conformation with decreased stability. Chemical analyses of the fragments purified by reverse-phase HPLC revealed that there was a cleavage site in the disulfide-reduced form of ovalbumin, at least at the amino-terminal side of Cys⁷³, in addition to the well-known cleavage sites in plakalbumin.     

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。     

Subtilisin cleavage of tubulin heterodimers and polymers.

Native pig brain tubulin in heterodimer or polymer form was subjected to limited proteolysis by subtilisin, which is known to cleave at accessible sites within the last 50 amino acids of the highly variable carboxyl-termini of the alpha and beta subunits. Heterodimeric tubulin or tubulin polymerized in the presence of 4 M glycerol or taxol was used in these experiments. Digested tubulin was purified by cycles of polymerization and depolymerization, ammonium sulfate precipitation, or ion-exchange chromatography in the absence or presence of nonionic detergent; however, smaller cleaved products of about 34,000 to 40,000 MW remained associated with the major cleaved subunits, alpha' and beta', under all purification conditions. In order to determine the effect of subtilisin cleavage on tubulin heterogeneity, purified native or subtilisin-cleaved tubulin was subjected to isoelectric focusing, followed by SDS-PAGE. The total number of isotypes was reduced from 17-22 for native alpha,beta tubulin to 7-9 for subtilisin-cleaved alpha',beta' tubulin. When tubulin heterodimers were cleaved, a single major beta' isotype was evident; however, when tubulin polymerized in 4 M glycerol was cleaved, two major beta' isotypes were found. Monoclonal antibodies that recognize a beta carboxyl-terminal peptide, residues 410-430, reacted with both major beta' isotypes, indicating that subtilisin cleavage occurred within the last 20 of the 450 amino acids. In order to establish whether this difference was in fact associated with polymer or heterodimer forms of tubulin, digestion was carried out in the presence of taxol, which stabilizes tubulin polymers. A single major beta' isotype different from the cleaved heterodimer, but coincident with one of the bands of the cleaved glycerol-induced polymers, was found when taxol-treated tubulin was digested. This result suggests the presence of more than one subtilisin site in the beta subunit, near residues 430-435, with different accessibility to the enzyme in the heterodimer and polymer form.     

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.     

抗氧化型枯草杆菌碱性蛋白酶高产工程菌的构建——Ⅰ.抗氧化型碱性蛋白酶的提纯及其主要性质的研究

枯草芽孢杆菌(Bacillus Subtilis)B135工程菌能产生抗氧化型碱性蛋白酶,粗酶经硫酸铵分级沉淀,CM-52层析,Sephadex G-100层析,得到凝胶电泳均一样品,比活达到1700U/mg,是粗酶比活的7.69倍.该酶在60℃时酶活力最高,最适pH为10.2,在50℃时,温浴10min后,酶活降低到原来的50%.该酶受1M H_2O_2作用20min后,仍保持96%的酶活     

Subtilisin of Leishmania amazonensis as Potential Druggable Target: Subcellular Localization,In Vitro Leishmanicidal Activity and Molecular Docking of PF-429242, a Subtilisin Inhibitor

Subtilisin proteases, found in all organisms, are enzymes important in the post-translational steps of protein processing. In Leishmania major and L. donovani, this enzyme has been described as essential to their survival; however, few compounds that target subtilisin have been investigated for their potential as an antileishmanial drug. In this study, we first show, by electron microscopy and flow cytometry, that subtilisin has broad localization throughout the cytoplasm and membrane of the parasite in the promastigote form with foci in the flagellar pocket. Through in silico analysis, the similarity between subtilisin of different Leishmania species and that of humans were determined, and based on molecular docking, we evaluated the interaction capacity of a serine protease inhibitor against both life cycle forms of Leishmania. The selected inhibitor, known as PF-429242, has already been used against the dengue virus, arenaviruses, and the hepatitis C virus. Moreover, it proved to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro. Here, PF-429242 significantly inhibited the growth of L. amazonensis promastigotes of four different strains (IC₅₀ values = 3.07 ± 0.20; 0.83 ± 0.12; 2.02 ± 0.27 and 5.83 ± 1.2 µM against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC₅₀ = 170.30 µM). We detected by flow cytometry that there is a greater expression of subtilisin in the amastigote form; however, PF-429242 had a low effect against this intracellular form with an IC50 of >100 µM for intracellular amastigotes, as well as against axenic amastigotes (94.12 ± 2.8 µM for the LV78 strain). In conclusion, even though PF-429242 does not affect the intracellular forms, this drug will serve as a tool to explore pharmacological and potentially leishmanicidal targets.     

Increased Thermostability of Cross-Linked Enzyme Crystals of Subtilisin in Organic Solvents

Cross-linked enzyme crystals(CLECs) of subtilisin display the improved thermostability in organic solvents, compared to free subtilisin. CLECs are more stable than the free enzyme in octane with a half life of 200 days at 45°C, while that of free enzyme is 5.4 days. CLECs in octane is more stable than in acetonitrile.     

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。