Free energy perturbation studies on binding of A-74704 and its diester analog to HIV-1 protease

Free energy simulations have been employed to rationalize thebinding differences between A-74704, a pseudo C₂- symmetricinhibitor of HIV-1 protease and its diester analog. The diesteranalog inhibitor, which misses two hydrogen bonds with the enzymeactive site, is surprisingly only 10-fold weaker. The calculatedfree energy difference of 1.7 ± 0.6 kcal/mol is in agreementwith the experimental result. Further, the simulations showthat such a small difference in binding free energies is dueto (1) weaker hydrogen bond interactions between the two (P₁and P₁) NH groups of A-74704 with Gly27/Gly27' carbonyls ofthe enzyme and (2) the higher desolvation free energy of A-74704compared with its ester analog. The results of these calculationsand their implications for design of HIV-1 protease inhibitorsare discussed.     

产高温碱性蛋白酶菌株筛选及酶学性质研究

从高温堆肥土样中筛选到一株高产蛋白酶的菌株,经鉴定为Bacillus subtilis,命名为I15.对最适产酶发酵条件进行了优化,并研究了发酵产蛋白酶的酶学性质.结果表明,玉米淀粉和豆饼粉是该菌发酵产蛋白酶的最适经济碳氮源;该蛋白酶属高温碱性蛋白酶,具有很强的热稳定性和酸碱稳定性.另外,突出的性质是该蛋白酶对表面活性剂和温和型去垢剂具有超强的耐受性,将具有广阔的市场前景.     

碱性蛋白酶水解豌豆蛋白的条件优化

采用碱性蛋白酶对豌豆蛋白进行水解,通过单因素试验,分析了温度、pH、酶与底物浓度比、底物浓度、水解时间对水解度影响,并在单因素试验的基础上,利用响应面分析法,优化确定的豌豆蛋白酶水解最佳条件为:温度54.5℃,pH7.9,酶与底物浓度比1.5%,底物浓度2.9%,水解时间3.0 h,最佳酶水解条件下的水解度为13.42%.     

低温海洋微生物产碱性蛋白酶菌株的筛选

从连云港黄海海域的鱼类、贝类和海水等样品中分离到近150株产碱性蛋白酶的低温菌。并通过正交试验确定菌株的最佳培养基为干酪素平板培养基,水解酪蛋白试验可对菌株的产酶情况进行定性研究,酶活较高的菌株都能产生较大的水解圈。干酪素分离培养基配合水解酪蛋白试验,可以提高产碱性蛋白酶菌株筛选效率。对其中3株产酶活性较高的菌株(ASG,YSG,ZG)进行了耐盐性试验和培养温度试验,菌株ASG,YSG的最适生长温度、时间以及培养基的最适ω(NaCl)分别为:15℃,36 h,5％;20℃,30 h,3％。     

碱性蛋白酶对酪蛋白水解的最适宜条件

蛋白质酶解是改善蛋白质特性，便于人体吸收的有效途径。我们对富含于乳制品中的酪蛋白，采用碱性蛋白酶进行水解实验。通过对主要影响因素的分析及回归方法，确定碱性蛋白酶对酪蛋白水解的最佳工艺条件：温度55℃，酶—底物比为7％，底物质量分数为6％，pH值为8。     

虾头的内源蛋白酶酶解及复合酶解研究

实验对虾头内源蛋白酶酶解，以及对内源蛋白酶与A1calase2．4L、F1avourzyme、Kojizyme、木瓜蛋白酶、中性蛋白酶、Trypsin和Protamex的复合酶解进行了比较研究。说明利用虾头内源蛋白酶可以对虾头进行有效的酶解；A1calase2．4L及F1avouzyme与虾头内源蛋白酶复合使用后，能较大程度地提高酶解产物中游离氨基酸的含量；实验中所用的7种蛋白酶与虾头内源蛋白酶复合酶解，难以明显地提高酶解产物中TCA可溶性蛋白(短肽)的含量。     

碱性蛋白酶对酪蛋白水解的最适宜条件

蛋白质酶解是改善蛋白质特性,便于人体吸收的有效途径。我们对富含于乳制品中的酪蛋白,采用碱性蛋白酶进行水解实验。通过对主要影响因素的分析及回归方法,确定碱性蛋白酶对酪蛋白水解的最佳工艺条件:温度55℃,酶-底物比为7%,底物质量分数为6%,pH值为8。     

Construction of the mutant strain in Aspergillus oryzae 3.042 for abundant proteinase production by the N+ ion implantation mutagenesis

Because of secreting large amounts of enzymes, Aspergillus oryzae was widely used in the fermentation of soy sauce in many Asian countries. Here, N⁺ ion implantation mutagenesis was applied to improve the ability of A. oryzae to secrete acid protease. Several mutants were screened using three kinds of plates (Casein medium, Czapek’s medium and carboxymethyl cellulose medium agar plates). Compared with the other mutants, the mutant A100‐8 could secrete the most protease. Acid protease activity of A100‐8 was enhanced about 44.1% at 36 h by koji fermentation test. In addition, A100‐8 was stable by periodically subculturing and maintaining on the agar slant. The mRNA expression levels of two kinds of acid protease (serine carboxypeptidase and aspartyl protease) were measured by RT‐PCR at different periods. Serine carboxypeptidase and some kinds of aspartyl protease of A100‐8 were significantly (P < 0.01) expressed higher than the control at all times.