Stereochemical modeling of disulfide bridges. Criteria for introduction into proteins by site-directed mutagenesis

A computer modeling procedure for assessing the stereochemicalsuitability of pairs of residues in proteins as potential sitesfor introduction of cystine disulfide crosslinks has been developed.Residue pairs with C – C distances of 6.5 Å andC^beta;–C^ß distances of 4.5 Å are chosenfor geometrical fixation of S atoms using the program MODIP.The stereochemistry of the modeled disulfides is evaluated usinglimits for the structural parameters of the various torsionangles and S–S bond length in the disulfide bridge. Theability of the procedure to correctly model disulfides has beenchecked with examples of cystine peptides of known crystal structuresand 103 disulfide bridges from 25 available protein crystalstructures determined at 2 Å resolution. An analysis ofresults on three proteins with engineered disulfides, T4 lysozyme,dihydrofolate reductase and subtilisin, is presented. Two positionsfor the introduction of ‘stereochemically optimal’disulfides are identified in subtilisin.     

Replacement of cysteine-107 of Saccharomyces cerevisiae iso-1-cytochrome c with threonine: improved stability of the mutant protein

Site-directed mutagenesis has been used to change the codonfor cysteine-107 of Saccharomyces cerevisiae iso-1-cytochromec to a threonine codon. The resulting protein is active in vivo,is methylated as in the wild-type protein and has optical propertiesindistinguishable from those of the wild-type protein. The threonine-107iso-1-cytochrome c demonstrated fully reversible electrochemicalbehaviour and a mid-point reduction potential of 272 mV versusNHE. In addition, this mutant does not demonstrate a tendencyto autoreduce or to dimerize as does the wild-type protein.These properties of the threonine-107 mutant establish thatit will provide a useful background in which to make subsequentmutations for mechanistic and physical studies of yeast iso-1-cytochromec.     

Characteristics of spontaneous revertants in haploid yeast

The appearance dynamics of spontaneous revertants in adenine- or leucine-auxotrophic haploid Saccharomyces cerevisiae strains has been studied using mathematical simulation methods. In the case of adenine auxotrophs an increase in the number of revertants with decreasing metabolite content is found to result mainly from increasing the rate of intragenic suppressor mutations. In the case of leucine auxotrophs revertants result from increasing the appearance of mutants formed at similar rates under different cultivation conditions. In the latter case the appearance of mutant colonies increases with decreasing size of colonies of the initial auxotrophic cells. The last can simulate so-called adaptive mutagenesis. The heterogeneity of revertants appearing in different time periods is described.     

衣康酸生产菌的激光诱变育种及其产酸条件

本文利用纯铜蒸气激光对土曲霉ＡＴ１进行诱变育种。获得突变株ＡＴ８９－４的遗传性状稳定。在初始葡萄糖为１０ｇ／１００ｍｌ的摇瓶培养条件下，产衣康酸大于５ｇ／１００ｍｌ，糖转化率达６０％左右。     

Introduction of sulphydryl groups into pea vicilin: Formation of intra-and inter-polypeptide disulphide bonds

Site directed mutagenesis was used to introduce two cysteine residues into the hydrophilic regions of the pea vichlin polypeptide previously expressed in Saccharomyces cerevisiae. The mutated polypeptide was expresed and the resultant protein was characterised by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. The presence of both intra- and inter-polypeptide sulphydryl bonds was indicated by bands of different mobility from those of the native polypeptide. The association of mutated vicili polypeptides into disulphide-bonded aggregates within the yeast was not a random process, trimers being the major aggregate produced. When the mutated vicilin was extracted from yeast and purified, random aggregates of the vicilin polypeptide were formed.     

Rational Biosynthetic Engineering for Optimization of Geldanamycin Analogues

Tailor made : We report the rational biosynthesis of C15 hydroxylated non‐quinone geldanamycin analogues by site‐directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post‐PKS tailoring genes. Rational biosynthetic engineering allowed the generation of geldanamycin derivatives, such as DHQ3 illustrated in the figure, which had superior pharmacological properties in comparison to the parent compound.

     

Probing the Role of Backbone Hydrogen Bonding in a Critical β Sheet of the Extracellular Domain of a Cys‐Loop Receptor

Probing the sheet : The network of hydrogen bonds formed in the outer β sheet of the nicotinic acetylcholine receptor (nAChR; see figure) is fairly robust and tolerates single amide‐to‐ester mutations throughout. However, eliminating two proximal hydrogen bonds completely destroys receptor function; this adds further support to gating models that ascribe important roles to these β strands of the nAChR extracellular domain.

     

紫外诱变筛选高耐性低产高级醇优良啤酒酵母及其高浓发酵后啤酒风味的研究

以啤酒酵母泥X为出发菌株,经筛选,得到高耐渗,高耐酒精的酵母菌Y,并与以诱变得到高耐性低产高级醇菌株MS-5,经研究发现些菌株在高耐性发酵条件下能有效降低高级醇含量。对该菌株在28°P麦芽汁培养基中的发酵性能和发酵终止时啤酒的风味物质含量进行分析,与出发菌种X及菌株Y作对比,菌株MS-5更适合于高浓酿造。     

西索米星生产菌株的诱变选育及工艺优化

利用原生质体常压室温等离子体（ARTP）诱变方法选育西索米星高产菌株,经诱变筛选获得一株高产菌株I4-10,其西索米星的生物效价达到1 389 U/mL,较原始菌株提高了35.4%。通过对高产菌株I4-10进行碳源、氮源优化,确定可溶性淀粉和牛肉粉是突变菌株I4-10的最适碳源和氮源。进一步采用响应面实验设计方法优化发酵培养基,结果表明黄豆饼粉和DL-蛋氨酸的添加量为显著影响因素,经优化其最适添加质量浓度分别为32.78 g/L和1.75 g/L。在此最适工艺下,西索米星的生物效价提高至1 849 U/mL,较原始菌株提高了80%。最后对原始菌及高产突变株中西索米星代谢途径及菌株生长相关的关键酶进行转录水平比较分析,初步解析了突变菌株I4-10高产西索米星的机制。     

总状毛霉紫外诱变及发酵提取壳聚糖初探

为了提高壳聚糖产量,通过对紫外诱变处理总状毛霉及发酵条件的研究,初步确定了不同发酵培养条件对总状毛霉产壳聚糖的影响,结果表明:发酵液培养基的蔗糖为50 g/L、酵母膏为2.0 g/L、硝酸钠为3.5 g/L、磷酸氢二钾为1.0 g/L、硫酸镁为0.2 g/L和硫酸铁为0.2 g/L,发酵温度控制在30℃,培养时间为48 h,壳聚糖产量为1.12 g/L。