雅致放射毛霉蛋白酶SP1基因的克隆及序列分析

Cloning and Sequence Analysis of Protease(SP1) Gene from Actinomucor elegans

雅致放射毛霉蛋白酶具有较好的脱苦味和水解豆类蛋白的能力.为了揭示其结构和功能的关系,促进其开发应用,文中通过RACE和PCR技术克隆获得丝氨酸蛋白酶SP1基因.根据生物信息学中的理论和方法,对SP1进行了较全面的预测和分析.结果表明:SP1基因由4个外显子和3个内含子组成,编码454个氨基酸残基.SP1属于蛋白酶K家族,与Rhizopus microsporus var.chinensis的丝氨酸蛋白酶同源性最高,亲缘关系最近.以蛋白酶K晶体结构(PDB code:1IC6)作为模板进行同源建模,通过对模拟结构的分析可知:SP1分子内无二硫键,具有1个Ca2+结合位点;SP1与蛋白酶K在底物结合区域、氢键、盐键和二硫键等方面均存在差异,这些差异可能就是SP1具有独特的水解作用和脱苦能力的原因.

Proteases produced from Actinomucor elegans （A. elegans） are of preferable debittering effect and high hydrolysis ability to soybean proteins. In order to reveal the relationship between the structure and the function and accelerate the development and application of the proteases, serine protease （ SP1 ） gene from A. elegans was obtained by means of PCR and RACE techniques, and was then predicted and analyzed in detail based on the bioinformatics theory and technology. The results show that SP1 gene, which contains 4 exons and 3 introns, encodes 454 amino acid residues, and that SP1 belongs to Proteinase K family and exhibits the closest genetic relationship to Rhizopus microsporus vat. chinensis with the highest homology. Moreover, according to the SP1 structure modeled via homology modeling with Proteinase K （PDB code： 1IC6） as the crystal coordinate, it is tbund that SP1 has no disulfide bonds and possesses a Ca^2＋ binding site, and that the differences in substrate-binding region, hydrogen bond, salt bridge and disulfide bond between SPI and Proteinase K are probably responsible for the special hydrolysis ability and debittering effect of SPI.