摘要
在过去五十年里,许多学者对纤维素酶进行了大量的研究,试图转化地球上大量、可再生的纤维素资源为可利用的生物能源,以代替日益枯竭的石化能源。然而,纤维素酶生产的高成本严重制约了纤维素生物转化产业的发展,解决这一问题的关键就是寻找新的高产菌株或通过分子生物学技术改造纤维素酶的催化活性,以此来提高酶的生物转化效率。
本研究从450株海洋菌以及陆地样品中,分别筛选出了46株和99株产纤维素酶的菌株。通过鉴定发现,其中大部分归类于芽胞杆菌属、盐单胞菌属和假单胞菌属。从中克隆了8株芽胞杆菌属菌株的内切葡聚糖酶(EG)基因,进化树分析表明它们在核苷酸和蛋白质水平上分属不同的分支,具有一定的多样性。其中含有来自枯草芽胞杆菌内切葡聚糖酶基因cel5A的克隆在纤维素平板上显示出较高的水解圈活性。
采用易错PCR和DNA shuffling的体外定向进化技术,本研究建立了来源于Bacillus subtilis BME-15的内切葡聚糖酶基因cel5A突变体库。利用刚果红染色法对突变体库中的71000株克隆进行筛选,得到了7株高活性突变株。其中内切葡聚糖酶突变体M44-11,S75和S78水解羧甲基纤维素钠的活性分别是野生型的2.03、2.54和2.68倍;此外,M44-11的酸碱耐受性和热稳定性也得到提高。通过同源模建,用Swiss-model构建了重组内切葡聚糖酶和各种突变酶的三维结构图。通过对酶分子结构的分析,发现M44-11,S75,S78的大部分突变位点氨基酸残基位于保守区域之外,只有S75的V255A突变位点处于活性中心,且毗邻于亲核基团Glu257附近。
为了进一步研究突变位点在酶突变体中所起的作用,本研究通过环式PCR法分别构建了120位赖氨酸残基和272位天冬氨酸残基的突变酶。通过检测突变酶的水解圈活性发现,120位赖氨酸残基突变为谷氨酸和272位天冬氨酸残基突变为甘氨酸都可以部分提高酶的活性。这一现象表明,在突变酶S78和M44-11中可能存在多个突变位点的协同叠加作用而促进了酶的催化效率,而不是单个氨基酸残基突变产生的作用。同时这一结果也揭示了处于非活性中心或非结合位点的突变位点可能通过改变整个酶分子的空间位阻、催化残基之间的氢键网络形成来提高酶的催化特性。此外,对可能与底物相互作用的69位色氨酸残基和263丙氨酸-264丝氨酸-265甘氨酸残基的饱和突变结果表明,69位色氨酸残基的改变对酶活性的影响较小,而263丙氨酸-264丝氨酸-265甘氨酸残基的突变则会导致突变酶的活性严重丧失,这说明了该位点对底物结合起着非常关键的作用。
本项研究为采用定向进化的方法改造第五家族糖基水解酶提供了有价值的理论参考。
In the past 50 years much effort had gone into the studies of cellulases as a potential means to obtain sustainable biobased products to replace depleting fossil fuels from an abundant, renewable energy resource, plant biomass. However, the high cost of cellulases production seemed to be a very important and difficult challenge in the cellulose bioconversion process. The way to increase enzyme volumetric productivity was to isolate hyperproducers and to improve the necessary characteristics of cellulases.
In this research, from more than 450 marine microorganisms and terrestrial soil samples, 46 and 99 isolates were found positive for cellulase production, respectively.Among these, cellulase-producing strains were mainly identified as Bacillus、Halomonasand Pseudomonas. Eight endo-β-1, 4-glucanase (EG) genes were cloned from Bacillus strains and showed different homology by DNA or protein phylogenetic tree analysis. Furthermore, the colony which harbored Bacillus subtilis BME-15 EG gene (cel5A) showed the highest halo-forming activity on CMC plates and was chose to be studied.
Using directed evolution techniques of error-prone PCR and DNA shuffling, several Cel5A variants with improved catalytic activity had been screened from the mutant library, which contained 71,000 colonies. Compared with the wild-type enzyme, the variants (M44-11, S75 and S78) showed 2.03 to 2.68 folds increased activities toward sodium carboxymethyl cellulose (CMC), while the M44-11 also exhibited a wider pH tolerance and higher thermostability. Structural models of M44-11, S75, S78 and WT proteins revealed that most of the substitutions were not located in the strictly conserved regions, except the mutation V255A of S75, which was closed to the nucleophile Glu257 in the catalytic center of the enzyme.
In order to study the functions of substitutions in the mutants, site-directed mutagenesis of K120 and D272 were constructed by using cycled PCR. By analyzing the halo-forming activities of the mutants and parents, substitutions K120E and D272G were found to show slight increased activity compared with wild type enzyme. This phenomenon implied that the increased activity of S78 and M44-11 was not due to the contribution of the single substitution, but due to the synergistic effect of multi-site substitutions. And it also revealed that mutations outside of the catalytic center or the binding sites resulted in increased catalytic activity by making new hydrogen bonds and repositioning of catalytic residues in the active site. Moreover, saturation mutagenesis of W69 and A263-S264-G265 sites were also constructed to study the reaction between Cel5A and substrate. The result showed that substitutions of W69 slight decreased the halo-forming activity of the enzyme, while substitutions of A263-S264-G265 resulted in most activity losing and this revealed that A263-S264-G265 played some very important fuction in binding substrate.
This study provided useful references for directed evolution of the enzymes belonged to glycoside hydrolase family 5 (GH5).
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