【作者】 丁海涛；

【导师】 赵宇华；

【作者基本信息】 浙江大学 ， 微生物， 2010， 博士

【摘要】 葡萄糖脱氢酶由于具有活性高和双辅酶特异性等优点,在辅酶再生领域具有广阔的应用前景。我们首先从若干株芽孢杆菌及其近缘种中克隆到若干个葡萄糖脱氢酶基因,选择出其中活性最高的来源于Lysinibacillus sphaericus G10中的葡萄糖脱氢酶(LsGDH)进行深入研究。对重组LsGDH进行了酶学性质和耐碱性机制的研究；通过理性设计得到了稳定性显著提高的突变体；并发明了直接从土壤宏基因组中一步克隆葡萄糖脱氢酶全长序列的方法。论文的主要研究结果如下：1) LsGDH基因的克隆、表达与性质研究利用简并PCR和染色体步移从L. sphaericus G10中克隆到葡萄糖脱氢酶基因全序列。该基因大小为786 bp,编码262个氨基酸。重组LsGDH在25℃活性为205.7±6.9 U/mg。经SDS-PAGE和排阻层析,确定LsGDH为同源四聚体。该酶的最适温度和最适pH分别是50℃和9.5；其热稳定性较差,只在35℃以下稳定；但是该酶的耐碱性较好,在pH 6.5-10.5之间稳定。LsGDH在直链烷醇中的稳定性可以用Gaussian分布模型来描述(R2≥0.94)。2) LsGDH耐碱机制的初步探讨通过将理论分析和功能实验相结合,从分子的角度初步阐明了LsGDH的耐碱机制。定点突变研究结果表明,第170位的氨基酸类型对于葡萄糖脱氢酶的pH稳定性起到了至关重要的作用,第114位的氨基酸对其稳定性有一定贡献,约为20%。对LsGDH由单体形成四聚体过程中的静电相互作用能和疏水相互作用能的变化分析结果表明,影响LsGDH四聚体在碱性pH下稳定的主要因素是静电相互作用,而非亚基间疏水相互作用。3)通过理性设计提高LsGDH的热稳定性通过对LsGDH经同源建模后的三维结构分析,结合定点突变技术,在LsGDH四聚体AC和BD亚基间各引入一对二硫键,获得了一株热稳定性显著提高的突变体DS255。DS255经亲和层析纯化过柱过程中有约60%蛋白被空气氧化形成二硫键,在4℃放置一周后经空气自然氧化后几乎全部蛋白都形成二硫键。DS255的Tm值为86.7℃,比野生型提高了40.5℃。DS255对于底物和辅酶亲和力下降了3倍左右,但是其kcat则提高了50%左右,而可溶性表达量尽管降低,但仍比较可观。这说明我们通过在LsGDH四聚体亚基间引入二硫键提高其热稳定性的策略是成功的,极大的提升了LsGDH的实用价值。4)从土壤宏基因组中一步克隆葡萄糖脱氢酶的方法发明了从土壤宏基因组中通过一步PCR方法扩增出葡萄糖脱氢酶全长序列的方法。对扩增条件的研究结果表明,土壤基因组的纯度和复杂性对能否成功扩增出目的基因至关重要。此外,用于扩增的引物简并性要尽量低且待扩增DNA片段长度尽量小于2 kb,目标片段越小,扩增效率越高。该方法省却了后续繁琐的染色体步移的步骤,由该方法克隆出的DNA片段混合物,可不经测序直接用于DNA改组介导的蛋白质定向进化,由于土壤微生物的多样性导致所获得片段的多样性,因而筛选到目的性状的成功率也大为提高。更多还原

【Abstract】 Glucose dehydrogenase could potentially be used for coenzyme regeneration in practical applications due to the advantages of its high activity and dual-cofactor specificity. We have cloned several genes encoding glucose dehydrogenase from strains of Bacillus and related species, and glucose dehydrogenase of highest activity from Lysinibacillus sphaericus G10 was chosen for further study. The enzymatic properties of recombinant LsGDH and alkali resistance mechanism was studyed; a mutant with improved thermal stability was obtained by rational design; and a strategy for one-step cloning of glucose dehydrogenase directly from the soil metagenome was invented. The main results are as follows:1) Cloning, expression and characterization of LsGDHThe full-length nucleotide sequence of LsGDH from L. sphaericus G10 was obtained by using degenerate PCR and chromosome walking. The gene constituted by 786 bp nucleotides, which encoding 262 amino acids. The activity of recombinant LsGDH was 205.7±6.9 U/mg at 25℃. LsGDH was determined to be a homologous tetramer by SDS-PAGE and size exclusion chromatography. The recombinant LsGDH exhibited maximum activity at pH 9.5 and 50℃; the enzyme was stable just at the temperature below 35℃, but very stable at a wide pH ranging from 6.5 to 10.5. The stability of LsGDH in the chain alkanol can be described by the Gaussian distribution model (R2≥0.94).2) The alkali-resistant mechanism of LsGDHThe alkali-resistant mechanism was investigated by theoretical analysis and functional experiment. The results of site-directed mutagenesis showed that the type of the 17th amino acid played a crucial role for the pH stability of LsGDH and the 114th amino acids accounted for about 20% contribution to the pH stability. The analysis of electrostatic interaction energy and hydrophobic interaction energy after energy minimization showed that electrostatic interactions was the main factor affected the pH stability of LsGDH, rather than the hydrophobic interactions.3) The rational design to improve the thermal stability of LsGDHA mutant with signicicantly improved thermal stability, DS255, has been designed by introducing disulfide bonds at the subunits intersurface. About 60% of the mutant formed disulfide bonds during the purification process, and almost all the DS255 formed disulfide bonds through the natural air oxidation after placing at 4℃for one week. The Tm value of DS255 is 40.5℃higher than wild-type LsGDH. The affinity to substrate and coenzyme of DS255 was about three times lower than wild-type, but the kcat value increased about 50%, meanwhile the soluble expression was little lower than wild-type. These results indicated that the strategy for increasing the thermal stability of LsGDH by introduction of disulfide bonds between subunits was successful, and greatly enhanced the practical value of LsGDH.4) one-step strategy for cloning of glucose dehydrogenase from soil metagenomic DNA.A strategy for one-step cloning of glucose dehydrogenase directly from soil metagenomic DNA was invented. Study on the conditions of the amplification process showed that the purity and complexity of the metagenomic DNA is essential to amplication. Furthermore, the degeneracy of the primers should be as low as possible and the desired length of the amplified fragment had better less than 2 kb. This method obviates the cumbersome chromosome walking process, and the obtained DNA fragments can be directly used for directed evolution mediated by DNA shuffling without sequencing.更多还原