To enhance the enantioselectivity of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1 (APE1547), a directed evolution approach is employed to generate mutant library from the native enzyme. A mutation (TBC26) is identified after one round of epPCR. The enantioselectivity of TBC26 is increased up to 2.6-fold compared to that of wild type enzyme. TBC26 contains five amino acid substitutions (R11G, L36P, V223A, I551L, A564T). The five mutation sites are spatially distant to the catalytic center. According to the published crystal structure ofWTand considering the changes of secondary and tertiary structure, here we try to explain the change of enantioselectivity of the TBC26. The results suggest that the change of enantioselectivity of enzyme has a close relationship to the configuration of the enzyme.

An open reading frame of the hyperthermophilic archaeon Aeropyrum pernix K1 APE2325, which composed of 474 bases, was cloned and expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL. The recombinant protein was puriWed by Ni-chelation aYnity chromatography. It showed a single band with a molecular mass of 18kDa in SDS–PAGE. The puriWed enzyme exhibited both phospholipase A2 and esterase activities with the optimal catalytic temperature at 90℃. The enzyme activity was Ca2+-independent. Kinetic analysis revealed its Km, kcat, and Vm for the p-nitrophenyl propionate substrate were 103μM, 39s-1, and 249μmol/min/mg, respectively. The recombinant protein was thermostable and its half-life at 100℃ was about 1h.

嗜热菌组蛋白基因序列中精氨酸密码子在大肠杆菌中的表达率极低，限制了组蛋白的大量获得。运用分子生物学技术，在传统PCR定点突变技术的基础上进行改进，参照古核嗜热菌组蛋白基因序列和大肠杆菌偏爱密码子表，设计了一对含有5个精氨酸突变密码子的单链DNA，全长为115nt，在3'端有20nt互补序列。将单链DNA在94℃变性，45℃复性，使其互补结合，72℃进行延伸反应，得到含有突变位点的组蛋白基因；再以此基因为模板设计相应的一对引物进行PER扩增反应，得到大量组蛋白突变基因。该研究为一些序列已知，片段较小基因的多点突变提供了一种简单、易行的方法。

A homolog to the eubacteria inorganic pyrophosphatase (PPase, EC 3.6.1.1) was found in the genome of the hyperthermophilic archaeon Pyrococcus horikoshii. This inorganic pyrophosphatase (Pho-PPase) grows optimally at 88℃. To understand the structural basis for the thermostability of Pho-PPase, we have determined the crystal structure to 2.66 A resolution. The crystallographic asymmetric unit contains three monomers related by approximate threefold symmetry, and a hexamer is built up by twofold crystallographic symmetry. The main-chain fold of Pho-PPase is almost identical to that of the known crystal structure of the model from Sulfolobus acidocaldarius. A detailed comparison of the crystal structure of Pho-PPase with related structures from S. acidocaldarius, Thermus thermophilus, and Escherichia colishows significant differences that may account for the difference in their thermostabilities. A reduction in thermolabile residues, additional aromatic residues, and more intimate association between subunits all contribute to the larger thermophilicity of Pho-PPase. In particular, deletions in two loops surrounding the active site help to stabilize its conformation, while ion-pair networks unique to Pho-PPase are located in the active site and near the C-terminus. The identification of structural features that make PPases more adaptable to extreme temperature should prove helpful for future biotechnology applications.

首先采用紫外线与亚硝基胍两种传统微生物诱变方法对干酪乳杆菌进行诱变，经低pH平板、碳酸钙平板和摇瓶试验获得了5株耐酸性提高的突变菌株。以获得的突变菌株为出发菌株，应用灭活双亲原生质体融合后致死损伤得到互补获得活性融合子的方法，对其进行基因组改组，经过低pH平板、碳酸钙平板和摇瓶筛选，获得4株可以在pH3.8平板上旺盛生长且产酸量较高的改组菌株。将改组菌株与原始菌株分别于pH 3.8和3.4的YE液体培养基中培养，改组菌株能够在原始菌株无法生存的pH条件（pH 3.4）下生长。在pH 3.8的条件下，对改组菌株与原始菌株的发酵特征进行比较，37℃发酵48小时后，改组菌株产酸量为原始菌株的2.4倍，表明基因组改组技术能有效提高多基因调控表型的进化。