The development of recombinant DNA has made it feasible to produce a wide range of valuable protein products in the bacterium Escherichia coli. Extraction of intracellular protein from E. coli is traditionally achieved by mechanical, chemical or enzymatic disruption technology. In this study, thermolysis, which differs from the traditional ones, is presented for disruption of E. coli cells to release recombinant thermostable enzyme. Heat treatment of E. coli at 80℃ is highly effective to destroy the integrity of the bacterial cell wall and release the recombinant thermostable enzyme. At the same time of disruption, the recombinant thermostable enzyme was partially purified. Moreover, thermolysis was carried out in fermentation broth in situ, which may make it a more applicable approach for industrial-scale processes.

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.

The histone protein HPhA from the hyperthermophilic archaeon Pyrococcus horikoshii, shows hyperthermostability, as required for optimal growth of the organism at 95℃. The structure of recombinant P. horikoshii HPhA has been determined to 2.3A resolution by molecular replacement, and refined to Rwork and Rfree values of 20.7% and 27.3%, respectively. The HPhA monomer structure is characterized by the histone fold and assembles into a homodimer like other archaeal histones. There are four anions found in the dimer structure, giving rise to clues as to where DNA might bind. A detailed comparison of four known structures of archaeal histones, which evolve and exist under different temperatures, shows that the thermophilic archaeal histone HPhA has a larger hydrophobic contact area, an increased number of hydrogen bonds and a reduced solvent-accessible area. We also observe a unique network of tyrosine residues located at the crossover point of the two HPhA monomers, which locks them together and stabilizes the dimer. These factors together account for the increased thermal stability.

A histone-like gene, PHS051 from hyperthermophilic archaeon Pyrococcus horikoshii OT3 strain, was cloned, sequenced, and expressed in Escherichia coli. The recombinant histone, HPhA, encodes a protein of 70 amino acids with a molecular weight of 7868 Da. Amino acid sequence analysis of HPhA showed high homology with other archaeal histones and eukaryal core histones. The HPhA was purified to homogeneity by heat precipitation and affinity chromatography. Gel electrophoresis mobility shift assays demonstrate that the purified HPhA has high affinity to DNA. The complex of the HPhA and DNA allows DNA to be protected from cleavage by the restriction enzyme TaqI at 65℃. Circular dichroism spectra reveal that the conformation of the recombinant histone HPhA becomes looser when temperatures increase from 25 to 90℃. The HPhA has inherited a remarkable thermostability especially in the presence of 1M KCl and retained DNA binding activity at extreme temperature, which is consistent with our previous report about its structure stability analyzed by X-ray crystallography.

A novel plasmid, pBSR2, was constructed by incorporating a strong lipase promoter and a terminator into the original pBD64. A mature lipase gene from Bacillus subtilis strain IFFI10210, an existing strain for lipase expression, was cloned into the plasmid pBSR2 and transformed into B. subtilis A.S.1.1655. Thus, an overexpression strain, BSL2, was obtained. The yield of lipase is about 8.6mg protein/g of wet weight of cell mass and 100-fold higher than that in B. subtilis strain IFFI10210. The recombinant lipase was puriWed in a three-step procedure involving ammonium sulfate fractionation, ion exchange, and gel Wltration chromatography. Characterizations of the puriWed enzyme revealed a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, maximum activity at 43℃ and pH 8.5 for hydrolysis of p-nitrophenyl caprylate. The values of Km and Vm were found to be 0.37mM and 303 μmolmg-1·min-1, respectively. The substrate speciWcity study showed that p-nitrophenyl caprylate is a preference of the enzyme. The metal ions Ca2+, K+, and Mg2+ can activate the lipase, whereas Fe2+, Cu2+, and Co2+ inhibited it. The activity of the lipase can be increased about 48% by sodium taurocholate at the concentration of 7mM and inhibited at concentrations over 10mM.