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.

The gene APE1547 of the aerobic thermophilic Aeropyrum pernix K1 encoding 582 amino acid residues was cloned into Escherichia coli. BL21 (DE3) by using vector pET11a with a T7 promoter. An alignment of similarity analysis of APE1547 with protein sequences from A. pernix K1 databank revealed that it showed a lipase motif and low homology with the known thermophilic esterases. However, it had a high degree homology with several acyl amino acid-releasing enzymes. After purified by ion exchange chromatography and gel filtration chromatography, the recombinant protein showed both esterase activity and acylamino acid-releasing enzyme (AARE) activities. The optimum of temperature and pH of the esterase activity are 90℃ and 8.0, respectively. The recombinant protein showed the hydrolytic activity for a wide range of substrates, such as p-nitrophenyl alkanoate esters of varying alkyl chain lengths, pNA-labelled amino acid and peptide. The highest activity was observed for the substrate p-nitrophenyl caprylate. The recombinant enzyme was extremely stable and protein concentration-dependent. Its half-life at 90℃ was over 160h. at the concentration of 2.14 mg/ml, which renders this new esterase very attractive for biotechnological applications.

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.

It has been shown that highly conserved residues that form crucial structural elements of the catalytic apparatus may be used to account for the evolutionary history of enzymes. Using saturation mutagenesis, we investigated the role of a conserved residue (Arg526) at the active site of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 in substrate discrimination and catalytic mechanism. This enzyme has both peptidase and esterase activities. The esterase activity of the wild-type enzyme with p-nitrophenyl caprylate as substrate is 7 times higher than the peptidase activity with Ac-Leu-p- nitroanilide as substrate. However, with the same substrates, this difference was increased to 150-fold for mutant R526V. A more dramatic effect occurred with mutant R526E, which essentially completely abolished the peptidase activity but decreased the esterase activity only by a factor of 2, leading to a 785-fold difference in the enzyme activities. These results provide rare examples that illustrate how enzymes can be evolved to discriminate their substrates by a single mutation. The possible structural and energetic effects of the mutations on kcat and Km of the enzyme were discussed based on molecular dynamics simulation studies.

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.