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.

The enzyme (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine from the corresponding racemic methyl ester. Reaction conditions are optimized to enhance the enantioselectivity. The effects of various racemic alkyl esters, substrate concentration, operating temperature, pH of the aqueous medium and organic solvents on activity and enantioselectivity of BSL2 for kinetic resolution are also studied. A high enantiomeric ratio (E=60.7) is reached in diisopropyl ether/water (10%, v/v) and the enantioselectivity is about 22-fold higher than that in pure buffered aqueous solution. The results show that the reaction medium greatly influences BSL2 reaction and its enantioselectivity in the hydrolysis of racemic methyl ester.

Acylpeptide hydrolases (APH; also known as acylam-ino acid releasing enzyme) catalyze the removal of an N-acylated amino acid from blocked peptides. The crystal structure of an APH from the thermophilic arch-aeon Aeropyrum pernix K1 to 2.1 A resolution confirms it to be a member of the prolyl oligopeptidase family of serine proteases. The structure of apAPH is a sym-metric homodimer with each subunit comprised of two domains. The N-terminal domain is a regular seven-bladed β-propeller, while the C-terminal domain has a canonical α/β hydrolase fold and includes the active site and a conserved Ser445-Asp524-His556 catalytic triad. The complex structure of apAPH with an organo- phosphorus substrate, p-nitrophenyl phosphate, has also been determined. The complex structure unam- biguously maps out the substrate binding pocket and provides a basis for substrate recognition by apAPH. A conserved mechanism for protein degradation from archaea to mammals is suggested by the structural features of apAPH.

Inorganic pyrophosphatase (PPase; EC 3.6.1.1) from the hyperthermophile Pyrococcus horikoshii was crystallized by the hanging-drop vapour-diffusion method at pH 5.0 using polyethyleneglycol 4000 as the precipitant. The crystal belongs to space group P21212, with unitcell parameters a=71.7, b=86.5, c=92.5 A, α=β=γ=90°. There are two molecules in the asymmetric unit. The crystals were stable during exposure to X-rays and a full set of X-ray diffraction data was collected to 2.7 A Ê resolution in-house.

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.