The aim of this work was to evaluate the capability of corn steep liquor being a low cost substrate on the recombinant protein production by cultivating recombinant Escherichia coli and increasing the production of hyperthermophilic esterase (HE). The effect of corn steep liquor, mineral salt and trace metals on hyperthermophilic esterase production was investigated by means of a five-level three-factor central composite rotatable design. Optimized values of the factors were determined and a maximum hyperthermophilic esterase production of 251.39 U/ml was obtained. This value equaled the yield by yeast extract and peptone medium on the whole.

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.

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.

极端酶具有超常的生物学稳定性，能够在极端温度、pH、压力和离子强度下表现出生物学活性，因此极端酶为生物催化和生物转化提供了良机。新的极端物种的发现、基因组序列的确定及基因工程技术的应用，加快了发现和制备新酶的进程。蛋白质工程和定向进化技术进一步改善酶的活性和特异性，促进了极端酶的工业应用。对极端酶的研究加深了人们对酶稳定性机制的理解，丰富了分子进化理论,

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.