| The great potential of enzymes in organic synthesis is well documented. But enzymatic catalysis has not been adopted on wide scale due to four major problems: Poor operational stability, inadequate selectivity for unnatural synthetic substrates, low volumetric productivity and high cost. This paper dealt with making the novel catalyst in form of Cross-Linked Enzyme Crystals (CLECs) combined with crystallization of enzyme and chemical cross-linking crystals. CLECs offer a set of properties such as retaining its activity in harsh environments and high stereoselectivity in organic synthesis.Crude lipase from Candida rugosa (CRL) is a poorly to moderately enantioselective catalyst for chiral carboxylic acid. We report a simple 2-propanol treatment increase the activity and enantioselectivity of crude CRL by converting the closed conformational form to the open conformational form.The CRL-CLEC production process consists of two major steps: batch crystallization of enzymes, and chemical cross-linking crystals. Both steps are equally critical in ensuring the stability and good mechanical properties of catalyst. The enzyme crystals obtained by the vapor diffusion method independently using 2-methyl-2,4-pentanediol as a precipitant. The better conditions are as follow: the concentration of 2-methyl-2,4-pentanediol 40%, pH5.0-5.5, temperature 10℃. The crystals of CRL cross-linked with a bifunctional agent such as glutaraldehyde. The conditions are as follow: the concentration of glutaraldehyde 1%, pH5.0, temperature 25 ℃ and reactive time 60min. CLECs not only increase stability of temperature and pH, but also attain the activity in aqueous-organic mixtures such as 50% tetrahydrofuran and 50% dimethyl sulfoxide.Studies attempting to relate the molecular architecture of an enzyme to its function are confronted by a fundamental problem. We study the catalytic kinetics of enzyme in the crystalline state. The apparent kinetic constant of CRL-CLECs is...
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