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Abstract: The chemical modification and immobilisation of lipase B from Candida antarctica (CalB) onto three different types of mesoporous silicate (MPS) were undertaken. Soluble CalB was modified by two bifunctional reagents, ethylene glycol bis(succinimidyl succinate) (EGNHS) and glutaraldehyde, and by the monofunctional citraconic anhydride. Both chemically modified and untreated enzyme were then immobilised onto SBA-15-, CNS- and MCM-type MPS by adsorption. Thermal stabilities of chemically modified CalB in solution and of the immobilised preparations were evaluated and compared. Citraconic anhydride dramatically reduced the stability of CalB whereas both bifunctionals yielded an eightfold increase in stability over the native free CalB at 70°C. Following immobilisation of the EGNHS-treated preparation onto CNS-MPS, the stability gain increased to over 60-fold and this combination proved to be the most effective stabilisation strategy. CalB also showed a preference for MPS with larger pores, namely SBA-15. Immobilisation of CalB in alginate beads was also stabilising. [Copyright &y& Elsevier]

Abstract: Laccase from two different source organisms, Myceliophthora thermophila and Trametes hirsuta, were subjected to chemical modification in solution by (i) two bifunctional reagents, ethylene-glycol-N-hydroxy succinimide (EGNHS) and glutaraldehyde and (ii) by the monofunctional citraconic anhydride. The untreated and chemically modified forms of both enzymes were then immobilised onto three different types of mesoporous silicate (MPS) particle (MCM, CNS and SBA-15). Thermal stabilities of native, modified-soluble and immobilised laccases were then evaluated. Although the two laccases have similar lysine contents, those of M. thermophila are clearly more amenable to chemical modification. Treatment of the M. thermophila enzyme with EGNHS led to a 8.7-fold increase in thermal stability over the free soluble enzyme while glutaraldehyde gave a 5.7-fold increase. Increased activity of M. thermophila laccase occurred only with citraconic anhydride modification (a 3-fold increase), while the glutaraldehyde modification marginally increased the activity of the T. hirsuta enzyme (by 1.2-fold). Upon immobilisation onto MPS, the greatest increase in stability was for the glutaraldehyde-treated M. thermophila preparation on SBA-15 (24-fold over the soluble enzyme). Chemical modification of laccase from T. hirsuta with both glutaraldehyde and EGNHS gave only a 2-fold increase in stability, increasing >4-fold upon immobilisation onto SBA-15 and MCM-41/98. [Copyright &y& Elsevier]

Abstract: Proline-specific dipeptidyl peptidase-like (DPP IV; EC 3.4.14.5) activity in bovine serum has attracted little attention despite its ready availability and the paucity of useful proline-cleaving enzymes. Bovine serum DPP IV-like peptidase is very tolerant of organic solvents, particularly acetonitrile: upon incubation for 1h at room temperature in 70% acetonitrile, 47% dimethylformamide, 54% DMSO and 33% tetrahydrofuran (v/v concentrations) followed by dilution into the standard assay mixture, the enzyme retained half of its aqueous activity. As for thermal performance in aqueous buffer, its relative activity increased up to 50°C. Upon thermoinactivation at 71°C, pH 8.0 (samples removed periodically, cooled on ice, then assayed under optimal conditions), residual activities over short times fit a first-order decay with a k-value of 0.071±0.0034min−1. Over longer times, residual activities fit to a double exponential decay with k 1 and k 2 values of 0.218±0.025min−1 (46±4% of overall decay) and 0.040±0.002min−1 (54±4% of overall decay), respectively. The enzyme''s solvent and thermal tolerances suggest that it may have potential for use as a biocatalyst in industry. Kinetic analysis with the fluorogenic substrate Gly-Pro-7-aminomethylcoumarin over a range of pH values indicated two pK values at 6.18±0.07 and at 9.70±0.50. We ascribe the lower value to the active site histidine; the higher may be due to the active site serine or to a free amino group in the substrate. [Copyright &y& Elsevier]