PDF(Pubmed)
Abstract：
Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.
摘要：
仅报道了少数产生羧酸酯酶的嗜盐古细菌。对古细菌酯酶的生物催化特性的有限研究主要是由于它们在天然生物中的产量非常低。克隆了一个编码盐碱菌NRC-1羧酸酯酶的基因，并在Haloferax火山中成功表达。重组羧酸酯酶(rHsEst)经亲和层析纯化,收率为81%,通过SDS-PAGE(33kDa)估算其分子量。rHsEst的最佳动力学参数是使用对硝基苯基戊酸酯作为底物（KM=78µM，kcat=0.67s-1)。rHsEst对大多数测试的金属离子和一些溶剂(乙醚，正己烷,正庚烷)。使用硅藻土545有效地固定纯化的rHsEst。通过底物特异性研究证实了rHsEst的酯酶活性。rHsEst活性位点中丝氨酸残基的存在通过用PMSF抑制来揭示。游离rHsEst的最佳活性的pH为8，而对于固定化rHsEst，最大活性是在8至10之间的pH范围内。rHsEst的固定化增加了其热稳定性，嗜盐和对EDTA等抑制剂的保护，BME和PMSF。值得注意的是,固定化rHsEst在高达5M的NaCl浓度下稳定且有活性。固定化rHsEst的这些生化特性揭示了其作为生物催化剂用于工业应用的潜力。
