Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
Bioresour Technol. 2011 Jul;102(13):6972-81. doi: 10.1016/j.biortech.2011.03.083. Epub 2011 Mar 29.
The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65°C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50°C for more than 150 min.
成熟的 ARM 脂肪酶基因被克隆到 pTrcHis 表达载体中，并在大肠杆菌 TOP10 宿主中过表达。在诱导后 18 小时用 1.0mM IPTG 进行培养，可获得最佳的脂肪酶表达，其酶活比野生型高约 1623 倍。通过使用固定化金属亲和层析，可以快速、高效地一步纯化 His 标记的重组脂肪酶，回收率为 63.2%，纯化倍数为 14.6。纯化的脂肪酶被鉴定为一种高活性（7092 U mg(-1)）、丝氨酸水解酶、热稳定、耐有机溶剂的 1,3-特异性脂肪酶，分子量约为 44 kDa。该酶是一种单体，其结构中含有二硫键，但不是金属酶。ARM 脂肪酶在较宽的温度和 pH 范围内具有活性，最适脂肪酶活性在 pH 8.0 和 65°C。该酶在 pH 6.0-7.0、50°C 下保留 50%的残余活性超过 150 分钟。
