南海深海新颖低温脂肪酶的克隆、表达及酶学性质鉴定

Cloning,Expression and Characterization of a Novel Psychrophile Lipase from the Deep Sea of the South China Sea

从南海深海放线菌Pseudonocardia antitumoralis SCSIO 01299中克隆一个脂肪酶基因lipase B5。lipase B5基因片段大小为972bp,编码的蛋白质具有323个氨基酸残基并与Pseudonocardia sp.HH130629-09中假定脂肪酶有98%的同源性。以p ET28a（＋）为载体,将重组质粒转化到E.coli BL21（DE3）中,实现lipase B5高效表达,并利用Ni-NTA亲和层析纯化lipase B5。Lipase B5最适反应底物为对硝基苯酚癸酸酯（p-NPD）,最适温度为30℃,最适反应p H为7.5。Lipase B5催化p-NPD水解反应的活性达到140.14U/mg,Vmax和Km分别为109.8μmol/min、0.976mmol/L,催化橄榄油的活力为32.019 7U/mg。Lipase B5在p H7.5~8.5保持良好的p H稳定性;在10～20℃低温下保持较高的酶活力,在10~40℃内具有温度稳定性。Lipase B5对大部分金属离子有很好的耐受性,低浓度的Li^＋、Mg^2＋对该酶活性有促进作用。Lipase B5对高浓度的Na Cl有很好的耐受性,在100mmol/L的Na Cl存在下,酶活性大约保持在103%。Lipase B5对多种短链醇类有机溶剂和表面活性剂有很好的耐受性,Triton X-100、Tween-80和Tween-20对lipase B5酶有激活作用。

A gene lipase B5 encoding a lipase,was cloned from the genome of Pseudonocardia antitumoralis SCSIO 01299 isolated from the deep sea of the South China Sea. Lipase B5 contains 972 bp and encodes a putative lipase of 323 amino acids. Lipase B5 exhibits 98% similarity with a putative lipase from Pseudonocardia sp.HH130629-09. Lipase B5 was cloned into expression vector p ET28a（ ＋）,expressed in E. coli BL21（ DE3） and further purified using Ni-NTA affinity chromatography. The optimal substrate tested of lipase B5 was p-NPD,the optimal working temperature was 30℃,the optimal working p H was 7. 5. Vmaxand Kmwere 109. 8μmol / min and0. 976 mmol / L,respectively. The hydrolysis activity of lipase B5 toward olive oil was 32. 019 7U / mg. Lipase B5 could remain high activities under p H 7. 5 ~ 8. 5 and 10 ~ 20℃. Lipase B5 was thermal-stable at 10 ～ 40℃.Multiple metal ions could not affect the activities of Lipase B5 and low-concentration Li＋and Mg2 ＋could stimulate the activity of Lipase B5. Lipase B5 could stand high concentration of Na Cl and could still remain 103% of its original activity in the presence of 100 mmol / L Na Cl. Lipase B5 could also stand many short-chain alcohols and surfactants. Triton X-100,Tween-80 and Tween-20 could even stimulate the activities of Lipase B5.