来源于青霉XMZ-9两个低温脂肪酶的基因克隆、原核表达与性质测定

Gene cloning,expression and characterization of two cold-adapted lipases from Penicillium sp.XMZ-9

低温脂肪酶在低温条件下仍具有较高活性,在食品添加剂、洗涤添加剂及有机合成等产业具有非常独特的应用前景。从低温菌株中分离低温脂肪酶基因是开发新的低温脂肪酶的有效手段。首先利用油脂同化平板与三丁酸甘油酯-维多利亚蓝平板从冰川土样中筛选分离获得一株具有较高脂肪酶活性的真菌,18S rDNA鉴定其属于青霉属,命名为Penicillium sp.XMZ-9。根据真菌脂肪酶多序列比对获得的保守区,设计简并引物,利用降落PCR与染色体步移的方法从Penicillium sp.XMZ-9中克隆到2个完整的脂肪酶基因,分别记为LipA与LipB。LipA全长1 014 bp,无内含子,编码337个氨基酸。而LipB全长1 232 bp,cDNA长1 122 bp,含有2个内含子,编码373个氨基酸。将两基因的cDNA序列克隆到pET30a(+)载体上,转化大肠杆菌Escherichiacoli BL21(DE3)。经低温诱导表达后,LipA大部分表达为包涵体,包涵体经复性后具有脂肪酶活性,并表现出低温适应性;LipB则大部分表达为可溶性蛋白,Ni-亲和层析柱纯化后,其亦具有低温脂肪酶活性。青霉菌株XMZ-9的获得与低温脂肪酶的克隆表达研究,为研究低温菌株与低温酶的适冷机制提供了宝贵的资源,也为进一步开发利用低温脂肪酶奠定了基础。

Cold-adapted lipases are attractive biocatalysts that can be used at low temperatures as additives in food products, laundry detergents, and the organic synthesis of chiral intermediates. Cold-adapted lipases are normally found in microorganisms that survive at low temperatures. A fungi strain XMZ-9 exhibiting lipolytic activity was isolated from the soil of glaciers in Xinjiang by the screening plates using 1% tributyrin as the substrate and Victoria blue as an indicator. Based on morphological characteristics and phylogenetic comparisons of its 18S rDNA, the strain was identified as Penicillium sp. The partial nucleotide sequences of these two lipase related genes, LipA and LipB, were obtained by touchdown PCR using the degenerate primers designed according to the conservative domains of lipase. The full-length sequences of two genes were obtained by genome walking. The gene lipA contained 1 014 nucleotides, without any intron, comprising one open reading frame encoding a polypeptide of 337 amino acids. The gene lipB comprised two introns （61 bp and 49 bp） and a coding region sequence of 1 122 bp encoding a polypeptide of 373 amino acids, eDNA sequences of both lipA and lipB were cloned and expressed in Escherichia coli BL21 （DE3）. The recombinant LipA was mostly expressed as inclusion bodies, and recovered lipase activity at low temperature after in vitro refolded by dilution. Differently, the recombinant LipB was expressed in the soluble form and then purified by Ni-NTA affinity chromatography Column. It showed high lipase activity at low temperature. These results indicated that they were cold-adapted enzymes. This study paves the way for the further research of these cold-adapted lipases for application in the industry.