地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增、克隆及序列测定

在地衣芽孢杆菌NCIB 6816菌株碱性蛋白酶基因已知序列的基础上,通过设计合适的引物,利用PCR(Polymerase Chain Reaction)技术从地衣芽孢杆菌2709菌株的柒色体DNA中扩增了2709碱性蛋白酶的编码序列。对两个克隆的PCR片段的全序列分析结果显示,2709碱性蛋白酶的编码序列同相应的NCIB 6816序列相比有3％左右的碱基组成差异。由此推定的2709碱性蛋白酶的氨基酸序列肯定了2709碱性蛋白酶属典型的subtilisin Carlsberg类,同时还表明来源于不同地衣芽孢杆菌菌株的subtilisin Carlsberg存在着若干氨基酸组成上的差异。

植物根际芽胞杆菌菌种资源采集及其具有过水解催化活性水解酶基因的克隆

[背景]芽胞杆菌源枯草杆菌蛋白酶(subtilisin carlsberg)、乙酰基木聚糖酯酶(acetyl xylan esterase)和头孢菌素乙酰水解酶(cephalosporin acetyl hydrolase)具有较高的过水解催化活性,有商业开发价值。[目的]挖掘芽胞杆菌菌株中具有过水解酶催化活性的水解酶蛋白基因,为后续制备过水解酶及酶法合成过氧乙酸奠定基础。[方法]利用定向筛选培养基,从植物根际及纳豆产品中筛选产蛋白酶芽胞杆菌候选菌株,并利用RFLP及16S rRNA基因对其进行鉴定。从蛋白酶高产芽胞杆菌菌株中克隆枯草杆菌蛋白酶、乙酰木聚糖醋酶和头孢菌素乙酰水解酶的全长基因。[结果]从植物根际土壤及纳豆产品中共分离到85个候选菌株,RFLP及16S rRNA基因鉴定结果表明候选菌株均为芽胞杆菌,分别属于Bacillus subtilis、Bacillus cereus、Bacillus pumilus和Bacillus megaterium四个类群。从B.subtilis NSYT-3克隆的枯草杆菌蛋白酶基因编码的多肽链全长381个氨基酸,从B.pumilus OSLJ-3克隆得到的乙酰基木聚糖酯酶基因编码的多肽链全长320个氨基酸,从B.subtilis NSYT-3克隆的头孢菌素乙酰水解酶基因编码的多肽链全长318个氨基酸,3D结构模拟表明这3个酶蛋白均具有α/β水解酶折叠家族蛋白结构特点。[结论]芽胞杆菌源具过水解催化活性水解酶基因的克隆,为后续开发酶法合成过氧乙酸工艺奠定了基础。

Reverse micelles extraction ofnattokinase:From model system to real system

Nattokinase is a novel fibrinolytic en- zyme, which is homologous to Subtilisin Carlsberg. In this paper, Subtilisin Carlsberg was taken as a model protein of nattokinase. Effects of pH, ionic strength, concentration of isopropanol on the extraction of Subtilisin Carlsberg with AOT/isooctane reverse mi- celles system were investigated. Further, the process of reverse micelles extraction of nattokinase from fermentation broth was studied. By taking the reverse micelles of AOT/isooctane as extractant to perform a full extraction cycle, it was found that about eighty percent of the total activity of nattokinase in the fer- mentation broth could be recovered and the purifica- tion factor was about 2.5. Homologous protein could be reasonably used as model protein of a target protein.