黑曲霉内切聚半乳糖醛酸酶A基因在大肠杆菌中的表达及产物酶学性质研究  

Expression of Aspergillus niger endo-polygalacturonase A gene in Escherichia coli and the hydrolytic properties

作　　者：刘明启[1] 戴贤君[1] 白兰芳[1] 

机构地区：[1]中国计量学院生命科学学院,浙江杭州310018

出　　处：《中国计量学院学报》2012年第4期403-409,共7页Journal of China Jiliang University

基　　金：浙江省科技计划资助项目(No.2012C22079);浙江省自然科学基金资助项目(No.Y3090503);杭州市科技发展计划资助项目(No.20110232B81)

摘　　要：采用Trizol法提取黑曲霉(Aspergillus niger JL-15)的总RNA,RT-PCR扩增到黑曲霉内切聚半乳糖醛酸酶A基因(pgaA),pgaA全长1113bp,编码370个氨基酸残基.pgaA经EcoRI和NotI双酶切,定向插入到表达载体pET-32a(+)中,并转化Escherichia coli Rosetta-gami B(DE3),重组子经IPTG诱导,其表达产物(rePGA)果胶酶活性最高为637.0U/mg.酶学性质研究表明,rePGA的最适温度为60℃,热稳定性较差,最适pH为pH 5.0;Ca2+对其活性具有显著促进作用,而Fe3+和Mg2+对其活性具有显著抑制作用;SDS-PGAE结果表明,rePGA的相对分子质量约为40 500Da;rePGA的米氏常数(Km)和最大反应速度(Vmax)分别为4.05mg/mL和39.37μmol/(mL.min),rePGA能快速降低果胶溶液的粘度,符合其内切作用模式特点.The complementary DNAs(cDNAs) were synthesized from total RNA from Aspergillus niger JL-15 by Reverse Transcription.The endo-polygalacturonase A gene(pgaA) was amplified from the cDNAs and sequenced.The open reading frame(ORF) of pgaA was 1113 bp,encoding a polypeptide of 370 amino acids with the predicted molecular weight of 38.8 kDa.The pgaA was cloned into pUCm-T vector and digested by EcoRI and NotI.The recovered pgaA was ligated into pET-32a(+) and the obtained recombinant plasmid was transformed into competent Escherichia coli Rosetta-gami B(DE3).After induction by IPTG,the highest activity of the recombinant pectinase(rePGA) in culture supernatant was 637.0 U/mg.The optimum temperature and pH of the rePGA were 60 ℃ and pH 5.0,respectively.Ca2+ could enhance the activity of the enzyme,but Fe3+and Mg2+ could inhibit the activity of the enzyme significantly.SDS-PAGE analysis showed that the molecular mass of rePGA was about 40.5 kDa.The rePGA exhibited Km and Vmax values of 4.05 mg/mL and 39.37 μmol/(mL·min),respectively.The rePGA cleaved pectin and caused a rapid decrease in viscosity,which revealed it is an endo-acting pectinase.

关 键 词：黑曲霉 内切聚半乳糖醛酸酶 克隆 表达 酶学性质 

分 类 号：TQ925[轻工技术与工程—发酵工程]