摘要
以乙醇耐受力较强的酿酒酵母为受体菌,构建了能够分泌菊粉酶的基因工程菌并进行了菊芋粉的生料发酵。首先,以马克斯克鲁维酵母Kluyveromyces marxianus中的基因组DNA为模板,PCR扩增菊粉酶编码基因inu,分别使用菊粉酶自身启动子和酵母磷酸甘油激酶（Phosphoglycerate kinase,pgk）启动子,构建重组表达质粒HO/p-inu和HO/pgk-inu。经NotⅠ线性化后,采用电击法转化酿酒酵母工业菌株Saccharomyces cerevisiae 6525,分别得到含菊粉酶基因的阳性菌株HI6/1~HI6/10及HPI6/1~HPI6/3。实验结果表明HI6/6及HPI6/3的菊粉酶活力较高,分别为86.0 U/mL和23.8 U/mL,是出发菌株的4.6倍和1.5倍。进而以粗菊芋粉生料为底物进行了乙醇发酵,当浓度为200 g/L时,重组菌株HI6/6和HPI6/3的发酵终点乙醇浓度分别为55 g/L和52 g/L,糖醇转化率分别为0.495和0.453,达到理论值的96.9%和88.6%。这些研究工作为非粮作物菊芋生产燃料乙醇奠定了良好的基础。
Ethanol fermentation from Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae strains expressing the inulinase gene（inu） from Kluyveromyces marxianus was investigated.The inu native and pgk promoters were used to drive the expression of the inu gene,and the inulinase was expressed as an extracellular enzyme.All positive clones（confirmed by PCR） were able to express inulinase as measured by enzyme activity in the culture supernatant,among which two clones HI6/6 and HPI6/3 were selected,and their inulinase activity and ethanol fermentation performance were compared with their wild type.The inulinase activities of 86 and 23.8 U/mL were achieved,which were 4.6-fold and 1.5-fold higher than that of the wild type.Furthermore,ethanol fermentation was carried out with the recombinants and medium containing 200 g/L raw Jerusalem artichoke meal,and ethanol concentrations of 55 g/L and 52 g/L were obtained,with ethanol yields of 0.495 and 0.453,respectively,equivalent to 96.9% and 88.6% of the theoretical value.
出处
《生物工程学报》
CAS
CSCD
北大核心
2011年第7期1032-1039,共8页
Chinese Journal of Biotechnology
基金
国家自然科学基金(No.20806014)
辽宁省优秀人才计划(No.2008RC57)资助~~
关键词
菊粉酶基因
整合表达
酿酒酵母
菊芋
乙醇
inulinase gene
integrative expression
Saccharomyces cerevisiae
Jerusalem artichoke
ethanol
