D 乳酸高产菌菊糖芽胞乳杆菌Y28磷酸果糖激酶基因在大肠杆菌中的克隆和表达

摘要

The gene encoding phosphofructokinase ( pfk ) amplified by PCR using Sporolactobacillus inulinus Y2-8 genomic DNA as a template was 960 bp.Alignments of the amino acid sequences revealed that compared with phosphofructokinase ( PFK) from other representative lactic acid bacteria,PFK from S.inulinus Y2-8 had strictly conserved substrate binding sites,but different allosteric sites.The pfk gene was cloned into the vector pSE380,producing recombinant strain E-pSE-pfk.After optimization,the PFK specific activity of recombinant strain reached 4.89 U/mg, thus it was 4.79-fold higher than that of primary expression condition.The result showed that low-temperature induction was helpful to the soluble expression of PFK from S.inulinus in E.coli.%以D 乳酸高产菌菊糖芽胞乳杆菌Y28基因组DNA为模板，通过PCR扩增得到960 bp的磷酸果糖激酶基因（ pfk）。氨基酸序列比对分析表明，该磷酸果糖激酶（ PFK）与其他乳酸菌PFK具有保守的底物结合位点，但是其变构效应物结合位点存在差异。将pfk基因克隆到表达载体pSE380上，获得重组菌E pSE pfk。进一步通过诱导条件的优化，重组菌的PFK比酶活达到4�89 U/mg，是优化前的4�79倍。采用低温诱导策略有助于实现菊糖芽胞乳杆菌pfk基因在大肠杆菌中可溶性表达。