The gene of MTSase (maltooligosyltrehalose synthase) from \%Sulfolobus acidocaldarius\% ATCC49426 was amplified by PCR.The primers were designed according to the published sequence of homologous gene from \%Sulfolobus...The gene of MTSase (maltooligosyltrehalose synthase) from \%Sulfolobus acidocaldarius\% ATCC49426 was amplified by PCR.The primers were designed according to the published sequence of homologous gene from \%Sulfolobus acidocaldarius \%ATCC33909.This gene was inserted into the plasmid pBV220 and the resultant recombinant plasmid pBV220\|GT was transformed to \%E.coli\% DH5α.The activity of recombinant enzyme was about 10u/g(wet cell).In order to improve the expression level of target protein,some nucleotides in the 3′ and 5′ of the gene were modified to optimize the second structure of mRNA by PCR amplification using the new primers devised according to the biosoftware GOLDKEY2.0.As a result,the activity of recombinant enzyme increase to 19.8u/g(wet cell).Then,the helping plasmid pUBS520 which carried the gene encoding the tRNA of rare codons AGG and AGA was transformed to the recombinant strain.But it took little effect.展开更多
文摘The gene of MTSase (maltooligosyltrehalose synthase) from \%Sulfolobus acidocaldarius\% ATCC49426 was amplified by PCR.The primers were designed according to the published sequence of homologous gene from \%Sulfolobus acidocaldarius \%ATCC33909.This gene was inserted into the plasmid pBV220 and the resultant recombinant plasmid pBV220\|GT was transformed to \%E.coli\% DH5α.The activity of recombinant enzyme was about 10u/g(wet cell).In order to improve the expression level of target protein,some nucleotides in the 3′ and 5′ of the gene were modified to optimize the second structure of mRNA by PCR amplification using the new primers devised according to the biosoftware GOLDKEY2.0.As a result,the activity of recombinant enzyme increase to 19.8u/g(wet cell).Then,the helping plasmid pUBS520 which carried the gene encoding the tRNA of rare codons AGG and AGA was transformed to the recombinant strain.But it took little effect.
文摘来源于古细菌嗜酸热硫矿硫化叶菌(Sulfolobus acidocaldarius ATCC 33909)的麦芽寡糖基海藻糖水解酶(maltooligosyltrehalose trehalohydrolase,MTHase)是双酶法生产海藻糖的关键酶。对1株蛋白表达量提高1.9倍的突变株L202P/L218D/Y323G进行酶学性质方面的测定和海藻糖转化。对突变株L202P/L218D/Y323G Sa MTHase进行最适温度、最适pH值、pH稳定性和60℃半衰期的测定,发现该突变株的酶学性质未发生较大变化。将麦芽四糖基海藻糖为底物,测定突变株L202P/L218D/Y323G Sa MTHase酶动力学相关参数。将麦芽糊精(DE值5~7)作为底物,利用嗜酸热硫矿硫化叶菌的麦芽寡糖基海藻糖合成酶(maltooligosyl trehalose synthase,MTSase)与突变株L202P/L218D/Y323G Sa MTHase以不同比例作用于底物生产海藻糖,海藻糖的转化率最高达到76.0%。
