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.
A novel α amylase gene was amplified from Sulfolobus shibatae by using PCR technique. The amplified 1.7kb DNA fragment was inserted into an expression vector pBV220 to yield the recombinant plasmid pSBAM. The novel α amylase gene in pSBAM was expressed in E. coli . The production of the novel α amylase activity reached over 8 units/100mL of the culture. The molecular weight of this enzyme was about 61kD by SDS PAGE. The expressed novel α amylase protein in E.coli DH5α accounted for about 20% of the total protein in the recombinant cell. The cooperative action of the novel α amylase and the maltooligosyltrehalose synthase from Sulfolobus shibatae was investigated and trehalose was detected by using HPLC analysis when using amylose and partial starch hydrolysates as substrates.
