The Cloning Of Maltooligosyl Trehalose Synthase Genes And Expression In The Ecoli. Cells And Analysis Of Enzymology Character

The genes encoding for maltooligosyl trehalose synthase(MTSase)were amplified from the total DNA of Sulfolobus shibatae B12 by the polymerase chain reaction(PCR),ligated the fragment with T-easy—vector by T4 DNA ligase and transformemed the competent cells Ecoli DH5α.The transformants were screened on amplicillin/Xgal-IPTG on plate.Identifying the fragment by clony PCR,plasmid amplifying PCR,restriction enzyme digest analysis and sequencing.Restriction endonucleases Nde I and BamH 1 were used to cleave both T-easy—vector-MTSase and PET-21 a,cloned the object gene fragment to expression vector by , T4 DNA ligase,identified by enzyme digest,transformed recombinant plasmid to E.coli BL21(DE3)by heat shock,induced the recombinant plasmids PET-21a-GT by the addition of IPTG,expressed MTSase protein.The recombinant protein were shown to be 16.8%of the total bacterial protein. The molecular weight of expressed MTSase detected by SDS-PAGE was 74 kDa..The enzyme activity was 38.53U of the total wet bacterial substance.The conditions of affecting the recombinant enzyme expression were optimized. Findings showed:The nucleotide sequence of the MTSase gene consisted of 2,187bp open reading frame which encoded for a protein with 729 amino acid.The overall nucleotide sequence and amino acid sequence homologies between MTSase of Sulfolobus shibatae B12 and Sulfolobus solfataricus KMl is 96%.