Isolation Of Enzyme-Producing Microflora, Cloning And Expression Of Starch-Degrading Enzyme Genes From Frozen Soil At Root Of Saussureae Involucratae In Tianshan, Xinjiang

Using the frozen soil at the root of Saussureae Involucratae Kar.et Kir.et Maxim.from TianShan in XinJiang province as material,we isolated the microflora under different cluture conditions at low temperature,and indentified the microflora by 16S or 18S rDNA sequence.The results display that Pseudeomonas,Flavobacterium and Arthrobacter are preponderant microflora in cold environment.By using selective media,the enzyme-producing microflora were screened, including amylase,mannanase,xylanase,cellulase and proteinase ect. There are severeal microflora producing kinds of enzymes.It is conclud that there are lots of novel unexploited microflora in cold environments.There are four conserved regions in sequences of alpha-amylase family.We designed a pair of degenerate primers(AmyF / AmyR) according to theâ… andâ…¡consered regions.By using the primers,we got 14 amylase gene fragments from the isolated cold adapted microfloras. The identity between the fragments is very low that proves there are abundant novel amylase genes existing in cold adapted microfloras. The full-length genes were isolated further from three strains which show high starch degrading activity,using TAIL-PCR based on the fragment sequences obtained.Three glycoside hydrolase genes,including alpha amylase(amyA),glycoside hydrolase family 43(ghB)and alpha-N-arabinofuranosidase(abfC)were cloned from psychrophilic bacteria of Flavobacterium sp.M-2.Two glycogen debranching enzymes (glgD and glgE)were cloned from psychrophilic bacterias of Proteobacterium sp.DB-18 and Arthrobacter sp.G-7 respectively.The genes amyA,glgD and glgE are relate to the degradation of starch.The amino acid and nucleotide sequences of the genes show low identity to the genes reported,indicating that they were novel glycoside hydrolase genes.The genes amyA and glgD were transformed into Escherichia coli BL21(DE3)and expressed heterologously,when the transformants were induced by IPTG at the concentration of 0.4 mmol·L^-1,and cultured for 24-48 hours at 18-20℃,while they could not be expressed at 37℃.The amylase activity of 1.1 U·mL¹ and 0.9 U·mL^-1were detected in cell lysate of E.coli transformed by amyA and glgD,respectively.The results display the difference of expression in mesophilic host between psychrophilic genes and mesophilic genes.The starch-degrading enzyme genes could be candidate for industrial utilizations.