Molecular Cloning Of Inulinase Gene And Expression In Pichia Pastoris

Inulinases are a family of glycoside hydrolase, the enzyme that hydrolyze the (3-2,1-glycosidic linkage to produce fructose and fructo oligosaccharide(FOS). Inulinase has been found in a wide variety of plants and microorganisms including fungi, yeast, bacteria as well as in the root of some plants. It has been found that microorganisms can hydrolyze inulin in1920’s, inulinase gene INU1was cloned in1991, and inulinases’ structure, function and properties stimulated the interest of many scientists. However, the complete function and structure of the inulinase remains unkown.Microbial inulinases can be divided into exo-and endo-acting enzymes by their modes of action on inulin. The products of hydrolyzing inulin can be used in industry. Inulinase production by using recombinant pichia pastoris and high cell density fermentation technology is a more efficient, more industrialized and cheaper way.Genomic DNA of Kluyveromyces marxianus was amplified by polymerase chain reaction(PCR) getting DNA fragments including ORF of gene INU1. ORF of gene INUl was amplified from DNA fragments as template. A plasmid pEASY T3was used for TA cloning in E.coli DH5a. Recombinant pEASY T3-INU1was cleaved by restriction endonucleases and then cloned in plasmid pPIC9.In the second part of this article, the recombinant plasmid pPIC9-INUl was linearized, then transformed into Pichia pastoris GS115. Homologous recombination occured between the alcohol oxidase gene (AOX1) of the vector and chromosome. The transformants with gene HIS4was cultivated on MD medium without histidine. Some of the Pichia pastoris transformants were selected and checked for DNA fragment of the INU1into the chromosomes by PCR. Recombinant protein was detected by SDS-PAGE, which showed that the protein was secreted out of the cell. Inulinase activity was assayed. Enzyme activity of recombinant Pichia pastoris in the fermentation liquid was10.168U within163h. The recombinant Pichia pastoris was cultivated in5L fermentator, Enzyme activity reached12.458U, which almost was higher amount of the enzyme by two times than kluyveromyces marxianus (4.874U). In a word, Expression of gene INU1in Pichia pastoris was researched by gene engineering and recombinant Protein Expression. The advantage and yield of expressing protein in Pichia Pastoris was clarified that provided references for the application of the recombinant Pichia Pastoris in industry.