Secreted Expression Of The Thermostable Xylanase Gene In Two Yeasts

In this study, Two mutated xylanase genes were obtained from PCR by using the genomic DNA of Thermotoga maritima MSB8 as template, termed as mxynB(64) and mxynB(577), which represented that the bases of the 64^th and 577^th sites of the gene xynB muftated from A to G and from T to C and the amino acids mutated correspondingly from Asn²² to Asp and from Tyr¹⁹³ to His respectively, by comparing with the xylanase gene published in GenBank with the registration number of AE001693. With the assay of the advanced structure of xylanaseB by bioinformatics, vaxynB(64) was cloned into E. coli expression vector pET-28a (+) , and was successfully expressed in E. coli BL21 as solublef protein product. The recombinant mXynB₍₆₄₎ in E. coli showed great enzyme activity which reach to 121.6U/ml (tested by RBB-xylans methods), and it showed high activity in 90℃. The thermostability of this protein offered it to be purified easily. But the recombinant mXynB₍₆₄₎ expressed in E. coli could not be secreted out of the cell which would be an obstacle for it to be applied in practice.Both of the two xylanase genes, vaxynB_<sub>(64) and mxynB₍₅₇₇₎ were inserted into the shuttle and integrated expression vector pPIC9k respectively and expressed by Pichia pastoris as an active extracellular product efficiently. The two recombinant xylanases expressed in P. pastoris still show extreme thermostability and pH stability. mXynB₍₆₄₎ and mXynB₍₅₇₇₎ were optimally active at 90℃ and 80℃ respectively and both quite stable over the pH range of pH 5.0-10.8 at 70℃, from which that the mutated amino acid in mXynB₍₅₇₇₎ only affected the thermostability of this enzyme but none on its pH properties could be suggested. In the conditions of high cell density cultivation, P. pastoris recombinant strain GS115-9K- mxynB₍₆₄₎ II could secreted the recombinant protein of mXynB₍₆₄₎ efficiently, which indicated that it was of great use in a variety of industrial and agricultural applications.Another methylotrophic yeast, Hansenula polymorpha, expression system was also established in this study. Three promoter sequences were obtained by PCR, and integrated into the four H. polymorpha shuttle and integrated expression vectors with the xylanase expression cassette, among which pMOX_p-mxynB₍₆₄₎ (10.6Kb) , pFMD_p-mxynB₍₆₄₎ (9.7Kb) , pAOX1_p-mxynB₍₆₄₎ (9.8Kb) were of inducible promoters, while pPMA1_p-mxynB₍₆₄₎ (11.1Kb) , constitutive promoter.. The four H. polymorpha expression vectors were used to heterologously express mxynB₍(64) gene in H. polymorpha A16. After cultivation in shake flash for 108 hours, the H. polymorpha recombinant strain of A16-pMOX_p-mxynB₍₆₄₎ showed the highest intercellular xylanase activity of 0.25U/ml. High cell fermentation of H. polymorpha recombinant strain, A16- pPMA1_p-mxynB₍₆₄₎, showedthat the relatively simple fermentation process would make better sense in future industrial application.