| Xylan,which is the main component of plant hemicelluloses and the second largest renewable resource in nature,is a kind of polypentose.The key enzyme for xylan hydrolysis is endo-?-1,4-xylanase,which has great potential for use in fodder,food and biomass energy development.Hence,it would be highly desirable to construct high-yield xylanase engineered strain and reduce the cost of xylanase production.In previous study,a 606-bp xylanase encoding gene xynHB was cloned from Bacillu pumilus HBP8 by a shot-gun method and expressed in Pichia pastoris.SDS-PAGE showed that there are two protein bands with molecular weight 32.2 kDa and 29.6 kDa caused by the difference of glycosylation extent.The enzymology properties analysis showed that the optimal pH and temperature of the enzyme was pH 6-9 and 40-60?,respectively.But the thermostability of this enzyme at 60? is unsatisfactory for the half-life of it only last for 12 min.Then,the amino acid N of the third glycosylation site at position 187 was mutated into A via site-directed mutagenesis and the thermostability of the mutant remarkably increased.The present studies consist of two parts.Part one is the expression of xynHB in Saccharomyces cerevisiae by rDNA integration and development of the xylanase that can be used in bakery.Part two is to screen P.pastoris genetic strain with high-yield xylanase though codon optimization and dose effect so that xylanase cen be used in pulp bleaching.The detailed results are as follows:1.Expression of xylanase encoding gene xynHB in S.cerevisiae and application in baking.Due to P.pastoris is not a GRAS microorganism approved by FDA,it can't be used in food industry while S.cerevisiae is a GRAS and has been used in food industry for decades.This research focused on the expression of xylanase encoding gene xynHB in S.cerevisiae by an rDNA-mediated integration vector pHBM367H which was constructed previously.Firstly,an rDNA-mediated vector pHBM367H-xynHB was constructed and introduced into S.cerevisiae INV-Scl competent cell.Secondly,a strain named A13 that had the ability to form the largest halo was screened and maximal xylanase activity of XynHB from A13 strain reached 2.5 U/mL induced in SC medium for 72 h and 11.2 U/mL in YEPD medium for 48 h,respectively.Thirdly,the copy number of A13 strain evaluated by Real time-PCR reached 13.64 copies per cell.Xylanase activities of A13 strain remain unchanged with the orginal strain after about 106-time serial subcultivations which indicate that strain A13 possesses excellent genetic stability.At last,XynHB secreted by A13 strain was applied into breadmaking as food additives.It can reduce kneading time and increase dough volume when the additive amount of the xylanase is 1,200 ppm.Meanwhile,the volume,skin and shape,springiness and texture of the xylanase-additive bread are all better than the control.2.Construction of P.pastoris genetic strain with high-yield xylanase.Optimized sequence xynHBs and its primer sequence of the mutant sequence xynHBN187A were designed via software DNAworks based on the codon bias of P.pastoris.Optimized gene xynHBs was synthetized by overlapping extension PCR and P.pastoris secretory expression vector to get plasmid pHBM905A-xynHBs.Then,recombinant plasmid was introduced into competent cell of P.pastoris GS115 after digestion by SalI.Single copy number strain of both GS15/pHBM905A-xynHBs and GS115/pHBM905A-xynHB were screened on the plate with RBB-xylan.Xylanolytic activity of Shake flask fermentation of recombinant contained optimized sequence xynHBs is as much as 39.5%higher than original one.Then a high-yield xylanase producing strain Y16 was sceened from thousands of transformants by the plate of substrate.Shake flask fermentation showed that xylanolytic activity reached more than 6,000 U/mL.Copy number of Y16 strain was evaluated by Real time-PCR,reaching 9 copies per cell. |
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