Expression Of Endoxanthanase In Pichia Pastoris And Co-culture For Low-molecular-weight Xanthan Gum Preparation

Xanthan gum is an extracellular polysaccharide composed of glucose,glucuronic acid,and mannose with molecular weight ranging from 2×10⁶ to 5×10⁷ Da.Xanthan gum is widely used in food,medicine,agriculture and other fields because of its excellent stability,safety,and rheology.Low-molecular-weight xanthan gum has higher biological activity and broad application prospects.Enzymatic hydrolysis has been applied for low-molecular-weight xanthan gum preparation because of its mild conditions and environmentally friendly process.However,the high-purity enzyme is hard to obtain,and the high viscosity of xanthan gum aqueous solution results in low hydrolysis efficiency.To solve the above problems,in this study,endoxanthanase derived from Microbacterium sp.XT11was expressed in Pichia pastoris.The endoxanthanase can effectively hydrolyze xanthan gum to produce low-molecular-weight xanthan gum.Thereafter,the co-culture system of P.pastoris and Xanthomonas campestris was established.Xanthan gum produced by X.campestris was degraded by endoxanthanase secreted by P.pastoris to prepare low-molecular-weight xanthan gum directly.Finally,the structure and bioactivity of low-molecular-weight xanthan gum were studied.The main research results are as follows:（1）The endoxanthanase derived from Microbacterium sp.XT11 was expressed in P.pastoris GS115.The fermentation conditions of recombinant P.pastoris were optimized in the shake flask,and the enzymatic properties of endoxanthanase secreted by P.pastoris were studied.The results showed that the optimal fermentation conditions of recombinant P.pastoris with GAP as the promoter were 30℃,pH 6.0,220 r·min^-1,and 5%inoculation amount.The highest activity of recombinant endoxanthanase reached 1230.25 U·L^-1 in a 7-L bioreactor.The optimal reaction conditions of recombinant endoxanthanase are pH 6.0 and 40℃.It can directly act on the main chain of xanthan gum to obtain low-molecular-weight xanthan gum with average molecular weight of 1400 Da.（2）Recombinant P.pastoris GS115-p GAP9K-EX was used to establish a co-culture system with X.campestris,and the fermentation conditions of the co-culture system were optimized.X.campestris was cultured for 36 h with pH of 7.0;then P.pastoris was inoculated into the co-culture system with the suitable inoculation ratio of X.campestris to P.pastoris（1:3）.Bean sprouts was added as the nitrogen source and pH was adjusted to 6.0 for subsequent fermentation.The highest total sugar concentration in the co-culture fermentation system reached 5.79 g·L^-1 in the shake flask,and the proportion of low-molecular-weight xanthan gum（1485 Da）was 78%.In a 7-L bioreactor,the concentration of the total sugar reached 12.12 g·L^-1,and the proportion of low-molecular-weight xanthan gum（1093 Da）was 91%.（3）The low-molecular-weight xanthan gum prepared by the co-culture system in the 7-L bioreactor was extracted and purified,then the structure was identified by UV spectra,MALDI-TOF MS analysis,monosaccharide composition analysis,FT-IR spectrum,and ¹H-NMR analysis.It was determined that the co-culture system can produce low-molecular-weight xanthan gum with degree of polymerization of 4,5,and 8.（4）Biological activity of low-molecular-weight xanthan gum was analyzed.The antioxidant activity experiment showed that low-molecular-weight xanthan gum had good antioxidant property.The scavenging rate of DPPH radical reached 61.2%when the concentration of low-molecular-weight xanthan gum was 5 g·L^-1,and the scavenging rate of hydroxyl radicals reached 77.6%.In vitro probiotics fermentation experiments showed that low-molecular-weight xanthan gum can effectively promote the growth of human intestinal probiotics and the secretion of lactic acid and short-chain fatty acids were beneficial to intestinal health.