Study On The Fermentation And Application Of Recombinant Xylanase

Xylanase, the main component of xylanolytic enzyme systerm, can be produced in submerged liquid culture by many microorganisms and widely used in various industrial fields, such as brewing, feed, food, medicine, pulp and paper, energy and environment. In order to solve the drawbacks of low yield and instability of xylanase, the production condition, characterization and application of recombinant xylanase were studied.The conditions for shake flask culture of recombinant Pichia pastoris were analysed by one-factor test, including seed age, inoculum content, initial pH of seed phase, carbon source, nitrogen source and incubation time. Prominent factors were screened from ten elements in induction phase by Plackett-Burman experiment design. The principal factors which have effect on the xylanase activity are the yeast extract, induction pH and shaking revolution by analysis of variance. Then these factors were optimized by Box-Behnken which is one kind of the response surface analysis, and the optimum activity reached 262.8 U/mL. When the optimal shake flask cultural conditions were applied in 7 litres fermenter, high-density expression of the xylanase was achieved with an activity of 2054.9 U/mL.Dialyzed enzyme solution was separated and purified by anion exchange chromatography and gel filtration chromatography successively. There were two proteins in partly purified xylanase proved by SDS-PAGE, and their molecular weight are 35 kDa and 31 kDa respectively. The optimum pH and temperature of partly purified xylanase were 6 and 60℃independently, and its thermal and pH stability keep well. The relative activity still retain more than 60% after kept for 1 hour in pH 4.0⁷.5 and 52% after maintained 2 min in 80℃. The activity was promoted by low concentration of Na⁺, Ca²⁺ and high concentration of Fe²⁺, Mn²⁺, SDS, while restrained by K⁺, Cu²⁺ and low concentration Zn²⁺ and it was barely influenced by EDTA. The parameters of enzyme kinetics are a Km of 20.07 mg/mL and a V_max of 714.29μmol/mL/min.The rate of filtration of wort was accelerated, the content of PAX was dropped and the concentration of extract and soluble nitrogen were increased when the xylanase was applied in mashing course of whole malt and wheat and rice as the auxiliary materials. The quality of malt was improved by using enzyme solution in the process of malting. The total xylan from barley, wheat, bran, corn flour were reduced by xylanase and degradation of crude protein was promoted in vitro.