Recombinant Pichia Pastoris Xylanase Produced By Fermentation Conditions And Its Enzymatic Properties

Xylan is the chief ingredient of hemicellulose as well as abundant reproducible resource of nature. Xylanase can degrade xylan into xylose or Xylo-oligosaccharide. So it is widely used in feed ,food , weave and paper making industries, and it is one of the hotspots of research on modern enzymology . this paper studied on fermentation conditions for xylanase production by combined Pichia pastoris and its enzymic properties. The chief researching results were as follows:Primary grope for fermentation conditions of strain YDL2006 was undertaken by shake flask fermentation, which made certain that the best seeding time was 16 hours, the inoculum concentration was 4%, the growth pH was 4.8, inducing pH was 5.0 and the methanol concentration was 1%, the induction was undertaken for 96 hours, the enzymic activity could reach as high as above 4500 u/mL.Analysis of the fermentation process was also done in a 50 litres fermenter. Systemic analysis and optimization of fermentation parameter such as dissolving oxygen, pH value, stirring speed , ventilatory capacity and feeding process were proceeded, which ascertained the following process: the inoculum concentration was 4%, the initial stirring speed was 300rpm. Ventilatory was 1.5 m³/h,an additional glucose of 3500mL was added as supplement, its concentrations was 50%, the wet weight was 290g/L.Adjustion of stirring speed,ventilatory capacity, fermenter pressure and the feeding speed of glucose maintained DO above 20%.The methanol concentration was keeped under 1% by methanol concentration sensor in the feeding process.The enzymic activity was maximal with the average activity 52700u/mL and wet weight 290g/L by maintaining feeding time 110h with methanol volume 6000mL.All the fermentation time was about 160h.This paper also studied enzymic properties of xylanase produced by combined Pichia pastoris. Deoxidization of sugar was used for its basic properties. Enzymic activity was mensurated in buffer solutions of different pH values. Results indicated that the enzyme had good stability in pH3.5 to 6.0 and its enzymic activity could maintain above 80%, and the most appropriate pH was 4.5; mensuration of enzymic activity was also done under different temperatures, which indicated that appropriate temperature for the reaction was 45-60℃while the most appropriate temperature for the reaction was 55℃.The enzyme's relative activity was rather low when the temperature was either lower than 30℃or higher than 70℃.For example, the relative enzymic activity was 9.3% when enzyme was put in environmental temperature of 80℃.Putting enzyme solutions in 55℃,65℃or 75℃for different time, Enzymic activity could maintain as high as more than 90% when it was put in 55℃for 120 minutes while the enzymic activity decreases much by heat treatment of 65℃or 75℃.Treatment of 65℃for 120 minutes or 75℃for 100 minutes still lead the enzymic activity to above 50%. And it had good stability toendure heat. Mg²⁺,Zn²⁺,Ca²⁺ could promote xylan's enzymic activity while Cu²⁺,Na⁺,Fe³⁺ inhibited its activity. Mensuration results of TLC and HPLC indicated that product of xylanase were chiefly Xylobiose and xylotetraose the content of xylose was quite low, this xylan was a relative simplex incision enzyme.