Optimal Production Process Of Xylanase Powder By Recombinant Pichia Pastoris

Xylanase is an important hemicellulase,which is widely used in food,feed,paper,textile and other industries.A strain of recombinant Pichia pastoris was obtained in the laboratory.In this paper,xylanase expressed by this engineering strain is designed to improve xylanase yield and reduce production cost through optimization of fermentation and spray drying.The details and results are as follows:（1）Optimization and amplification of high density fermentation processIn the induction period,mixed flow carbon source is the commonly used expression method of heterologous protein in Pichia pastoris fermentation.This paper compares 3 kinds of assisted carbon source of the Pichia pastoris:glucose,glycerol,sorbitol.The result shows that:glucose,glycerol,sorbitol can effectively promote the expression of xylanase,the glycerol had the best effect in the induction period by adding 0.5%glycerol that can make the expression of xylanase increased by 2.4 times,better than sorbitol by 1.9 times and glucose by 2.2 times.Using dissolved oxygen as the monitoring index,the dissolved oxygen of the fermentation broth was controlled in 20⁴0,and the fermentation scale was directly expanded by 2000 times to 60m³.The result of the fermentation was in conformity with the expected result.（2）Optimization of spray drying processTemperature,filling agent and protective agent is the most important factor in spray drying.This subject has optimized the spray drying process of xylanase.Firstly,by the indexes of inlet and outlet temperature,we using single factor experiment to determine the optimal xylanase outlet temperature is 70℃,then the filler added in proportion to the crude enzyme and spray drying,using relative residual enzyme activity,relative weight and sample moisture absorption as performance index,determination"8%starch+12%light calcium carbonate"as the best filler;the protective agent is added to the crude enzyme solution,5min and 70℃water bath,then using the relative residual enzyme activity as index,determine the 1%trehalose as the best protective agent,and through spray drying verification;the above technique was verified in the production workshop.The relative residual enzyme of 5batches was 83.64%.（3）Characterization of xylanaseThe enzymatic properties of xylanase have great influence on its application field.The enzymatic properties of xylanase were identified in this project.The results showed that the optimum temperature of xylanase was 60℃and the optimum pH was5.5.This enzyme has high activity under 37℃,in the activity of pH4.5⁶.5>80%;the enzyme has wide thermal stability,60℃water bath after 30min had higher enzyme activity;stability of the enzyme under acidic condition is good,suitable for the storage of pH 4.5⁶.0,pH too high or too low will lead to inactivation of the enzyme;different metal ions on xylanase has different effects.Mg²⁺,Ca²⁺and Zn²⁺have a trace of the promoting effect on the enzyme,Mn²⁺and Fe³⁺have little inhibitory effect on the enzyme,Cu²⁺can strongly inhibit xylanase enzyme activity,The enzymatic reaction of the enzyme does not require the participation of polyvalent metal ions.