High-density Fermentation Technology Of Thermophilic Xylanase In Recombinant Yeast And Biological Characteristics Of Thermophilic Xylanase

The aim of this research was to establish technology of recombinant xylanase expressed in Pichia pastoris in high cell-density fermentation,study degradation of wheat bran with recombinant xylanase and tap fibrous feed such as wheat bran functional potential.Expt.1 was conducted to get high active recombinant xylanase expressed in Pichia pastoris by batch fermentation.The recombinant xylanase activity was improved 12.2%,7.4%,12.0%and 9.9%by supplementing the Pichia pastoris culture with 20 g/L wheat bran,5 mg/L L-histidine,10 mg/L L-tryptophan and 10 mg/L L-methionine in shake flasks,respectively.In treatment I,1%?v/v?glycerol was fed at 9 h and 17 h,and 0.5%?v/v?methanol was fed at 24 h,32 h,48 h and then every 8 h to the end in 1-L bioreactors;in treatment II,0.5%?v/v?methanol was fed every 24 h in 1-L bioreactors.The maximal DCW and recombinant xylanase activity in treatment I were 1.69 times and 1.97 times as much as those in treatment II,which showed that glycerol and methanol feeding had significant effect on DCW and recombinant xylanase activity.It also provided the basis for enhancement of recombinant xylanase activity by glycerol and methanol feeding strategies in following experiment.Expt.2 was conducted to get high active recombinant xylanase expressed in Pichia pastoris by fed-batch fermentation.Novel glycerol fed-batch fermentation including three stages and four-stage methanol fed-batch fermentation with a stepwise increase in methanol feeding rate were developed in 50-L bioreactor.The DCW reached 53.48 g/L,and maximal xylanase activity was 560.7 U/mL,which was 7.05 times of that in shake flask.In the initial 72 h of methanol induction,the linear dependence of recombinant xylanase activity on methanol intake was observed.Recombinant xylanase was separated and concentrated by membrane technology,the ratio of it passed through ceramic membrane was 85.32%,and then was concentrated for 7.21 times by nanofiltration membrane,and the ratio was 94.97%.Novel glycerol and methanol feeding strategies were developed,and recombinant xylanase activity was significantly improved in this experiment.Expt.3 was conducted to study recombinant xylanase biological characteristics.The optimal temperature and pH of recombinant xylanase were 80? and 8.0,respectively.Recombinant xylanase retained 98.03%and 82.52%of its initial activity after pre-incubation at 80? for 10 min and 50 min,respectively.Recombinant xylanase retained 97.40%and 89.01%of its initial activity after pre-incubation at pH 11.0 for 2 h and 11 h,respectively.This recombinant xylanase showed thermoalkaliphilic property.Expt.4 was conducted to study degradation of wheat bran by recombinant xylanase,determine bioactive substance in the enzymatic hydrolyzate,and tap fibrous feed such as wheat bran functional potential.Enzymatic hydrolyzate contained 614.0 ?g/mL reducing sugars,and xylose,arabinose and glucose were 23.8 ?g/mL,15.6 ?g/mL,and 17.3 ?g/mL,respectively.Xylooligosaccharides were analyzed by HPLC,and xylobiose and xylotriose were 213.3 ?g/mL and 174.0?g/mL.Total phenolic contents were 129.44 ?g/mL.Phenolic acids were analyzed by LC-ESI-MS,and ferulic acid was 13.1?g/mL.The enzymatic hydrolyzate showed total antioxidant activity with a value of 141.83 mg ascorbic acid equivalents g^-1 crude xylan and Fe²⁺ chelating activity with a value of 195.67 mg EDTA equivalents g^-1 crude xylan,and the highest DPPH radical scavenging activity reached 92.65%.Bioactive substances including xylobiose,xylotriose and ferulic acid were produced by degradation of wheat bran,which showed favorable antioxidant capacity.In conclusion,we established technology of recombinant xylanase expressed in Pichia pastoris in high cell-density fermentation,got high active recombinant xylanase with thermoalkaliphilic property,established method to degrade wheat bran with recombinant xylanase to produce bioactive substances,and further confirmed functional potential of fibrous feed.