Study On The Co-surface Display Of Three NSP Enzymes And Degradation Of Wheat Bran

Wheat has been widely used in poultry feeding as an energy feed instead of corn.However,wheat contains non-starch polysaccharides(NSP)such as arabinoxylan,glucan,pectin,etc.,which make wheat possess certain anti-nutritional properties,which will reduce the digestibility of feed and affect the growth of animals.Adding NSP enzyme in the diet can degrade wheat NSP to some extent,thus reducing the anti-nutritional effect,and the effect of adding compound enzyme is better than that of single enzyme.At present,most of the NSP complex enzymes are produced in the form of compound.First,multiple single enzymes need to be produced,which is more complicated.In addition,the high temperature in the process of pelletizing in feed production greatly affects the activity and stability of enzymes.Yeast surface display system is developing rapidly and widely used a eukaryotic expression system in recent years,most of enzymes can be expressed on the surface of yeast cells as whole-cell catalyst,it has the characteristic and advantage of immobilized enzyme,and the preparation method is simple,easy to recycle and reuse,and display enzyme was more better in thermal stability and p H stability than the free enzyme,so the use of yeast surface display technology to express a variety of NSP enzymes for preparation of high performance composite NSP enzymes provides a new train of thought.In this study,three kinds of NSP enzymes were surface displayed in Pichia pastoris.On this basis,the surface co-display system of the three enzymes was constructed,then display and co-display enzyme were applied to the degradation of wheat bran NSP.The main research contents and results are as follows:(1)The anchoring protein had a great effect on the efficiency of the surface display enzyme,Six Pichia pastoris GPI cell wall proteins were used to express three NSP enzymes in Pichia pastoris respectively,including xylanase DSB,β-glucanase EGⅡ and polygalacturonase PG5,among them,GCW61 as the anchor protein display DSB,and the maximum enzyme activity reached 13601 U/g at the shaking flask level;With GCW51 as the anchor protein,the expression of EGⅡ was the highest,and the maximum activity reached 2350 U/g at the shaking flask level.GCW21 was used as the anchor protein to surface display PG5,and the maximum activity reached 5724 U/g at the shaking flask level.The analysis of enzymatic properties showed that the three display enzymes were all high temperature enzymes and had good heat resistance,which were suitable for the industries that need high temperature treatment in the process of processing.The optimum p H values of the three display enzymes were 6.5,4.8 and 3.5,respectively,which could maintain high activity in acidic environment and were suitable for industrial application in acidic environment.(2)Three NSP co-display plasmids were constructed by using the method of in vitro multicopy recombinant plasmid construction,and then transformed into Pichia pastoris to obtain coexpression recombinant strain X33/DEP,the recombinant strain can efficiently express three enzymes at the same time,realizing "one strain with multiple enzymes".The maximum enzyme activity of DSB,EGⅡ and PG5 was up to 12583 U/g,1984 U/g and 5932 U/g after induced horizontally by shaking flask.The analysis of heat resistance and acid resistance showed that the co-display enzyme do better,and there was no significant difference between display enzyme and co-display in heat resistance and acid resistance.In addition,the enzyme activity of DSB display enzyme and co-display increased by 67.3% and 60.3% respectively after irradiation,the activity of EGⅡ display enzyme and co-display was increased by 5.9% and 16.7%respectively,the activity of PG5 display enzyme co-display were decreased by 3.3% and 5.2%respectively.(3)Co-display enzyme was applied to the degradation of NSP in wheat bran,and compared with three display enzymes with the same activity unit and a mixture of three display enzymes with the same activity unit(complex enzyme),the reducing sugar content of wheat bran substrate treated by co-display enzymes and complex enzymes was significantly higher than that treated by three display enzymes alone,and was greater than the sum of the reducing sugar content treated by three display enzymes,these results indicated that the three enzymes had synergistic promoting effects on the degradation of wheat bran substrate NSP.