小麦饲料中木聚糖酶抑制蛋白对外加木聚糖酶的作用研究

Wheat is increasingly used in feed for poultry and livestock. To cope with the anti-nutritional effect caused by high amount of arabinoxylan in wheat, xylanase supplementation has become an effective way to improve the nutrient availability of a wheat-based diet. However, xylanase inhibitor proteins have been recently found existing in wheat, which exclusively inhibit exogenous xylanases. It has been proved that these xylanase inhibitor proteins obviously have negative effect on the function of xylanases used in wheat flour breadmaking and gluten-starch separation. Then, it is reasonable to ask how degree of the xylanase inhibitor proteins can also impede the function of xylanases added to wheat-based diet. To answer this question will provide a new idea to further improve the efficiency of wheat feed. So, in this proposal we plan first to clone the xylanase inhibitor protein genes and heterologously express them in E. coli and/or in Pichia pastoris. Then with these xylanase inhibitor proteins as indicator, the insensitive xylanases to inhibitors will be screened out from all sorts of microbial xylanases. Meanwhile a sensitive xylanase from A. niger belonging to F/10 family will be subjected to mutagenesis to get an insensitive mutant. Finally these two kinds of insensitive xylanases, along with their sensitive counterparts will be valued in terms of their effectiveness by xylanase activity analysis in vitro, also by broilers’ feeding trials. The role of xylanase inhibitor proteins in wheat-based diet on added xylanase will then be clarified.

小麦在畜禽饲料中的应用越来越广泛。为解除小麦中高含量阿拉伯木聚糖的抗营养作用，添加木聚糖酶已成为提高小麦饲料养分利用率的有效途径。然而，近年在小麦中发现了专门抑制外源木聚糖酶作用的木聚糖酶抑制蛋白成分，已有证据表明这些抑制蛋白在面包焙烤、小麦谷朊粉-淀粉分离方面对施用的木聚糖酶的功效产生明显的不利影响，那么在小麦用作饲料条件下抑制蛋白对外加木聚糖酶造成多大程度的阻碍作用？回答这一问题将为进一步提高小麦饲料利用率提供崭新的思路。本项目拟克隆小麦抑制蛋白基因，并进行异源表达获得重组抑制蛋白，以此作为筛查者从众多微生物来源木聚糖酶中筛选对抑制蛋白不敏感的抗性木聚糖酶，同时对敏感性的黑曲霉F/10木聚糖酶进行定点突变获得抗性突变体酶，通过体外酶活测定和鸡饲喂试验评价上述两类抗性和敏感性木聚糖酶的添加效果，从而探明小麦饲料中的木聚糖酶抑制蛋白对外加木聚糖酶的影响作用。

饲用小麦中添加木聚糖酶能够解除阿拉伯木聚糖的抗营养作用。然而，在面包焙烤、小麦谷朊粉-淀粉分离方面，小麦中木聚糖酶抑制蛋白却对外加的木聚糖酶的功效产生明显的不利影响。本项目旨在研究麦型饲料中抑制蛋白对外加木聚糖酶造成多大程度的阻碍作用。首先从小麦中克隆三型木聚糖酶抑制蛋白基因，然后从瘤胃真菌新丽鞭毛菌中筛选并异源表达了对XIP抑制蛋白具有抗性的GH11木聚糖酶，XIP抑制蛋白与重组抗性酶按照6:1摩尔比例下反应时，XIP抑制蛋白对纯化的抗性木聚糖酶没有任何的抑制作用；该瘤胃真菌来源的抗性酶与真菌来源的木聚糖酶进化关系较远，却与细菌来源GH11木聚糖酶具有更近的进化关系。本试验还对GH10木聚糖酶中XIP结合位点进行了突变，结果表明，通过底物结合位点突变获得的木聚糖酶突变体的酶活力明显降低或是抑制性质不发生变化。最后，通过体外模拟消化试验对XIP抗性木聚糖酶在小麦饲料中的添加功效进行了初步评价，发现饲料中小麦比例为20%时添加抗性木聚糖酶产生的总糖相比敏感酶提高了8.36%，表明抗性酶比敏感酶降解小麦木聚糖更为有效。由此可以认为，在体外模拟消化试验条件下，小麦饲料中的木聚糖酶抑制蛋白对外加敏感性木聚糖酶具有一定的阻碍作用，而外加抗性木聚糖酶则可以提高木聚糖酶的用酶效果。