饲用耐酸抗蛋白酶β-甘露聚糖酶的筛选分离及酶学性质

摘要

β–甘露聚糖酶作为饲料添加剂，不仅可以有效降解并消除饲料中的抗营养因子，还可以进一步增强动物的免疫反应并调控动物胃肠道中的微生态平衡．然而目前已发现的大部分β–甘露聚糖酶的酸稳定性差，并且容易被动物肠道中的蛋白酶所降解，因此很难在实际应用中发挥理想的效果．本研究从一株高产蛋白酶的枯草芽孢杆菌(Bacillus sublitis)中分离纯化到一种新型β–甘露聚糖酶，并对其酶学性质进行了研究；结果显示：该酶的最适作用温度为50,℃，最适催化pH为5.5，Zn2+、Co2+、Cu2+、Fe3+对该酶有明显的激活作用．稳定性研究表明：采用pH 2.0的酸性缓冲液处理1,h 后，残余酶活仍能保持70%,以上；进一步采用20,U/mL 的猪胰蛋白酶处理2,h，残余酶活仍在60%,以上，表明该酶不仅具有较好的酸稳定性，还对来源于猪胰腺的蛋白酶具有一定的抗性．这些研究结果显示：该酶作为一种新发现的β–甘露聚糖酶，在饲料行业中具有较好的研究前景和应用价值．%β-mannanases,which can be used as feed additives could not only eliminate the antinutritional factors in feed,but also enhance animal immune response and balance the micro-ecology of animal gastrointestinal tract. However,most of the previously reported β-mannanases hardly show satisfactory efficacy in practical application due to their poor acid stability and susceptibility to digestive proteases. In the present research,a novel β-mannanase was isolated from the fermentation broth of a Bacillus subtilis strain,which can simutaneously overproduce proteases. The purifiedβ-mannanase exhibited op-timal activity at 50,℃and pH 5.5. It can also be activated by the metal ions of Zn2+,Co2+,Cu2+and Fe3+. In addition,theβ-mannanase showed high acid stability and resistance to trypsin digestion. After one hour incubation in the acid buffer(pH 2.0)and a succession of two-hour treatment with porcine pancreatic protease(20,U/mL),the remained activities of the β-mannanase still reached more than 70%, and 60%, of the highest activity. These superior properties make the newly isolatedβ-mannanase a good candidate for feed additive.