利用原生质体融合技术构建植酸酶纤维素酶枯草芽孢杆菌工程菌研究.docVIP

利用原生质体融合技术构建植酸酶纤维素酶枯草芽孢杆菌工程菌研究 　　摘要:以产植酸酶枯草芽孢杆菌(A5)复合诱变的再生突变株Z56和产纤维素酶枯草芽孢杆菌(B6)复合诱变的再生突变株X57为亲本,利用双亲灭活原生质体融合技术进行种内融合,构建可同时产植酸酶、纤维素酶的工程菌。结果表明,从构建的385个融合子中筛选到6株两种酶活性相对较高的工程菌,其中R4、R5的纤维素酶产量高于亲本,植酸酶产量也相对较高;粗酶液用90 ℃处理10 min后,纤维素酶剩余酶活分别为对照的62%和58%,植酸酶剩余酶活分别为对照的73%和71%。 　　关键词:枯草芽孢杆菌;植酸酶;纤维素酶;原生质体融合;工程菌 　　中图分类号:Q814文献标识码:A DOI编码:10.3969/j.issn.1006-6500.2010.05.007 　　 　　Study on the Application of Intraspecific Protoplast Fusion Technique to Construct Bacillus subtilis Engineering Bacteria with Phytase and Cellulase Activities 　　XIE Feng-xing, ZHANG Feng-feng, ZHOU Ke, ZHAO Yu-jie 　　(Tianjin Research Center of Agricultural Biotechnology,Tianjin 300192,China) 　　Abstract:In order to construct phytase and ellulose-producing engineering bacteria, intraspecific protoplast fusion between Bacillus subtilis Z56, phytase-producing strain obtained from protoplast combination mutagenesis, and Bacillus subtilis X57, ellulose-producing strain obtained from protoplast combination mutagenesis, was performed by inactivated protoplast fusion technique. The results showed that 6 fusants with high phytase and cellulose activities were selected from 385 regeneration colonies. The productions of ellulose of R4 and R5 were higher than the parental strain and phytase activities were relatively high. The enzyme thermal stability results showed that the remained ellulose activities of R4 and R5 kept 62% and 58%, while the remained phytase activities kept 73% and 71% when the crude enzymes were treated with 90℃ for 10 min. 　　Key words: Bacillus subtilis;phytase;ellulose;protoplast fusion;engineering bacteria 　　 　　植酸酶是催化植酸及植酸盐水解产生磷酸和肌醇的一类酶的统称[1]。植酸酶作为饲料添加剂,一方面可以有效的提高动物对饲料中磷的利用率,从而使饲料中添加的无机磷减少70%[2],降低饲料成本的同时节约磷矿资源,另一方面可将动物粪便中排磷量减少30%～50%[3],减轻环境污染,有利于生态农业的发展。纤维素酶是一类将纤维素水解成葡萄糖的一组酶的统称。它可破解富含纤维的细胞壁,使其包含的蛋白质、淀粉等营养物质释放出来并加以利用,同时又可将纤维降解为可被畜禽机体消化吸收的还原糖,从而提高饲料利用率[4]。虽然目前利用微生物发酵生产酶制剂已达到工业化水平,但外源酶制剂的热稳定性问题一直没有得到很好的解决,导致其在饲料加工的高温(70～90 ℃)制粒工艺中,酶的活性大幅度丧失[5]。枯草芽孢杆菌是一种能产内生芽孢的革兰氏阳性菌,有较强的热稳定性,同时也是农业部允许作