破壁方法对红球菌11-3胞内几丁质脱乙酰酶释放的影响  

Effects of Cell Wall Disruption Methods on the Release of Intracellular Chitin Deacetylasefrom Rhodococcus sp. 11-3

作　　者：肖宇 石文琪 于宏伟[1] 马爱进 桑亚新[1] 孙纪录[1] Xiao Yu;Shi Wenqi;Yu Hongwei;Ma Aijin;Sang Yaxin;Sun Jilu(College of Food Science and Technology,Hebei Agricultural University,Baoding 071000;Hebei 2College of Food Science and Technology,Hebei Normal University of Science&Technology,Changli 066600;Hebei3School of Food and Health,Beijing Technology and Business University,Beijing 100048)

机构地区：[1]河北农业大学食品科技学院,河北保定071000 [2]河北科技师范学院食品科技学院,河北昌黎066600 [3]北京工商大学食品与健康学院,北京100048

出　　处：《中国食品学报》2023年第7期68-79,共12页Journal of Chinese Institute Of Food Science and Technology

基　　金：河北省重点研发计划项目(19273201D);河北省现代农业产业技术体系淡水养殖创新团队建设项目(HBCT2018180206)。

摘　　要：红球菌11-3是一株高产几丁质脱乙酰酶(CDA)的菌株,几丁质在该酶的催化作用下可转化为壳聚糖,该酶在壳聚糖的生产中具有重要作用。红球菌11-3菌株所产CDA为胞内酶,成为催化反应的一大障碍。为了提高CDA的释放率,本研究首先利用不同的物理方法(反复冻融、超声、球磨、匀浆和液氮研磨)、化学方法(表面活性剂处理、氯仿处理)和生物学方法(溶菌酶处理)对红球菌11-3进行破壁处理,测定CDA酶活力和释放率,并通过扫描电子显微镜观察细胞形态变化。结果表明,不同方法的破壁效果存在很大差异,其中,液氮研磨法破壁效果最佳,菌体表面出现细密的孔洞,CDA释放率为45.13%,总酶活力损失率为2.03%;匀浆处理法次之,CDA释放率为16.00%,总酶活力损失率为9.18%。利用匀浆和液氮研磨联合处理红球菌11-3细胞,细胞表面产生更多、更大的孔洞,CDA释放率高达86.17%,总酶活力损失率为9.11%,上清液中CDA酶活力为480.2 U/mL,较液氮研磨法相比提高了1.48倍。结论:匀浆和液氮研磨联合处理可有效破坏红球菌11-3细胞壁,提高胞内CDA释放效率。本研究结果对红球菌11-3内CDA应用于壳聚糖的生产具有参考作用。Rhodococcus sp.11-3 is a high-yielding chitin deacetylase(CDA)strain.The enzyme catalyzes chitin to produce chitosan,which plays an important role in the green production of chitosan.However,the CDA produced by Rhodococcus sp.11-3 is an intracellular enzyme,which has become a major obstacle to the catalytic reaction.In order to improve the release efficiency of CDA,different physical methods(repeated freezing and thawing,ultrasounding,ball milling,homogenization and liquid nitrogen grinding),chemical methods(surfactants treatment,chloroform treatment)and biological method(lysozyme treatment)were used to disrupt the cell wall of Rhodococcus sp.11-3.The enzyme activity and release efficiency of CDA were determined,and the changes of cell morphology was observed by scanning electron microscope.The results showed that different methods had very different effects on the cell wall breaking.Among them,liquid nitrogen grinding was the best method.By using it,there were fine holes on the cell surface,the release efficiency of CDA was 45.13%,and the loss rate of total enzyme activity was 2.03%.Homogenization was next only to liquid nitrogen grinding,the release efficiency of CDA was 16.00%,and the loss rate of total enzyme activity was 9.18%.Then,Rhodococcus sp.11-3 cell was treated with homogenization and liquid nitrogen grinding in succession.The results showed that more and larger pores were produced on the cell surface,the release efficiency of CDA was up to 86.17%,the loss rate of total enzyme activity was 9.11%,and the CDA activity in the supernatant was 480.2 U/mL,which was 1.48 times higher than that of liquid nitrogen grinding.Therefore,the combined treatment of homogenization and liquid nitrogen grinding could effectively disrupt the cell wall of Rhodococcus sp.11-3 and improve the release efficiency of intracellular CDA.The results would promote the application of Rhodococcus sp.11-3 CDA in the production of chitosan.

关 键 词：破壁方法 红球菌 几丁质脱乙酰酶 液氮研磨 匀浆 

分 类 号：TS201.3[轻工技术与工程—食品科学]