产甲壳素酶菌株的发酵条件、酶的分离纯化及酶学性质研究  

Fermentation Conditions of a Chitinase-Producing Strain,Isolation and Purification and Characterization of the Chitinase

作　　者：韩宝芹[1] 伊金玲[1] 蔡文娣[2] 杨艳[1] 董文[1] 刘万顺[1] 

机构地区：[1]中国海洋大学海洋生命学院,山东青岛266003 [2]潍坊医学院,山东潍坊261042

出　　处：《中国海洋大学学报（自然科学版）》2010年第10期57-62,74,共7页Periodical of Ocean University of China

摘　　要：利用甲壳素为唯一碳源从土壤中筛选得到1株产甲壳素酶活力较高的菌株HD002,初步鉴定其为Massilia属。确定菌株产甲壳素酶的最适培养基组成为(g/L):(NH4)2SO45,K2HPO40.7,KH2PO40.3,MgSO4.7H2O 1,胶体甲壳素10;最适产酶培养条件为:培养基起始pH值6.0,培养时间192 h,发酵液酶活力达1.314 U/mL。采用70%饱和度硫酸铵盐析、DEAE-Sepharose F.F阴离子交换层析、Sephadex G-75凝胶过滤层析对该甲壳素酶进行纯化,SDS-PAGE证明达到电泳纯。该甲壳素酶的分子量为60.2 kDa,最适反应温度为55℃,最适反应pH值为5.0,Cu2+和Fe3+对酶活力有明显的抑制作用,Ca2+和Na+对酶活力有明显的促进作用。An effective chitinase-producing strain HD002,which was originally identified as Massilia sp.,was screened from soil.The optimal compositions of the medium for strain HD002 to produce chitinase are as follows（g/L）：（NH4）2SO4 5,K2HPO4 0.7,KH2PO4 0.3,MgSO4·7H2O 1,colloidal chitin 10.When strain HD002 is incubated at 30 ℃ for 192 h in the liquid medium with initial pH 6.0,the maximal activity of the chitinase in the fermentation liquid could reach 1.314 U/mL.The chitinase is extracted by 70% saturation of ammonium sulfate from the fermentation supernatant.Then,the chitinase is purified sequentially by DEAE-Sepharose F.F anion exchange chromatography and Sephadex G-75 gel filtration chromatography.Single band of the chitinase on SDS-PAGE gel proving this protein is purified.The molecular weight of the chitinase is estimated to be 60.2 kDa.The optimum reaction temperature and pH of the chitinase is 55℃ and 5.0,respectively.Metal ions affect the chitinase activity,too.The chitinase activity is extremely inhibited by Cu^2＋（1 mmol/L） and Fe^3＋（1 mmol/L）,especially Fe^3＋,whereas increased by Ca^2＋（1 mmol/L） and Na^＋（1 mmol/L）.

关 键 词：甲壳素酶 发酵条件 分离纯化 酶学性质 

分 类 号：Q555[生物学—生物化学]