块菌菌根土壤产酸性几丁质酶放线菌Streptomyces omiyaensis SCPW03的筛选及其几丁质酶酶学性质研究

Screening of Acidic Chitinase-production Actinomyces and Analysis of Enzymatic Properties from Streptomyces omiyaensis SCPW03

【目的】开展具有优良酶学性质的酸性几丁质酶放线菌研究为几丁质原料的高效转化提供重要的微生物资源。【方法】以四川攀枝花地区偏酸性块菌菌根土壤为材料,采用稀释平板法分离筛选、16S rDNA基因鉴定产酸性几丁质酶放线菌,并分析高产菌株的酶学性质。【结果】从分离到的17株降解几丁质放线菌中筛选到8株产酸性几丁质酶菌株,经16S rDNA序列测序分析表明均为链霉菌属(Streptomyces)。对其中的高产菌株Streptomyces omiyaensis SCPW03(30.2 U/mL)进行酶学性质表明,该菌所产酶分子大小约为54 kDa,最适反应温度和pH分别为35℃和4.0;该酶在温度25~35℃、pH 6.0的条件下保温60 min较稳定,酶活力保持在90%以上;酶活性受化学抑制剂EDTA、SDS及β-巯基乙醇和金属离子Cu^2+、Zn^2+、Ca^2+、K^+、Na^+的影响较小,而Fe^2+、Mn^2+则能够显著促进几丁质酶活性。【结论】研究表明四川攀枝花地区偏酸性块菌菌根土壤中含丰富的产酸性几丁质酶放线菌,菌株S.omiyaensis SCPW03具有高产酸性几丁质酶能力且所产酶具有较多的优良特性。

【Objective】This research was aimed to obtain actinomyces,which could produce excellent acidic chitinase,play an important role during the bioconversion of chitin materials.【Method】We took the Tuber indicum ectomycorrhizosphere soil in Sichuan as samples to isolate by plate streating and identify by 16 S rDNA sequence analysis.Then,enzymatic properties of the obtained high-yield strain were characterized.【Result】Eight acidic chitinase-producing actinomycetes strains were screened from 17 chitinolytic actinomycetes isolates and these strains belonged to Streptomyces based on the 16 S rDNA sequence analysis.Among them,S.omiyaensis SCPW03 possessed the highest yield of acid chitinase and the molecular masses of this enyme was about 54 kDa.According to the analysis of enzymatic properties,the optimum reaction temperature and pH value for this chitinase were 35℃and 4.0,respectively.Meanwhile,more than 90%of relative activity of this chitinase was retained after incubation for 60 min under the conditions of pH at 6.0 and temperature during 25-35℃,which showed its stable enzyme activity.Moreover,the chemical inhibitors(EDTA,SDS,β-mercaptoethanol)and metal ions(Cu^2+、Zn^2+、Ca^2+、K^+and Na^+)had no significant effects on the chitinase activities,while Fe^2+and Mn^2+could improve the enzyme activity.【Conclusion】This study demonstrated that the T.indicum ectomycorrhizosphere soil in Sichuan offered an enormous diversity for acidic chitinase actinomycetes,and the acidic chitinase produced by S.omiyaensis SCPW03 possessed more excellent characteristics and higher enzyme activity,which will have good potential in improving the bioconversion of chitin materials.