一株产葡萄糖氧化酶菌株的筛选、鉴定及酶学性质表征

Screening,identification and enzymatic characterization of a marine glucose oxidase strain

摘要:

【背景】海洋中蕴藏着大量未被开发利用的微生物种质资源，而且海洋微生物产的酶类因其具有耐低温、耐高压和耐高盐等明显区别于陆地微生物所产酶类的特点而备受关注。【目的】从渤海海域海泥样品中分离筛选产葡萄糖氧化酶的菌株，并研究其酶学性质。【方法】通过平板初筛和酶活复筛，确定产葡萄糖氧化酶的菌株；通过形态学鉴定和构建系统发育树分析，对产葡萄糖氧化酶的菌株进行酶学性质研究。【结果】筛选到一株产葡萄糖氧化酶的菌株(31号)，经生理生化特征和多序列分析确定为担子菌属(Basidiomycete)；31号菌株所产葡萄糖氧化酶(glucose oxidase，GOD)在25−35 ℃内有较高酶活，达20 U/mL；最适反应温度为30 ℃，而且在0 ℃时仍有酶活；在pH 7.0环境下酶活性最高，在pH 5.0−7.0之间活性较为稳定，酶活残余率较高；5 mmol/L的Ni²⁺、Na⁺和Zn²⁺对酶活的促进作用尤其显著，5 mmol/L的Mn²⁺对GOD酶活性有显著抑制作用；Cu²⁺、K⁺和Ca²⁺对酶活性有抑制作用，而且浓度越高抑制作用越显著，不同浓度的β-巯基乙醇、乙二胺四乙酸、曲拉通-100、次硫酸钠和二硫苏糖醇对GOD酶活性均有显著抑制作用。【结论】31号菌株是一株极具研究价值的产低温葡萄糖氧化酶的菌株，为葡萄糖氧化酶的生产提供了良好的种质资源，为后续研究积累了相关数据，同时也填补了利用该菌生产葡萄糖氧化酶的空白。

Abstract:

[Background] There are abundant undeveloped microbial germplasm resources in the ocean,and the enzymes produced by marine microorganisms have attracted attention because of their tolerance to low temperature,high pressure,and high salt,which are different from those produced by terrestrial microorganisms.[Objective] To isolate and screen out the strains producing glucose oxidase (GOD) from the mud samples of the Bohai Sea,and to study its enzymatic properties.[Methods] The GOD-producing strain was preliminarily screened with plate culture method and re-screened based on enzyme activity.The candidate was identified based on morphological,physiological,and phylogenetic characteristics.The enzymatic properties of the GOD produced by the strain screened out were further characterized in detail.[Results] strain No.31 that produced GOD was screened out and initially identified as Basidiomycete sp.based on morphological and phylogenetic characteristics.The GOD produced by strain No.31 had high activity reaching 20 U/mL at 25–35℃.This enzyme had the optimum reaction temperature at 30℃ and still had activity at 0℃.Its activity was stable between pH 5.0 and 7.0 and peaked at pH 7.0.Ni²⁺,Na⁺,Zn²⁺,Mg²⁺ can improve GOD activity,and high concentrations of Ni²⁺,Na⁺,and Zn²⁺ had significant effect.Different concentrations of β-mercaptoethanol,urea,EDTA,Triton-100,sodium hyposulfite,and DTT had significant inhibitory effect on GOD activity.[Conclusion]The experimental results prove that strain No.31 is a valuable strain producing GOD tolerant to low temperature,which provide a good strain resource for the production of GOD,accumulate relevant data for follow-up research,and fill the blank of using this fungus to produce GOD.