碱性木聚糖酶高产菌株的筛选鉴定、酶学性质检测及培养条件优化

Screening,identification,enzymatic characteristics and optimization of fermentation condition of a high alkaline xylanase-producing strain

目的 筛选碱性木聚糖酶的高产菌株,并对其进行分子鉴定、酶学性质检测及培养条件优化。方法 采集山西、河南和浙江农田土壤样品,通过富集培养及分离后进行16S rDNA鉴定,筛选碱性木聚糖酶的高产菌株,并检测其酶学性质。单因素试验优化发酵条件,包括培养基碳源(木聚糖、乳糖、可溶性淀粉、蔗糖、麸皮、葡萄糖)、氮源[草酸铵、蛋白胨、酵母粉、(NH_(4))_(2)SO_(4)、NH_(4)Cl、NaNO_(3)、尿素、牛肉膏、大豆粉、KNO_(3)、(NH_(4))_(2)HPO_(4)或以蛋白胨、酵母粉、牛肉膏、(NH_(4))_(2)HPO_(4)、大豆粉两两随机组合]、金属离子[CuCl_(2)、MgCl_(2)、ZnCl_(2)、Al_(2)(SO_(4))_(3)、CoCl_(2)、MnCl_(2)、Ag NO_(3)、NaCl、CaCl_(2)、FeCl_(2)、BaCl_(2)、FeCl_(3)]、pH(3.0~10.0)及发酵温度(25、28、30、33、35、37、40℃),响应面法优化培养基组分含量[木聚糖、(NH_(4))_(2)HPO_(4)和大豆粉]。采用优化的培养条件培养碱性木聚糖高产菌株,检测木聚糖酶酶活,并与模型预测结果进行对比。结果筛选出1株碱性木聚糖酶高产菌株,命名为SX6-18,经16S rDNA鉴定为类芽孢杆菌(Paemibacillus)。SX6-18菌株产生的碱性木聚糖酶在温度为25~55℃和pH 6.0~10.0的范围内可保持70%以上的相对活性;Mg^(2+)可促进碱性木聚糖酶活,Fe^(3+)、Mn^(2+)和Cu^(2+)抑制酶活。确定最适碳源为木聚糖,氮源为大豆粉和(NH_(4))_(2)HPO_(4)组合,金属离子为Fe^(3+),pH为8.0,发酵温度为30℃。确定最适培养基组分含量为:15.00 g/L木聚糖、3.03 g/L(NH_(4))_(2)HPO_(4)、3.28 g/L大豆粉。采用优化的培养基及发酵条件下生产的碱性木聚糖酶酶活达701.08 U/mL,与预测值(693.96 U/mL)相近。结论 成功筛选出了1株碱性木聚糖酶高产菌株SX6-18,在碱性条件下可保持较高酶活,本实验为木聚糖酶在造纸及洗涤等领域的应用奠定了基础。

Objective To screen a high alkaline xylanase-producing strain,subject to molecular identification and characterization of enzymatic property,and optimize its fermentation condition.MethodsThe farmland soil samples from Shanxi,Henan and Zhejiang Provinces were collected aseptically,from which the high xylanase-producing strains were screened by enrichment culture,isolation and identification of 16S r DNA,and determined for enzymatic properties.The carbon source(xylan,lactose,soluble starch,sucrose,bran and glucose),nitrogen source[ammonium oxalate,peptone,yeast powder,(NH_(4))_(2)SO_(4),NH_(4)Cl,Na NO_(3),urea,beef extract,bean powder,KNO_(3),(NH_(4))_(2)HPO_(4)or random mixture of two of peptone,yeast powder,beef extract,(NH_(4))_(2)HPO_(4)and bean powder],metal ion[CuCl_(2),MgCl_(2),ZnCl_(2),Al_(2)(SO_(4))_(3),CoCl_(2),MnCl_(2),AgNO_(3),NaCl,CaCl_(2),FeCl_(2),BaCl_(2) and FeCl_(3)],pH value(3.0~10.0)in medium and the fermentation temperature(25,28,30,33,35,37 and 40℃)were optimized by single factor test,while the contents of components[xylan,(NH_(4))_(2)HPO_(4)and bean powder]by response surface method.The high alkaline xylanase-producing strain was fermented by using the optimized medium under the optimized condition,and determined for xylanase activity,and the result was compared with that predicated in model.ResultsA high alkaline xylanase-producing strain named as SX6-18 was screened and identified as Paemibacillus based on 16s rDNA sequence analysis.The xylanase produced by the strain maintained more than 70%of relative activity at temperatures of 25~55℃and pH 6.0~10.0.Mg^(2+)promoted while Fe^(3+),Mn^(2+)and Cu^(2+)inhibited the activity of xylanase.The optimal carbon source,nitrogen source and metal ion of the medium were xylan,bean powder+(NH_(4))_(2)HPO_(4) and Fe^(3+),while the optimal p H value and temperature for fermentation were 8.0 and 30℃,respectively.However,the optimal component contents were 15.00 g/L xylan,3.03 g/L(NH_(4))_(2)HPO_(4)and 3.28 g/L bean powder.The activity of xylanase cultured in optimized medium under opti-mized condition for fermentation reached 701.08 U/m L,which was closed to the expected value(693.96 U/m L).ConclusionA high alkaline xylanase-producing strain SX6-18 was successfully screened,which maintained relatively high enzyme activity in alkaline condition.This study laid a foundation of application of xylanase in papermaking and washing fields.