【作者】 刘晶；

【导师】 兰时乐；

【作者基本信息】 湖南农业大学 ， 生物工程（专业学位）， 2022， 硕士

【摘要】 葡萄糖氧化酶（glucose oxidase）系统名称为β-D-葡萄糖氧化还原酶（EC1.1.3.4）,简称GOD。葡萄糖氧化酶在有氧条件下能专一催化β-D-葡萄糖生成葡萄糖酸和过氧化氢。葡萄糖氧化酶在面制品加工、食品保鲜、动物饲养、动物疾病防治、医学诊断和啤酒工业等领域应用广泛。目前,葡萄糖氧化酶的制备主要是采用黑曲霉、点青霉、特异青霉通过液体深层通风发酵。本文采用Fiedure.K.J显色法从土壤中分离筛选葡萄糖氧化酶产生菌,通过形态学观察,并结合ITS序列分析对分离菌株进行了鉴定,采用单因素试验法及响应面法研究了葡萄糖氧化酶产生菌的固体发酵培养基组成和发酵工艺条件,并研究了葡萄糖氧化酶酶催化动力学等相关酶学性质。主要研究结果如下:（1）采用Fiedure.K.J显色法,从采集土样中分离纯化得到葡萄糖氧化酶产生菌,命名为LJ-1。经过形态学观察并基于ITS-r DNA序列分析构建系统发育树,将菌种LJ-1鉴定为褐红篮状菌LJ-1（Talaromyces pinophilus LJ-1）。（2）通过单因素试验,研究了葡萄糖添加量、氮源种类及添加量、KH₂PO₄添加量、Mg SO₄·7H₂O添加量、Ca Cl₂添加量、Zn SO₄添加量和固水比、发酵温度、培养基初始p H值、接种量、装量和发酵时间对菌株LJ-1固体发酵产葡萄糖氧化酶活力的影响,在单因素试验结果的基础上,采用响应面法分别对发酵培养基组成和发酵工艺条件进行了优化。结果表明,适宜的固体发酵培养基组成为:麦麸100%、葡萄糖14.245%、Na NO₃0.644%、KH₂PO₄ 0.2%、Mg SO₄·7H₂O 0.25%、Ca Cl₂0.197%、Zn SO₄0.25%、吐温80 1%;适宜的发酵工艺条件为:固水比1:0.78,温度32℃,初始p H值6.68,固体装量20g,接种量6%。在此发酵条件下,葡萄糖氧化酶酶活力可达47.40U/g·干基。（3）分析了褐红篮状菌LJ-1（Talaromyces pinophilus LJ-1）葡萄糖氧化酶的酶学性质。其最适反应p H值为6.4,在p H5.6-8.0范围内具有较强的稳定性;最适反应温度为40℃,在20-80℃温度范围内酶活稳定。浓度为0.01mol/L金属离子Fe³⁺对酶活力有明显促进作用,Al³⁺对酶活力有轻微促进作用,而Zn²⁺对酶活有显著的抑制作用,Ca²⁺、K⁺、Mg²⁺、Na⁺对酶活力影响不大。采用Lineweaver-Burk方程双倒数法作图得到菌株LJ-1葡萄糖氧化酶酶促反应的米氏常数Km为118mmol/L,Vmax为5.07mol/（L·min）。更多还原

【Abstract】 Glucose oxidase（glucose oxidase）system name isβ-D-glucose oxidoreductase（EC1.1.3.4）,referred to as GOD.Glucose oxidase can specifically catalyzeβ-D-glucose to gluconic acid under aerobic conditions.Glucose oxidase is widely used in flour processing,food preservation,animal feeding,animal disease prevention and control,medical diagnosis and beer industry.At present,the preparation of glucose oxidase mainly adopts Aspergillus niger,Penicillium punctatum,Penicillium notatum through liquid deep ventilation fermentation.Fieldure K.J chromogenic method was used to isolate and screen glucose oxidase producing strain from soil in this paper.The isolated strains were identified by morphological observation and ITS sequence analysis.The composition of solid fermentation medium and fermentation process conditions of glucose oxidase producing strain were studied by single factor test and response surface methodology,and the catalytic kinetics of glucose oxidase and other related enzymatic properties were studied.The main results were as follows:（1）The glucose oxidase producing strain,which named LJ-1,was isolated and purified from the collected soil samples by using Fiedure K.J color development method.Strain LJ-1was identified as Talaromyces pinophilus LJ-1 through morphological observation and phylogenetic tree construction based on ITS-r DNA sequence analysis.（2）Effects of glucose content,the type and amount of nitrogen source,KH₂PO₄ addition,Mg SO₄.7H₂O addition,Ca Cl₂ addition,Zn SO₄ addition and solid water ratio,fermentation temperature,initial p H value,inoculation amount,loading and fermentation time on glucose oxidase activity by solid fermentation of strain LJ-1 through single factor test.The composition of fermentation medium and fermentation conditions were optimized by response surface methodology based on the results of single factor experiment.The results showed that the suitable composition of solid fermentation medium were:wheat bran 100%,glucose14.245%and Na NO₃ 0.644%,KH₂PO₄ 0.2%,Mg SO₄·7H₂O 0.25%,Ca Cl₂0.197%,Zn SO₄0.25%,Tween80 1%;The optimum fermentation conditions were as follows:solid water ratio1:0.78,temperature 32℃,initial p H 6.68,solid loading 20g and inoculation amount 6%.Under these fermentation condition,the glucose oxidase activity reached 47.40U/g·dry basis.（3）The enzymatic properties of glucose oxidase of Talaromyces pinophilus LJ-1 were analyzed.The optimal reaction p H of glucose oxidase was 6.4,and maintained strong stability at Ph5.6-8.0;The optimal reaction temperature of glucose oxidase was 40℃,which was stable in the temperature range of 20-80℃.The concentration of 0.01mol/L metal ion Fe³⁺could promoted the enzyme activity significantly and Al³⁺promoted the enzyme activity slightly,while Zn²⁺inhibited the enzyme activity significantly,and Ca²⁺,K⁺,Mg²⁺,Na⁺had little effect on the enzyme activity.The Michaelis constant km and Vmax of glucose oxidase enzymatic reaction of strain LJ-1 were 118 mmol/L and 5.07 mol/（L·min）.更多还原