高温碱性条件下漆酶活力测试方法的研究  

Studies on the Assay Method of Laccase Activity under Elevated Temperature and Alkaline Conditions

作　　者：孙见良 刘浩[1] 何焕清[2] 许向阳 

机构地区：[1]华南理工大学轻工科学与工程学院,广东广州510640 [2]广东省农业科学院蔬菜研究所,广东广州510640 [3]枣庄市杰诺生物酶有限公司,山东枣庄277100

出　　处：《造纸科学与技术》2017年第3期66-71,共6页Paper Science & Technology

基　　金：制浆造纸工程国家重点实验室基金项目(2017QN01)

摘　　要：用带控温装置的紫外可见光谱仪作为高温酶活检测平台,建立了以2,2’-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)二铵盐(ABTS)为底物的高温、碱性漆酶活力测试方法,着重探讨了ABTS游离基(ABTS·+)消减对测试的影响,结果表明,本测试系统可供进行83℃以下动力学光谱分析,可用于漆酶的耐温、耐碱性研究,宜采用低离子浓度(4 mmol/L)缓冲液做介质,测试时长1 min,按此条件,在pH 4、7和10时,因ABTS·+消减而产生的误差绝对值分别低于1%,1.5%和5%。使用该系统测得诺维信漆酶最适pH为4.5、最适温度为60℃,还从三种担子菌漆酶中筛选出具有较高耐温性的乌芝酶。A testing platform for enzyme activity at elevated temperature was established by connecting a UV -Vis spectroscopy with a temperature regulating device. The assay method of laccase towards 2,2＇-azino-bis （3-ethyl- benzothiazoline-6-sulphonic acid） （ABTS） was developed, in particular under heating and alkaline conditions. The impacts of ABTS·＋ radical scavenging on the testing was discussed. Results showed the platform allowed spectroscopic measurement at a temperature as high as 83℃, applicable in the research on heat - and base - stability of laccase. Recommended conditions included the use of buffer with low ion coneentration, i.e. 4 mmol/L, as testing media and a duration time within 1 min. Under these conditions, the absolute values of errors caused by ABTS·＋ radical scavenging were lower than 1% , 1.5% and 5% , respectively, for media with pH of 4, 7 and 10. Using this testing platform, the pH optima of Novozymes laccase was determined to be 4.5, and the optimal temperature was 60℃. Laccase from Fruetificatio Amaurodermatis Rudae was selected from three basidiomycete laccases due to its higher temperature adaptability.

关 键 词：漆酶 ABTS 活力测试 高温 碱性 

分 类 号：Q55[生物学—生物化学]