在葡萄糖氧化酶研发生产过程中,需要对发酵液进行酶活力检测。目前常用的方法有滴定法、电化学法和连续分光光度法,但这些方法均因其独立重复实验误差较大而缺乏实用性。因此建立一种操作简单、快速、重现性好、成本低的检测方法,是葡萄糖氧化酶研发生产中亟待解决的问题。该项研究在生产实践中,开发建立了一种新的葡萄糖氧化酶活性测定方法:碘-淀粉分光光度法。以β-D-葡萄糖为底物,利用葡萄糖氧化酶特异性催化作用,生成中间产物过氧化氢,再通过碘-淀粉分光光度法定量测定过氧化氢含量,进而计算葡萄糖氧化酶活力。结果表明:反应体系在葡萄糖氧化酶浓度为0.1~0.5 U/mL范围时,检测平行性良好,独立重复实验酶活力计算结果相对偏差小于5%,在统计学分析误差范围内。
Many methods, such as titration, electrochemical and continuous spectrophotometry have been developed for measuring the activity of glucose oxidase. However, they are regarded as impractical since they all have large errors in independent repeated experiments and a simple and fast method with better reproducibility and less costs is desperately needed. In this study, an iodine-starch spectrophotometry method for measuring the activity of glucose oxidase was developed. The method used β-D-glucose as the substrate which could be turned into gluconic acid and produce hydrogen peroxide at the same time when the reaction started and an iodine-starch spectrophotometry method was used to measure the content of hydrogen peroxide produced. The results showed that the new developed method had good parallelism within the activity range of 0.1-0.5 U/mL in the measurement system. The average relative deviation was less than 5%, which was within the statistical margin of error. To sum up, this innovative method for measuring glucose oxidase activity is promising in laboratory or industry application for its accurate and stable with good reproducibility and low cost.
[1] HATZINIKOLAOU D G, MACRIS B J. Factors regulating production of glucose oxidase by Aspergillus niger [J]. Enzyme Microb Tech, 1995, 17(6): 530-534.
[2] PLUSCHKELL S, HELLMUTH K, RINAS U. Kinetics of glucose oxidase excretion by recombinant Aspergillus niger [J]. Biotechnol Bioeng, 1996, 51(2): 215-220.
[3] HATZINIKOLAOU D G, HANSEN O C, MACRIS B J, et al. A new glucose oxidase from Aspergillus niger: Characterization and regulation studies of enzyme and gene [J]. Appl Microbiol Biot, 1996, 46(4): 371-381.
[4] 李蓉, 张庆芳, 迟乃玉. 微生物葡萄糖氧化酶的研究进展[J]. 中国酿造, 2018, 37(3): 1-5.
[5] 廖兆民, 蔡俊, 林建国. 微生物葡萄糖氧化酶的研究进展[J]. 食品与发酵工业, 2018, 44(7): 308-315.
[6] 郭贺楠, 杨勇智, 董冰, 等. 葡萄糖氧化酶研究进展[J]. 中国畜牧杂志, 2018, 54(4): 10-14.
[7] 刘颖, 陈雄, 廖蓓, 等. 工业酒精发酵污染菌的分离及新型抑菌剂的应用[J]. 食品与发酵工业, 2017, 43(8): 46-52.
[8] 黎芳, 刘佳, 王冉冉, 等. 葡萄糖氧化酶对全麦面团及全麦馒头品质改良的影响[J]. 食品工业科技, 2019, 40(14): 78-82;88.
[9] 刘春莹, 胡善松, 张庆芳, 等. 海洋低温葡萄糖氧化酶细菌选育及其酶学性质研究[J].食品与发酵工业,2019,45(13):52-58.
[10] DUBEY M K, ZEHRA A, AAMIR M, et al. Improvement strategies, cost effective production, and protential applications of fungal glucose oxidase(GOD): Current updates [J]. Frontiers in Microbiology, 2017, 8(8): 89-90.
[11] SISAK C, CSANADI Z, RONAY E, et al. Elimination of glucose in egg using immobilized glucose oxidase [J]. Enzyme and Microbial Technology, 2016, 39(5): 1 002-1 007.
[12] 郑伟萍, 王洪军, 张民, 等. 葡萄糖氧化酶和过氧化氢酶对肉鸡生长性能及养分代谢率的影响[J]. 饲料工业, 2019, 40(4): 35-39.
[13] 高立云. 一种快速葡萄糖氧化酶活性测定方法与应用效果研究[D]. 泰安:齐鲁工业大学, 2017.
[14] 任婷月, 周万里, 张利群, 等. 一种检测葡萄糖氧化酶活力的新方法[J]. 食品与发酵工业, 2015, 41(1): 212-215.
[15] 肖志明, 樊霞, 马东霞. 葡萄糖氧化酶的应用和检测方法研究进展[J]. 中国畜牧杂志, 2014, 50(18): 76-81.
[16] 李丕武, 刘瑜, 李瑞瑞,等. 两种葡萄糖氧化酶活力测定方法的比较[J]. 食品工业科技, 2013, (12): 71-75;80.
[17] QB/T 4614—2013, 工业用过氧化氢酶制剂[S]. 北京: 中国轻工业出版社, 2013.
[18] 董娜, 张爱菊, 张小林. 基于碘化钾-淀粉显色光度法测定过氧化氢酶活性[J]. 中国食品添加剂, 2019, 30(8): 150-153.
[19] 王宁丽, 魏鉴腾, 祝潇, 等. 基于碘-淀粉显色的α-淀粉酶抑制剂筛选方法研究及应用[J]. 中国海洋药物, 2017, 36(5): 75-78.
[20] 梁奇峰, 黄琼. 碘化钾-淀粉体系分光光度法测定食品中亚硝酸钠含量[J]. 化学试剂, 2016, 38(7): 649-652.
[21] 梁奇峰, 王东阳, 刘莉莉. 碘-淀粉体系褪色光度法测定蜂蜜中葡萄糖的含量[J]. 食品工业, 2014, 35(7): 275-277.
[22] GB/T 24401—2009, α—淀粉酶制剂[S]. 北京: 中国标准出版社, 2009.
