Please wait a minute...
 
 
食品与发酵工业  2020, Vol. 46 Issue (16): 99-104    DOI: 10.13995/j.cnki.11-1802/ts.023934
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
棘孢木霉(Trichoderma asperellum)产聚半乳糖醛酸酶的特性
王恒震, 李化强*, 吴菲菲, 陈琼, 张早明, 刘庆
邵阳学院 食品与化学工程学院, 湖南 邵阳,422000
Characterization of pectin degrading polygalacturonase produced by Trichoderma asperellum
WANG Hengzhen, LI Huaqiang*, WU Feifei, CHEN Qiong, ZHANG Zaoming, LIU Qing
Department of Food and Chemical Engineering, Shaoyang University, Shaoyang 422000, China
下载:  HTML   PDF (2506KB) 
输出:  BibTeX | EndNote (RIS)      
摘要 聚半乳糖醛酸酶是水解D-半乳糖醛酸α-1,4-糖苷键的酶,在食品工业特别是果蔬加工中具有重要意义。该文研究棘孢木霉(14636)所产聚半乳糖醛酸酶的酶学特性,采用硫酸铵沉淀法、透析袋透析,对粗酶液进行纯化,并以聚合烯酰胺凝胶电泳(sodium dodecyl sulfate polyamide gel electrophoresis,SDS-PAGE)确定其分子质量大小。实验结果表明,该酶的活性区域在29.29~50.46 kDa,聚半乳糖醛酸酶的最适反应pH为4.0,在pH 3.0~5.0稳定性较好,最适温度为40 ℃,具有一定的热稳定性;在乙酸乙酸钠缓冲溶液中活性较高。催化性能表明,果胶是聚半乳糖醛酸酶的最佳底物,Km值为0.74 mg/mL,Vmax为3 100 μg/min; Mn2+、Mg2+、Cu2+、Ca2+和Tritonx-100对酶有激活作用,Ca2+和Tritonx-100激活作用较强,Co2+、Zn2+、Ba2+、Li+、Fe2+、SDS和Tween-80对该酶有不同的抑制作用;贮藏特性研究表明,该酶即使在30 ℃下30 d仍保留70%以上酶活性。研究结果为果蔬清洁加工领域新型酶制剂的开发提供理论依据。
服务
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章
王恒震
李化强
吴菲菲
陈琼
张早明
刘庆
关键词:  棘孢木霉  聚半乳糖醛酸酶  酶学特性  纯化    
Abstract: Polygalacturonase is an enzyme that hydrolyzes D-galacturonic acid α-1,4- glucoside bond, which is of great significance in food industry, especially in fruit and vegetable processing. To exploit the biochemical properties of polygalacturonase produced from Trichoderma asperellum (14636), the polygalacturonase was isolated and purified by ammonium sulfate fractionation and dialysis method. Its molecular weight was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). According to the experimental results, the activation range of this enzyme was from 29.29 kDa to 50.46 kDa, the optimum pH was 4.0, it had good stability within pH 3.0-5.0; the optimum temperature was 40 ℃, and it showed certain thermal stability. The enzyme activity was higher in sodium acetate buffer. The catalytic properties showed that pectin was the optimal substrate for polygalacturonase, and the Km value was 0.74 mg/ml, Vmax was 3100 μg/min. Mn2+, Mg2+, Cu2+, Ca2+ and Tritonx-100 could activate this enzyme, and the activate effect of Ca2+ and Tritonx-100 were stronger; while Co2+, Zn2+, Ba2+, Li+, Fe2+, SDS and Tween-80 exerted different inhibitory effects on its activity. Storage characteristics showed that this enzyme retained more than 70% of its activity at 30 ℃ for 30 d. This study can provide a theoretical basis for the development of new enzyme preparations in the field of fruit and vegetable clean processing.
Key words:  Trichoderma asperellum    polygalacturonase    enzymatic characteristics    purification
收稿日期:  2020-03-12      修回日期:  2020-04-15           出版日期:  2020-08-25      发布日期:  2020-09-17      期的出版日期:  2020-08-25
基金资助: 邵阳学院研究生科研创新项目(CX2018SY041;CX2019SY037);湖南省教育厅科学研究一般项目(17C1433);优秀青年项目 (17B241);湖南省自然科学基金青年项目(2017JJ3284);湖南省科技厅“三区”科技人才项目(S2017NCTPSQ0044)
作者简介:  硕士研究生(李化强副教授为通讯作者,E-mail:biohqff@163.com)
引用本文:    
王恒震,李化强,吴菲菲,等. 棘孢木霉(Trichoderma asperellum)产聚半乳糖醛酸酶的特性[J]. 食品与发酵工业, 2020, 46(16): 99-104.
WANG Hengzhen,LI Huaqiang,WU Feifei,et al. Characterization of pectin degrading polygalacturonase produced by Trichoderma asperellum[J]. Food and Fermentation Industries, 2020, 46(16): 99-104.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.023934  或          http://sf1970.cnif.cn/CN/Y2020/V46/I16/99
[1] NAIDU G S N, PANDA T. Production of pectolytic enzymes:A review[J]. Bioprocess Engineering, 1998, 19(5):355-361.
[2] GUMMADI N S, PANDA T. Purification and biochemical properties of micro-bial pectinases:A review[J]. Process Biochemistry, 2003,38(7): 987-996.
[3] PAGÁN A, CONDE J, IBARZ A, et al. Albedo hydrolysis modelling and digestion with reused effluents in the enzymatic peeling process of grape fruits[J]. Journal of the Science of Food and Agriculture, 2010, 90(14):2 433-2 439.
[4] KASHYAP D, VOHRA P, CHOPRA S, et al. Applications of pectinases in the commercial sector: A review[J]. Bioresour Technol, 2001,77(3): 215-227.
[5] VAN BUREN J P. Causes and prevention of turbidity in apple juice[J]. Processed Apple Products, 1989:97-120.DOI:10.1007/978-1-4684-8225-6_5.
[6] DEY T B, ADAK S, BHATTACHARYA P, et al. Purification of polygalacturonase from Aspergillus awamori nakazawa MTCC 6652 and its application in apple juice clarification[J]. LWT-Food Science and Technology,2014,59(1): 591-595.
[7] 龙艳珍,吴菲菲,李化强,等.真空冷冻干燥棘孢木霉菌株工艺优化研究[J].食品研究与开发,2019,40(2):144-148.
[8] 尹锦辉,吴菲菲,赵良忠,等.酶法去除脐橙白皮层的产酶培养基优化研究[J].中国南方果树,2016,45(5):44-49;51.
[9] SANTOS-VILLALOBOS S D L, GUZMÁN-ORTIZ D A, GÓMEZ-LIM M A,et al. Potential use of Trichoderma asperellum (Samuels, Liechfeldt et Nirenberg) T8a as a biological control agent against anthracnose in mango (Mangifera indica L.)[J]. Biological Control, 2013, 64(1):37-44.
[10] ADE R, NASARUDDIN N, HENDARTO H, et al. Endophytic association of Trichoderma asperellum within Theobroma cacao suppresses vascular streak dieback incidence and promotes side graft growth[J]. Mycobiology, 2016, 44(3):180-186.
[11] MILLER G L. Use of dinitrosalycilic acid reagent for determination of reducing sugar[J]. Analytical Chemistry, 1959,31(3): 426-428.
[12] 王小敏,吴文龙,闾连飞,等.分光光度计法测定果胶酶活力的方法研究[J].食品工业科技,2007,28(5):227-229.
[13] BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72(12):248-254.
[14] 赵祥颖,刘建军,刘丽萍,等.硫酸铵对DNS试剂测定几丁质酶活力的影响[J].食品与生物技术学报,2009,28(4):555-558.
[15] RIED J L, COLLMER A. Activity stain for rapid characterization of pectic enzymes in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels[J]. Applied & Environmental Microbiology, 1985, 50(3):615-622.
[16] REHMAN H U, AMAN A, NAWAZ M A, et al. Characterization of pectin degrading polygalacturonase produced by Bacillus licheniformis KIBGE-IB21[J]. Food Hydrocolloids, 2015, 43:819-824.
[17] CHENG Z, CHEN D, LU B, et al. A novel acid-stable endo-polygalacturonase from Penicillium oxalicum CZ1028: Purification, characterization, and application in the beverage industry[J]. Journal of Microbiology and Biotechnology, 2016, 26(6):989.
[18] ZHOU M, WU J, WANG T, et al. The purification and characterization of a novel alkali-stable pectate lyase produced by Bacillus subtilis PB1[J]. World Journal of Microbiology and Biotechnology, 2017, 33(10):190.
[19] KANT S, VOHRA A, GUPTA R. Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323[J]. Protein Expression and Purification, 2013, 87(1):11-16.
[20] NITURE S K.Comparative biochemical and structural characterizations of fungal polygalacturonases[J]. Biologia, 2008, 63(1):1-19.
[21] JAYANI R S, SAXENA S, GUPTA R. Microbial pectinolytic enzymes: A review[J]. Process Biochemistry, 2005, 40(9):2 931-2 944.
[22] MCDERMID A S, MCKEE A S, MARSH P D. Effect of environmental pH on enzyme activity and growth of Bacteroides gingivalis W50[J]. Infection and Immunity, 1988, 56(5):1 096-1 100.
[23] 林建城,杨文杰,朱丽华,等.商品果胶酶(Aspergillus niger)的催化动力学研究[J].甘肃农业大学学报,2006,41(4):81-85.
[24] PEDROLLI D B, CARMONA E C. Purification and characterization of the exopolygalacturonase produced by Aspergillus giganteusin submerged cultures[J].Journal of Industrial Microbiology & Biotechnology,2010, 37(6):567-573.
[25] PEDROLLI D B, MONTEIRO A P, GOMES E, et al. Pectin and pectinases: Production, characterization and industrial application of microbial pectinolytic enzymes[J]. Open Biotechnology Journal, 2009, 3(1):9-18.
[26] KOBAYASHI T, HIGAKI N, YAJIMA N, et al. Purification and properties of a galacturonic acid-releasing exopolygalacturonase from a strain of Bacillus[J]. Bioscience Biotechnology and Biochemistry, 2001, 65(4):842-847.
[27] MEI Y, CHEN Y, ZHAI R, et al. Cloning, purification and biochemical properties of a thermostable pectinase from Bacillus halodurans M29[J]. Journal of Molecular Catalysis B: Enzymatic, 2013, 94:77-81.
[28] 张跃,钟芳,麻建国.商品果胶酶中PE和PG的热性质[J].哈尔滨商业大学学报(自然科学版),2007,23(6):736-739;746.
[29] SILVA D, MARTINS E D, LEITE R S, et al. Purification and characterization of an exo-polygalacturonase produced by Penicillium viridicatum RFC3 in solid-state fermentation[J]. Process Biochemistry,2007,42(8): 1 237-1 243.
[30] NAWAZ M, KARIM A, AMAN A, et al. Continuous degradation of maltose: Improvement in stability and catalytic properties of maltase (α-glucosidase) through immobilization using agar-agar gel as a support[J]. Bioprocess and Biosystems Engineering, 2015, 38(4):631-638.
[31] YUAN P, MENG K, LUO H, et al. A novel low-temperature active alkaline pectate lyase from Klebsiella sp. Y1 with potential in textile industry[J]. Process Biochem,2011,46(10): 1 921-1 926.
[1] 蔡燕, 田丹, 严鑫, 李百裕, 李宇杰, 于丽娟, 吴锦明. 一步法快速从脱脂豆粉中三相分离脂肪氧合酶[J]. 食品与发酵工业, 2021, 47(9): 149-153.
[2] 张晓晓, 柴智, 冯进, 崔莉, 李春阳, 李莹, 黄午阳. 牛蒡多糖的提取及生物活性研究进展[J]. 食品与发酵工业, 2021, 47(6): 280-288.
[3] 彭燕鸿, 苏爱秋, 黄伟文, 蓝素桂, 杨天云, 谭强. 微生物嗜热脂肪酶研究进展[J]. 食品与发酵工业, 2021, 47(6): 289-294.
[4] 胡冠华, 王德宝, 苏琳, 赵丽华, 田建军, 张忠海, 朱浩田, 靳烨. 食源性钙螯合肽的研究概况[J]. 食品与发酵工业, 2021, 47(3): 224-229.
[5] 武芸, 王春林, 王丽朋, 张腊腊, 胡浩斌. 黑果枸杞多酚吸附分离特性及抗氧化性研究[J]. 食品与发酵工业, 2021, 47(2): 70-77.
[6] 张强, 李伟华. 抗氧化肽的研究现状[J]. 食品与发酵工业, 2021, 47(2): 298-304.
[7] 李丹阳, 李宇辉, 高云云, 闫艺, 王俊钢, 卢士玲. 新疆哈萨克族风干肉中产蛋白酶乳酸菌的筛选及酶学特性研究[J]. 食品与发酵工业, 2020, 46(9): 57-63.
[8] 董振香, 顾秋亚, 李丹晨, 孙驰翔, 余晓斌. 不同分子质量魔芋甘露聚糖的制备及功效活性分析[J]. 食品与发酵工业, 2020, 46(8): 48-53.
[9] 伍燕, 朱家豪, 汪伟, 申利群. 暗褐网柄牛肝菌多糖AHP分离纯化和结构研究[J]. 食品与发酵工业, 2020, 46(8): 92-96.
[10] 李驰, 李春生, 杨贤庆, 戚勃, 赵永强, 王悦齐. 海洋细菌Vibrio fluvialis的分离鉴定、产琼胶酶条件优化及酶的分离纯化[J]. 食品与发酵工业, 2020, 46(7): 35-42.
[11] 刘卫宝, 余讯, 徐静静, 詹晓北, 张洪涛, 朱莉. 黄芪多糖的分离、结构表征及益生活性研究[J]. 食品与发酵工业, 2020, 46(7): 50-56.
[12] 裴若楠, 翟红蕾, 戚勃, 郝淑贤, 黄卉, 杨贤庆. 石花菜多糖的分离纯化及单糖组成分析[J]. 食品与发酵工业, 2020, 46(7): 57-62.
[13] 胡晓, 周雅, 杨贤庆, 吴燕燕, 陈胜军, 李来好, 马海霞. 食物蛋白源降尿酸活性肽的研究进展[J]. 食品与发酵工业, 2020, 46(4): 287-293.
[14] 范三红, 贾槐旺, 张锦华, 任嘉兴, 白宝清. 羊肚菌多糖纯化、结构分析及抗氧化活性[J]. 食品与发酵工业, 2020, 46(3): 65-71.
[15] 张梅娟, 钱朋智, 董力青, 韩杨, 汲广习, 冯镇. 农产品副产物半纤维素水解糖的分离纯化及单糖组成分析[J]. 食品与发酵工业, 2020, 46(20): 72-77.
No Suggested Reading articles found!
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
版权所有 © 《食品与发酵工业》编辑部
地址:北京朝阳区酒仙桥中路24号院6号楼111室
本系统由北京玛格泰克科技发展有限公司设计开发  技术支持:support@magtech.com.cn