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.
[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.
2020, Vol. 46 
