马立娟,佟文哲,杜丽平,崔馨予,马清,肖冬光

• 1:天津科技大学生物工程学院
• 2:工业发酵微生物教育部重点实验室

摘要(Abstract)：

裂解性多糖单加氧酶(LPMOs)辅助活性9家族蛋白(AA9)可有效促进纤维素酶降解纤维素。为研究N-糖基化对来源于黑曲霉的AA9蛋白AnLPMO15g与纤维素酶协同性的影响,采用定点突变的方法,将An LPMO15g中3个N-糖基化修饰位点(151、334、385)上的天冬酰胺用中性谷氨酰胺取代。结果表明,作用于微晶纤维素时,突变体N-151产生的还原糖量比AnLPMO15g提高了10.34%,突变体N-385产生的还原糖量降低了12.73%,突变体N-334变化不显著;作用于稻草粉时,3个突变体产生的还原糖量均高于An LPMO15g,提高幅度为7%～19%。当与纤维素酶共同作用于微晶纤维素时,只有突变体N-334产生的还原糖量较AnLPMO15g显著提高了38.89%;共同作用于稻草粉时,突变体N-334和N-385产生的还原糖量略高于AnLPMO15g。由此可见,N-糖基化修饰对AnLPMO15g及其与纤维素酶协同降解作用均有不同程度的影响,其中N-151和N-385位点的糖基化修饰对提高An LPMO15g与纤维素酶协同降解活性尤为重要。

关键词(KeyWords)： N-糖基化;纤维素酶;定点突变;协同性

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31770625);; 天津市自然科学基金重点项目(16JCZDJC31880)

作者(Author): 马立娟,佟文哲,杜丽平,崔馨予,马清,肖冬光

参考文献(References)：

• [1] ENGEL D, WEGENER G. Wood:Chemistry, ultrastructure, reactions[M]. Berlin:Springer Verlag, 1984.
• [2] KARIMI K, KHERADMANDINIA S, TAHERZADEH M J. Conversion of rice straw to sugars by dilute-acid hydrolysis[J].Biomass&Bioenergy, 2006, 30(3):247-253.
• [3] LYND L R, LASER M S, BRANSBY D, et al. How biotech can transform biofuels[J]. Nature Biotechnology, 2008, 26(2):169-172.
• [4]祝其丽,何明雄,谭芙蓉,等.木质纤维素生物质预处理研究现状[J].生物技术进展, 2015, 5(6):414-419.ZHU Q L, HE M X, TAN F R. et al. Progress on pretreatment technologies of lignocellulosic biomass[J]. Current Biotechnology,2015, 5(6):414-419.
• [5] HUMPULA J F, UPPUGUNDLA N, VISMEH R, et al. Probing the nature of AFEX-pretreated corn stover derived decomposition products that inhibit cellulase activity[J]. Bioresource Technology, 2014, 152(1):38-45.
• [6] TADESSE H, LUQUE R. Advances on biomass pretreatment using ionic liquids:An overview[J]. Energy&Environmental Science, 2011, 4(10):3913-3929.
• [7] SINGH J, SUHAG M, DHAKA A. Augmented digestion of lignocellulose by steam explosion, acid and alkaline pretreatment methods:A review[J]. Carbohydrate Polymers, 2015, 117:624-631.
• [8] HORN S J, VAAJEKOLSTAD G, BJ?RGE WESTERENG, et al. Novel enzymes for the degradation of cellulose[J]. Biotechnology for Biofuels, 2012, 5(1):45-45.
• [9] HU J, CHANDRA R, ARANTES V, et al. The addition of accessory enzymes enhances the hydrolytic performance of cellulase enzymes at high solid loadings[J]. Bioresource Technology, 2015, 186:149-153.
• [10]李欣,张丽丽,田莉,等.裂解多糖单加氧酶高效催化的研究进展[J].生物化学与生物物理进展, 2016, 43(10):970-979.LI X, ZHANG L L, TIAN L, et al. The advance of efficient catalysis of lytic polysaccharide monooxygenases[J]. Progress in Biochemistry and Biophysics, 2016, 43(10):970-979.
• [11] JOHANSEN K S. Lytic polysaccharide monooxygenases:The microbial power tool for lignocellulose degradation[J]. Trends in Plant Science, 2016, 21(11):926-936.
• [12] TANGHE M, DANNEELS B, CAMATTARI A, et al. Recombinant expression of Trichoderma reesei Cel61A in Pichia pastoris:optimizing yield and N-terminal processing[J]. Molecular Biotechnology, 2015, 57(11/12):1010-1017.
• [13]武婕,张晓雪,余河水,等.毕赤酵母工程菌高密度发酵研究与进展[J].中国生物工程杂志, 2016, 36(1):108-114.WU J, ZHANG X X, YU H S, et al. Research progress of high density fermentation process of Pichia pastoris[J]. China Biotechnology, 2016, 36(1):108-114.
• [14]詹洁,吴军.酵母N-糖基化工程研究进展[J].生物技术通讯, 2004, 15(3):272-274.ZHAN J, WU J. Advanced studies on the engineering of yeast asparagines-linked glycosylation[J]. Letters in Biotechnology, 2004,15(3):272-274.
• [15] MULLERSTEFFNER H, KUHN I, ARGENTINI M, et al. Identification of the N-glycosylation sites on recombinant bovine CD38expressed in Pichia pastoris:Their impact on enzyme stability and catalytic activity[J]. Protein Expression&Purification, 2010,70(2):151-157.
• [16] WEI Y D, LEE S J, LEE K S, et al. N-glycosylation is necessary for enzymatic activity of a beetle(Apriona germari)cellulase[J].Biochemical&Biophysical Research Communications, 2005, 329(1):331-336.
• [17] ALBERS J J, DAY J R, WOLFBAUER G, et al. Impact of site-specific N-glycosylation on cellular secretion, activity and specific activity of the plasma phospholipid transfer protein[J]. Biochimica et Biophysica Acta, 2011, 1814(7):908-911.
• [18] TIAN B, CHEN Y, DING S. A combined approach for improving alkaline acetyl xylan esterase production in Pichia pastoris, and effects of glycosylation on enzyme secretion, activity and stability[J]. Protein Expression&Purification, 2012, 85(1):44-50.
• [19] NAGAI K, IHARA Y, WADA Y, et al. N-glycosylation is requisite for the enzyme activity and Golgi retention of N-acetylglucosaminyltransferase III[J]. Glycobiology, 1997, 7(6):769-776.
• [20] YANG M, YU X W, ZHENG H, et al. Role of N-linked glycosylation in the secretion and enzymatic properties of Rhizopus chinensis lipase expressed in Pichia pastoris[J]. Microbial Cell Factories, 2015, 14(1):40.
• [21]马清,蔡瑞,姜风超,等. N-糖基化对β-甘露聚糖酶在毕赤酵母中异源表达的影响[J].食品科学, 2017, 38(16):86-91.MA Q, CAI R, JIANG F C, et al. Effect of N-glycosylation on the heterologous expression ofβ-mannanase in Pichia pastoris[J].Food Science, 2017, 38(16):86-91.
• [22] G. L. MILLER. Use of dinitrosalicylic acid reagent for determination of reducing sugar[J]. Analytical Biochemistry, 1959, 31(3):426-428.
• [23] TROMBETTA E S. The contribution of N-glycans and their processing in the endoplasmic reticulum to glycoprotein biosynthesis[J].Glycobiology, 2003, 13(9):77R-91R.
• [24] HAN M, WANG W, WANG X, et al. Enhanced expression of recombinant elastase in Pichia pastoris through the substitution of Thr for Ser in Asn-Xaa-Ser sequons[J]. Applied Biochemistry&Biotechnology, 2015, 175(1):428-435.