马立娟^1,2,佟文哲¹,杜丽平^1,2,崔馨予¹,马清¹,肖冬光^1,2

• 1:天津科技大学 生物工程学院，天津 300457
• 2:工业发酵微生物教育部重点实验室，天津 300457

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AnLPMO15g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AnLPMO15g，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AnLPMO15g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^1,2,佟文哲¹,杜丽平^1,2,崔馨予¹,马清¹,肖冬光^1,2

DOI: 10.3969/j.issn.1003-9015.2019.05.016

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, BJØRGE 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 CD38 expressed 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 of β-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.