Co-expression of lignocellulase from termite and their endosymbionts

DU Jiao 1 JIANG Shuzhe 1 WEI Jianhua 1 SHEN Yulong 1 NI Jinfeng1

(1.State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, China 266200)

【Abstract】Natural lignocellulosic materials contain cellulose, hemicellulose, and lignin. Cellulose hydrolysis to glucose requires a series of lignocellulases. Recently, the research on the synergistic effect of lignocellulases has become a new research focus. Here, four lignocellulase genes encoding β-glucosidase, endo-1,4-β-glucanase, xylanase and laccase from termite and their endosymbionts were cloned into pETDuet-1 and pRSFDuet-1 and expressed in Escherichia coli. After SDS-PAGE analysis, the corresponding protein bands consistent with the theoretical values were observed and all the proteins showed enzyme activities. We used phosphoric acid swollen cellulose (PASC) as substrate to measure the synergistic effect of crude extracts of co-expressing enzymes and the mixture of single enzyme. The co-expressed enzymes increased the degradation efficiency of PASC by 44% compared with the single enzyme mixture; while the degradation rate increased by 34% and 20%, respectively when using filter paper and corn cob pretreated with phosphoric acid as substrates. The degradation efficiency of the co-expressed enzymes was higher than the total efficiency of the single enzyme mixture.

【Keywords】 termite; lignocellulase; co-expression; synergism;

【Funds】 National Basic Research and Development Program of China (973 Program) (No. 2011CB707402) National Natural Science Foundation of China (Nos. 31272370, 30870085)

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(Translated by CHEN T)


    [1] Ohkuma M.Termite symbiotic systems: efficientbio-recycling of lignocellulose. Appl Microbiol Biotechnol, 2003, 61 (1): 1–9.

    [2] Li HJ, Yelle DJ, Li C, et al. Lignocellulose pretreatment in a fungus-cultivating termite. Proc Natl Acad Sci USA, 2017, 114 (18): 4709–4714.

    [3] Gardner KH, Blackwell J. The structure of native cellulose. Biopolymers, 1974, 13 (10): 1975–2001.

    [4] Woodward J, Wiseman A. Fungal and other β-D-glucosidases-their properties and applications. Enzyme Microbial Technol, 1982, 4 (2): 73–79.

    [5] Angelov A, Pham VTT, Übelacker M, et al. Ametagenome-derived thermostableβ-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148. Sci Rep, 2017, 7 (1): 17306.

    [6] Watanabe H, Tokuda G. Cellulolytic systems in insects. Ann Rev Entomol, 2010, 55 (1): 609–632.

    [7] Polizeli MLTM, Rizzatti ACS, Monti R, et al. Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol, 2005, 67 (5): 577–591.

    [8] Chávez R, Bull P, Eyzaguirre J. The xylanolytic enzyme system from the genus Penicillium. JBiotechnol, 2006, 123 (4): 413–433.

    [9] Shang TT, Si DY, Zhang DY, et al. Enhancement of thermoalkaliphilic xylanase production by Pichiapastoris through novel fed-batch strategy in high cell-density fermentation. BMC Biotechnol, 2017, 17: 55.

    [10] Raud M, Tutt M, Olt J, et al. Effect of lignin content of lignocellulosic material on hydrolysis efficiency. Agron Res, 2015, 13 (2): 405–412.

    [11] Donovan SE, Eggleton P, Bignell DE. Gut content analysis and a new feeding group classification of termites. Ecol Entomol, 2001, 26 (4): 356–366.

    [12] Nakashima K, Watanabe H, Saitoh H, et al. Dual cellulose-digesting system of the wood-feeding termite, Coptotermesformosanus Shiraki. Insect Biochem Molec Biol, 2002, 32 (7): 777–784.

    [13] Ni JF, Tokuda G, Takehara M, et al. Heterologous expression and enzymatic characterization ofβ-glucosidase from the drywood-eating termite, Neotermeskoshunensis. Appl Entomol Zool, 2007, 42 (3): 457–463.

    [14] Ni JF, Takehara M, Miyazawa M, et al. Random exchanges of non-conserved amino acid residues among four parental termite cellulases by family shuffling improved thermostability. Protein Eng Des Select, 2007, 20 (11): 535–542.

    [15] Ning N.Cloning and expression of lignocellulase genes from termites and their endosymbionts [D]. Ji’nan: Shandong University, 2017 (in Chinese).

    [16] Shi XY.Purification and cloning the bacterial xylanase from the hindgut of Macrotermesbarneyi and the microbial diversity in combs of fungus-growing termites [D]. Ji’nan: Shandong University, 2015 (in Chinese).

    [17] Martins LO, Soares CM, Pereira MM, et al. Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillussubtilis endospore coat. J Biol Chem, 2002, 277 (21):18849–18859.

    [18] Sawant SS, Salunke BK, Kim BS. Degradation of corn stover by fungal cellulase cocktail for production of polyhydroxyalkanoates by moderate halophile Paracoccus sp. LL1. Bioresource Technol, 2015, 194: 247–255.

    [19] Dong WL, Xue ML, Zhang Y, et al. Characterization of aβ-glucosidase from Paenibacillus species and its application for succinic acid production from sugarcane bagasse hydrolysate. Bioresource Technol, 2017, 241: 309–316.

    [20] Gao HY, Liu ZC, Duan SW, et al. Coexpression of β-mannanase and xylanase genes in Escherichia coli. Microbiology China, 2012, 39 (3): 344-352 (in Chinese).

    [21] Riou C, Salmon JM, Vallier MJ, et al. Purification, characterization, and substrate specificity of a novel highly glucose-tolerantβ-glucosidase from Aspergillusoryzae. Appl Environ Microbiol, 1998, 64 (10): 3607–3614.

    [22] Ni JF, Wu Y, Yun C, et al. cDNA cloning and heterologous expression of an endo-β-1, 4-glucanase from the fun-growing termite Macrotermesbarneyi. Arch Insect Biochem Physiol, 2014, 86 (3): 151–164.

    [23] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem, 1959, 31 (3): 426–428.

    [24] Geng AL, Wu J, Xie RR, et al. Characterization of a laccase from a wood-feeding termite, Coptotermesformosanus. Insect Sci, 2018, 25 (2):251–258.

    [25] Yu XX, Liu Y, Cui YX, et al. Measurement of filter paper activities of cellulase with microplate-based assay. Saudi J Biol Sci, 2016, 23 (S1): S93–S98.

    [26] Zhang YHP, Cui JB, Lynd LR, et al. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid:evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules, 2006, 7 (2): 644–648.

    [27] Banerjee G, Car S, Scott-Craig JS, et al. Alkaline peroxide pretreatment of corn stover:effects of biomass, peroxide, and enzyme loading and composition on yields of glucose and xylose. Biotechnol Biofuels, 2011, 4: 16.

    [28] Liu JM.Screening for cellobiohydrolase genes and the secretion of thermostable cellulase in Bacillussubtilis [D]. Shanghai: East China University of Science and Technology, 2011 (in Chinese).

    [29] Zhang YHP, Lynd LR. Toward an aggregated understanding of enzymatic hydrolysis of cellulose:noncomplexed cellulase systems. Biotechnol Bioeng, 2004, 88 (7): 797–824.

    [30] Fonseca-Maldonado R, Ribeiro LF, Furtado GP, et al. Synergistic action of co-expressed xylanase/laccase mixtures against milled sugar cane bagasse. Process Biochem, 2014, 49 (7):1152–1161.

    [31] Kumar S, Jain KK, Bhardwaj KN, et al. Multiple genes in a single host:cost-effective production of bacterial laccase (cotA), pectate lyase (pel), and endoxylanase (xyl) by simultaneous expression and cloning in single vector in E.coli. PLoS ONE, 2015, 10 (12): e0144379.

This Article


CN: 11-1998/Q

Vol 35, No. 02, Pages 244-253

February 2019


Article Outline


  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Discussion
  • References