Abstract
β-1,3-1,4-Glucanase (BGlc8H) from Paenibacillus sp. X4 was mutated by error-prone PCR or truncated using termination primers to improve its enzyme properties. The crystal structure of BGlc8H was determined at a resolution of 1.8 Å to study the possible roles of mutated residues and truncated regions of the enzyme. In mutation experiments, three clones of EP 2-6, 2-10, and 5-28 were finally selected that exhibited higher specific activities than the wild type when measured using their crude extracts. Enzyme variants of BG2-6, BG2-10, and BG5-28 were mutated at two, two, and six amino acid residues, respectively. These enzymes were purified homogeneously by Hi-Trap Q and CHT-II chromatography. Specific activity of BG5-28 was 2.11-fold higher than that of wild-type BGwt, whereas those of BG2-6 and BG2-10 were 0.93- and 1.19-fold that of the wild type, respectively. The optimum pH values and temperatures of the variants were nearly the same as those of BGwt (pH 5.0 and 40 °C, respectively). However, the half-life of the enzyme activity and catalytic efficiency (k cat/K m) of BG5-28 were 1.92- and 2.12-fold greater than those of BGwt at 40 °C, respectively. The catalytic efficiency of BG5-28 increased to 3.09-fold that of BGwt at 60 °C. These increases in the thermostability and catalytic efficiency of BG5-28 might be useful for the hydrolysis of β-glucans to produce fermentable sugars. Of the six mutated residues of BG5-28, five residues were present in mature BGlc8H protein, and two of them were located in the core scaffold of BGlc8H and the remaining three residues were in the substrate-binding pocket forming loop regions. In truncation experiments, three forms of C-terminal truncated BGlc8H were made, which comprised 360, 286, and 215 amino acid residues instead of the 409 residues of the wild type. No enzyme activity was observed for these truncated enzymes, suggesting the complete scaffold of the α6/α6-double-barrel structure is essential for enzyme activity.
Similar content being viewed by others
References
Adachi W, Sakihama Y, Shimizu S, Sunami T, Fukazawa T, Suzuki M, Yatsunami R, Nakamura S, Takénaka A (2004) Crystal structure of family GH-8 chitosanase with subclass II specificity from Bacillus sp. K17. J Mol Biol 343:785–795. doi:10.1016/j.jmb.2004.08.028
Anbar M, Gul O, Lamed R, Sezerman UO, Bayer EA (2012) Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis. Appl Environ Microbiol 78:3458–3464. doi:10.1128/AEM.07985-11
Anbar M, Lamed R, Bayer EA (2010) Thermostability enhancement of Clostridium thermocellum cellulosomal endoglucanase Cel8A by a single glycine substitution. ChemCatChem 2:997–1003. doi:10.1002/cctc.201000112
Bielecki S, Galas E (1991) Microbial β-glucanase different from cellulases. Crit Rev Biotechnol 10:275–305. doi:10.3109/07388559109038212
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Cho KM, Math RK, Hong SY, Asraful Islam SM, Kim JO, Hong SJ, Kim H, Yun HD (2008) Changes in the activity of the multifunctional beta-glycosyl hydrolase (Cel44C-Man26A) from Paenibacillus polymyxa by removal of the C-terminal region to minimum size. Biotechnol Lett 30:1061–1068. doi:10.1007/s10529-008-9640-6
Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66(Pt 4):486–501. doi:10.1107/S0907444910007493
Furtado GP, Ribeiro LF, Santos CR, Tonoli CC, Souza AR, Oliveira RR, Murakami MT, Ward RJ (2011) Biochemical and structural characterization of a β-1,3–1,4-glucanase from Bacillus subtilis 168. Process Biochem 46:1202–1206. doi:10.1016/j.procbio.2011.01.037
Gouet P, Courcelle E, Stuart DI, Métoz F (1999) ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15:305–308
Guérin DM, Lascombe MB, Costabel M, Souchon H, Lamzin V, Béguin P, Alzari PM (2002) Atomic (0.94 A) resolution structure of an inverting glycosidase in complex with substrate. J Mol Biol 316:1061–1069. doi:10.1006/jmbi.2001.5404
Hrmova M, Fincher GB (2001) Structure-function relationships of β-D-glucan endo- and exohydrolases from higher plants. Plant Mol Biol 47:73–91
Jeong YS, Na HB, Kim SK, Kim YH, Kwon EJ, Kim J, Yun HD, Lee JK, Kim H (2012) Characterization of Xyn10J, a novel family 10 xylanase from a compost metagenomic library. Appl Biochem Biotechnol 166:1328–1339. doi:10.1007/s12010-011-9520-8
Jia H, Li Y, Liu Y, Yan Q, Yang S, Jiang Z (2012) Engineering a thermostable β-1,3-1,4-glucanase from Paecilomyces thermophila to improve catalytic efficiency at acidic pH. J Biotechnol 159:50–55. doi:10.1016/j.jbiotec.2012.02.007
Kim SA, Cheng KJ, Liu JH (2002) A variant of Orpinomyces joyonii 1,3-1,4-beta-glucanase with increased thermal stability obtained by random mutagenesis and screening. Biosci Biotechnol Biochem 66:171–174
Krissinel E (2012) Enhanced fold recognition using efficient short fragment clustering. J Mol Biochem 1:76–85
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the sterochemical quality of protein structures. J Appl Crystallogr 26:283–291. doi:10.1107/S0021889892009944
Li S, Sauer WC, Huang SX, Gabert VI (1996) Effect of β-glucanase supplementation to hulless barley- or wheat-soybean meal diets on the digestibilities of energy, protein, beta-glucans and amino acid in young pigs. J Anim Sci 74:1649–1656
Liang C, Fioroni M, Rodríguez-Ropero F, Xue Y, Schwaneberg U, Ma Y (2011) Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile. J Biotechnol 154:46–53. doi:10.1016/j.jbiotec.2011.03.025
Lim WJ, Hong SY, An CL, Cho KM, Choi BR, Kim YK, An JM, Kang JM, Lee SM, Cho SJ, Kim H, Yun HD (2005) Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region. Appl Microbiol Biotechnol 68:46–52. doi:10.1007/s00253-004-1880-3
Lin L, Fu C, Huang W (2016) Improving the activity of the endoglucanase, Cel8M from Escherichia coli by error-prone PCR. Enzym Microb Technol 86:52–58
Lin L, Meng X, Liu P, Hong Y, Wu G, Huang X, Li C, Dong J, Xiao L, Liu Z (2009) Improved catalytic efficiency of endo-beta-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution. Appl Microbiol Biotechnol 82:671–679. doi:10.1016/j.enzmictec.2016.01.011
Mao S, Gao P, Lu Z, Lu F, Zhang C, Zhao H, Bie X (2016) Engineering of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 to improve its catalytic efficiency. J Sci Food Agric 96:109–115. doi:10.1002/jsfa.7066
Miller GL (1959) Use of dinitrosalicylic acid reagent for the determination of reducing sugar. Anal Chem 31:428–436. doi:10.1021/ac60147a030
Murshudov GN, Skubák P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Long F, Vagin AA (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr 67(Pt 4):355–367. doi:10.1107/S0907444911001314
Na HB, Jung WK, Jeong YS, Kim HJ, Kim SK, Kim J, Yun HD, Lee JK, Kim H (2015) Characterization of a GH family 8 β-1,3-1,4-glucanase with distinctive broad substrate specificity from Paenibacillus sp. X4. Biotechnol Lett 37:643–655 . doi:10.1007/s10529-014-1724-x Erratum 37:657–8
Niu D, Zhou XX, Yuan TY, Lin ZW, Ruan H, Li WF (2010) Effect of the C-terminal domains and terminal residues of catalytic domain on enzymatic activity and thermostability of lichenase from Clostridium thermocellum. Biotechnol Lett 32:963–967. doi:10.1007/s10529-010-0241-9
Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326. doi:10.1016/S0076-6879(97)76066-X
Park SR, Cho SJ, Kim MK, Ryu SK, Lim WJ, An CL, Hong SY, Kim JH, Kim H, Yun HD (2002) Activity enhancement of Cel5Z from Pectobacterium chrysanthemi PY35 by removing C-terminal region. Biochem Biophys Res Commun 291:425–430. doi:10.1006/bbrc.2002.6437
Pei H, Guo X, Yang W, Lv J, Chen Y, Cao Y (2015) Directed evolution of a β-1,3-1,4-glucanase from Bacillus subtilis MA139 for improving thermal stability and other characteristics. J Basic Microbiol 55:869–878. doi:10.1002/jobm.201400664
Planas A (2000) Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochim Biophys Acta 1543:361–382
Qiao J, Dong B, Li Y, Zhang B, Cao Y (2009) Cloning of a β-1,3-1,4-glucanase gene from Bacillus subtilis MA139 and its functional expression in Escherichia coli. Appl Biochem Biotechnol 152:334–342. doi:10.1007/s12010-008-8193-4
Schrodinger, L (2010) The PyMOL molecular graphics system, Version 1.3r1
Shin ES, Yang MJ, Jung KH, Kwon EJ, Jung JS, Park SK, Kim J, Yun HD, Kim H (2002) Influence of the transposition of the thermostabilizing domain of Clostridium thermocellum xylanase (XynX) on xylan binding and thermostabilization. Appl Environ Microbiol 68:3496–3501
Telke AA, Zhuang N, Ghatge SS, Lee SH, Ali Shah A, Khan H, Um Y, Shin HD, Chung YR, Lee KH, Kim SW (2013) Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates. PLoS One 8:e65727. doi:10.1371/journal.pone.0065727
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX woindows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882
Wang Y, Yuan H, Wang J, Yu Z (2009) Truncation of the cellulose binding domain improved thermal stability of endo-beta-1,4-glucanase from Bacillus subtilis JA18. Bioresour Technol 100:345–349. doi:10.1016/j.biortech.2008.06.001
Wen TN, Chen JL, Lee SH, Yang NS, Shyur LF (2005) A truncated Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase with improved enzymatic activity and thermotolerance. Biochemistry 44:9197–9205
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67(Pt 4):235–242. doi:10.1107/S0907444910045749
Woodward JR, Fincher GB, Stone BA (1983) Water-soluble (1 → 3), (1 → 4)-β-D-glucans from barley (Hordeum vulgare) endosperm. I Physicochemical properties Carbohydr polym 3:207–225. doi:10.1016/0144-8617(83)90004-8
Xu T, Zhu T, Li S (2016) β-1,3-1,4-glucanase gene from Bacillus velezensis ZJ20 exerts antifungal effect on plant pathogenic fungi. World J Microbiol Biotechnol 32:26. doi:10.1007/s11274-015-1985-0
Zhang XZ, Zhang YHP (2011) Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis. Microb Biotechnol 4:98–105. doi:10.1111/j.1751-7915.2010.00230.x
Acknowledgements
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (#2013R1A1A4A01013394) and by WTU Joint Research Grants of Konkuk University.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Seung Cheol Baek and Thien-Hoang Ho contributed equally to this work.
Rights and permissions
About this article
Cite this article
Baek, S.C., Ho, TH., Lee, H.W. et al. Improvement of enzyme activity of β-1,3-1,4-glucanase from Paenibacillus sp. X4 by error-prone PCR and structural insights of mutated residues. Appl Microbiol Biotechnol 101, 4073–4083 (2017). https://doi.org/10.1007/s00253-017-8145-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-017-8145-4