Cloning, Purification, and Characterization of a Heat- and Alkaline-Stable Endoglucanase B from Aspergillus niger BCRC31494
Abstract
:1. Introduction
2. Results and Discussion
2.1. Cloning, Sequencing, and Analysis of eglB
2.2. Expression, Purification, and Deglycosylation of EGLB
2.3. Optimal pH and Temperature of the Recombinant EGLB in Pichia pastoris
2.4. Substrate Specificity and Kinetic Parameters of EGLB
Substrate | Main linkage (monomer) | Specific activity (U/mg) | Km(mg/mL) | Vmax (U/min/mg) |
---|---|---|---|---|
β-D-glucan from barley | 1,3-1,4-β-(glucose) | 405 | 53.0 | 32.4 |
locust bean gum | backbone of (1-4) β-D-mannopyranosyl units branches of (1-6)-linked α-D-galactopyranosyl units | 171 | ||
cellobiose | 1,4-β-(glucose) | 78 | ||
CMC-Na | 1,4-β-(glucose) | 68 | 135 | 4.7 |
Laminarin | 1,3-β-(glucose) | 26 | ||
xylan from beechwood | 1,4-β-(xylose) | 19 | ||
Chitin | 1,4-β-(glucosamine) | 10 | ||
Salicin | β-(glucose) | 6 | ||
Avicel | 1,4-β-(glucose) | 0 | ||
p-nitrophyl-α-D-glucopyranoside | 0 | |||
p-nitrophyl-β-D-glucopyranoside | 0 |
2.5. Effects of Metal Ions and Chemicals on EGLB Activity
Compound added | Conc. (mM) | Relative acitivity(%) |
---|---|---|
None | 100 | |
CdCl2 | 1 | 102 |
5 | 88 | |
10 | 79 | |
FeCl2 | 1 | 84 |
5 | 85 | |
10 | 86 | |
MgCl2 | 1 | 87 |
5 | 84 | |
10 | 77 | |
CaCl2 | 1 | 88 |
5 | 85 | |
10 | 75 | |
CoCl2 | 1 | 141 |
5 | 175 | |
10 | 186 | |
ZnCl2 | 1 | 85 |
5 | 81 | |
10 | 68 | |
CuCl2 | 1 | 100 |
5 | 104 | |
10 | 117 | |
Triton X-100 | 10 | 201 |
Tween 80 | 10 | 205 |
SDS | 1 | 80 |
5 | 78 | |
10 | 1 | |
EDTA | 1 | 92 |
5 | 93 | |
10 | 110 |
3. Experimental
3.1. Strains, Plasmids, and Media
3.2. Total RNA, Genomic DNA, and Plasmid DNA Isolation
3.3. Construction of the eglB Library
3.4. RT-PCR of a cDNA Encoding eglB
3.5. Construction of the eglB Expression Vector
3.6. P. pastoris Transformation
3.7. P. pastoris Induction and Expression
3.8. Purification of the Recombinant EGLB from P. pastoris No. 31
3.9. pI Analysis
3.10. LC-MASS Assay
3.11. Assay of Endoglucanase Activity
3.12. Optimal Temperature and pH Determination
3.13. Determination of Kinetic Parameters
3.14. Effect of Metal Ions and Chemicals on Enzyme Activity
4. Conclusions
References
- Huang, H.; Yang, P.; Luo, H.; Tang, H.; Shao, N.; Yuan, T.; Wang, Y.; Bai, Y.; Yao, B. High-level expression of a truncated 1,3-1,4-β-D-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation. Appl. Microbiol. Biotechnol. 2008, 78, 95–103. [Google Scholar]
- Watanabe, H.; Tokuda, G. Cellulolytic systems in insects. Annu. Rev. Entomol. 2010, 55, 609–632. [Google Scholar] [CrossRef]
- Kitamoto, N.; Go, M.; Shibayama, T.; Kimura, T.; Kito, Y.; Ohmiya, K.; Tsukagoshi, N. Molecular cloning, purification and characterization of two endo-1,4-β-glucanases from Aspergillus oryzae KBN616. Appl. Microbial. Biotechnol. 1996, 46, 538–544. [Google Scholar]
- Horikoshi, K. Alkaliphiles: Some applications of their products for biotechnology. Microbiol. Mol. Biol. Rev. 1999, 63, 735–750. [Google Scholar]
- Jin, X.; Meng, N.; Xia, L.M. Expression of an endo-β-1,4-glucanase gene from Orpinomyces PC-2 in Pichia pastoris. Int. J. Mol. Sci. 2011, 12, 3366–3380. [Google Scholar] [CrossRef]
- Fujita, Y.; Ito, J.; Ueda, M.; Fukuda, H.; Kondo, A. Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. Appl. Environ. Microbiol. 2004, 70, 1207–1212. [Google Scholar] [CrossRef]
- Béguin, P.; Aubert, J.P. The biological degradation of cellulose. FEMS Microbiol. Rev. 1994, 13, 25–58. [Google Scholar] [CrossRef]
- Tomme, P.; Warren, R.A.J.; Gilkes, N.R. Cellulose hydrolysis by bacteria and fungi. Adv. Microb. Physiol. 1995, 37, 1–81. [Google Scholar] [CrossRef]
- van Petegem, F.; Vandenberghe, I.; Bhat, M.K.; van Beeumen, J. Atomic resolution structure of the major endoglucanase from Thermoascus aurantiacus. Biochem. Biophys. Res. Commun. 2002, 296, 161–166. [Google Scholar]
- Lynd, L.R.; Weimer, P.J.; van Zyl, W.H.; Pretorius, I.S. Microbial cellulose utilization: Fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 2002, 66, 506–577. [Google Scholar] [CrossRef]
- Wang, T.; Liu, X.; Yu, Q.; Zhang, X.; Qu, Y.; Gao, P.; Wang, T. Directed evolution for engineering pH profile of endoglucanase III from Trichoderma reesei. Biomol. Eng. 2005, 22, 89–94. [Google Scholar] [CrossRef]
- Qin, Y.; Wei, X.; Song, X.; Qu, Y. Engineering endoglucanase II from Trichoderma reesei to improve the catalytic efficiency at a higher pH optimum. J. Biotechnol. 2008, 135, 190–195. [Google Scholar]
- Mernitz, G.; Koch, A.; Henrissat, B.; Schulz, G. Endoglucanase II (EGII) of Penicillium janthinellum: cDNA sequence, heterologous expression and promoter analysis. Curr. Genet. 1996, 29, 490–495. [Google Scholar]
- Schuster, E.; Dunn-Coleman, N.; Frisvad, J.C.; van Dijck, P.W. On the safety of Aspergillus niger—A review. Appl. Microbiol. Biotechnol. 2002, 59, 426–435. [Google Scholar] [CrossRef]
- Quay, D.H.X.; Baker, F.D.A.; Rabu, A.; Said, M.; Lllias, R.M.; Mahadi, N.M.; Hassan, O.; Murad, A.M.A. Overexpression, purification and characterization of the Aspergillus niger endoglucanase, eglA, in Pichia pastoris. Afr. J. Biotechnol. 2011, 10, 2101–2111. [Google Scholar]
- van Peij, N.N.M.E.; Gielkens, M.M.C.; de Vries, R.P.; Visser, J.; de Graaff, L.H. The transcriptional activator XlnR regulates both xylanolytic and endoglucanase gene expression in Aspergillus niger. Appl. Environ. Microbiol. 1998, 64, 3615–3619. [Google Scholar]
- Hasper, A.A.; Dekkers, E.; van Mil, M. EglC, a new endoglucanase from Aspergillus niger with major activity towards xyloglucan. Appl. Environ. Microbiol. 2002, 68, 1556–1560. [Google Scholar]
- Hong, J.; Tamaki, H.; Akiba, S.; Yamamoto, K.; Kumagai, H. Cloning of a gene encoding a high stable endo-β-1,4-glucanase from Aspergillus niger and its expression in yeast. J. Biosci. Bioeng. 2001, 92, 434–441. [Google Scholar]
- Stricker, A.R.; Hain, K.G.; Würleitner, E.; Mach, R.L. Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina. Eukaryot. Cell 2006, 5, 2128–2137. [Google Scholar] [CrossRef]
- Meiler, S.; de Jongh, W.A.; Olsson, L.; Nielsen, J. Physiological characterization of cauB deletion in Aspergillus niger. Appl. Microbiol. Biotechnol. 2009, 84, 157–167. [Google Scholar]
- Yoshikazu, E.; Yokoyama, M.; Morimoto, M.; Shirai, K.; Chikamatsu, G.; Kato, N.; Tsukagoshi, N.; Kato, M.; Kobayashi, T. Novel promoter sequence required for inductive expression of the Aspergillus nidulans endoglucanase gene eglA. Biosci. Biotechnol. Biochem. 2008, 72, 312–320. [Google Scholar]
- Henrissat, B.; Callebaut, I.; Fabrega, S.; Lehn, P.; Mornon, J.P.; Davies, G. Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc. Natl. Acad. Sci. USA 1995, 92, 7090–7094. [Google Scholar] [CrossRef]
- Sakon, J.; Adney, W.S.; Himmel, M.E.; Thomas, S.R.; Karplus, P.A. Crystal structure of thermostable family 5 endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose. Biochemistry 1996, 35, 10648–10660. [Google Scholar]
- Kanokratana, P.; Chantasingh, D.; Champreda, V.; Tanapongpipat, S.; Pootanakit, K.; Eurwilaichitr, L. Identification and expression of cellobiohydrolase (CBHI) gene from an endophytic fungus, Fusicoccum sp. (BCC4124) in Pichia pastoris. Protein Expr. Purif. 2008, 58, 148–153. [Google Scholar] [CrossRef]
- Sreekrishna, K.; Brankamp, R.G.; Kropp, K.E.; Blankenship, D.T.; Tsay, J.T.; Smith, P.L.; Wierschke, J.D.; Subramaniam, A.; Birkenberger, L.A. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 1997, 190, 55–62. [Google Scholar] [CrossRef]
- Vidmar, S.; Turk, V.; Kregar, I. Cellulolytic complex of Aspergillus niger under conditions for citric acid production. Isolation and characterization of two β-(1→4)-glucan hydrolases. Appl. Microbiol. Biotechnol. 1984, 20, 326–330. [Google Scholar]
- Wonganu, B.; Pootanakit, K.; Boonyapakron, K.; Champreda, V.; Tanapongpipat, S.; Eurwilaichitr, L. Cloning, expression and characterization of a thermotolerant endoglucanase from Syncephalastrum racemosum (BCC18080) in Pichia pastoris. Protein Expr. Purif. 2008, 58, 78–86. [Google Scholar] [CrossRef]
- Lin, L.; Meng, X.; Liu, P.; Hong, Y.; Wu, G.; Huang, X.; Li, C.; Dong, J.; Xiao, L.; Liu, Z. Improved catalytic efficiency of Endo-β-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution. Appl. Microbial. Biotechnol. 2009, 82, 671–679. [Google Scholar]
- Ding, S.J.; Ge, W.; Buswell, J.A. Secretion, purification, and characterisation of a recombinant Volvariella volvacea endoglucanase expressed in the yeast Pichia pastoris. Enzyme Microb. Technol. 2002, 31, 621–626. [Google Scholar] [CrossRef]
- Singh, J.; Batra, N.; Sobti, R.C. Purification and characterization of alkaline cellulose produced by a novel isolate, Bacillus sphaericus JS1. J. Ind. Microbiol. Biotechnol. 2004, 31, 51–56. [Google Scholar] [CrossRef]
- Zheng, Y.; Pan, Z.; Zhang, R.; Wang, D.; Jenkins, B. Non-ionic surfactants and non-catalytic protein treatment on enzymatic hydrolysis of pretreated creeping wild ryegrass. Appl. Biochem. Biotechnol. 2008, 146, 231–248. [Google Scholar]
- Huang, X.M.; Yang, Q.; Liu, Z.H.; Fan, J.X.; Chen, X.L.; Song, J.Z.; Wang, Y. Cloning and heterologous expression of a novel endoglucanase gene egVIII from Trichoderma viride in Saccharomyces cerevisiae. Appl. Biochem. Biotechnol. 2010, 162, 103–115. [Google Scholar]
- Luo, H.; Yang, J.; Yang, P.; Li, J.; Huang, H.; Shi, P.; Bai, Y.; Wang, Y.; Fan, Y.; Yao, B. Gene cloning and expression of a new acidic family 7 endo-β-1,3-1,4-glucanase from the acidophilic fungus Bispora sp., MEY-1. Appl. Microbiol. Biotechnol. 2010, 85, 1015–1023. [Google Scholar] [CrossRef]
- Kim, S.B.; Kim, H.J.; Kim, C.J. Enhancement of the enzymatic digestibility of waste newspaper using tween. Appl. Biochem. Biotechnol. 2006, 130, 486–495. [Google Scholar] [CrossRef]
- Wu, S.; Letchworth, G.J. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 2004, 36, 152–154. [Google Scholar]
- Laemmli, U.K. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. [Google Scholar] [CrossRef]
- Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31, 426–428. [Google Scholar] [CrossRef]
- Sample Availability: Samples are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
Share and Cite
Li, C.-H.; Wang, H.-R.; Yan, T.-R. Cloning, Purification, and Characterization of a Heat- and Alkaline-Stable Endoglucanase B from Aspergillus niger BCRC31494. Molecules 2012, 17, 9774-9789. https://doi.org/10.3390/molecules17089774
Li C-H, Wang H-R, Yan T-R. Cloning, Purification, and Characterization of a Heat- and Alkaline-Stable Endoglucanase B from Aspergillus niger BCRC31494. Molecules. 2012; 17(8):9774-9789. https://doi.org/10.3390/molecules17089774
Chicago/Turabian StyleLi, Chien-Huang, Hsing-Ren Wang, and Tsong-Rong Yan. 2012. "Cloning, Purification, and Characterization of a Heat- and Alkaline-Stable Endoglucanase B from Aspergillus niger BCRC31494" Molecules 17, no. 8: 9774-9789. https://doi.org/10.3390/molecules17089774