A low-temperature active endo-β-1,4-mannanase from Bacillus subtilis TD7 and its gene expression in Escherichia coli

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Chen-Yang Li, Fang-Fang Liu, Jiang Ye, Jin-Feng Liu, Shi-Zhong Yang, Hui-Zhan Zhang, Bo-Zhong Mu


A low-temperature active endo-β-1,4-mannanase (YBMan) from Bacillus subtilis TD7 was isolated, characterized and successfully expressed in Escherichia coli to enhance the yield of mannanase for a potential application as a gel-breaker in guar gum-based fracturing fluids in oilfields. YBMan showed good compatibility with a wide temperature range and retained about 70% relative activity at 20°C compared to its optimal temperature (65°C). This is the highest relative activity among reported low-temperature active mannanases against guar gum. The gene (1104 bp) of strain TD7 coding a protein with 367 amino acid residues was cloned and its expression generated two recombinant mannanases, TBMan-1 and TBMan-2. Compared to the wild type, the protein yield of TBMan-1 from a one-liter shake flask broth increased 5.6-fold, and the specific activity (crude enzyme) increased 6.4-fold. The total enzyme activity increased 35.8-fold with a total activity of approximately 79550 U. Moreover, TBMan-1 had at 20°C still about 80% relative activity. The enzyme was evaluated also for its application as gel-breaker and showed excellent ability for viscosity reduction with guar gum at 20°C. Low-temperature activity and high yield make the recombinant β-mannanase attractive for applications with guar-based hydraulic fracturing fluids and other biotechnological aspects.


low-temperature active;β-mannanase;gel-breaker;

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Zhao W, Zheng J, Zhou HB. 2011. A thermotolerant and cold-active mannan endo-1,4-β-mannosidase from Aspergillus niger CBS 513.88: Constitutive overexpression and high-density fermentation in Pichia pastoris. Bioresour Technol, 102:7538-7547.

Saha BC. 2003. Hemicellulose bioconversion. J Ind Microbiol Biotechnol, 30:279-291.

Dhawan S, Kaur J. 2007. Microbial mannanases: an overview of production and applications. Crit Rev Biotechnol, 27:197-216.

Wang CH, Zhang JK, Wang Y, et al. 2016. Biochemical characterization of an acidophilic β-mannanase from Gloeophyllum trabeum CBS900.73 with significant transglycosylation activity and feed digesting ability. Food Chem, 197:474-481.

Srivastava PK, Kapoor M. 2014. Cost-effective endo-mannanase from Bacillus sp. CFR1601 and its application in generation of oligosaccharides from guar gum and as detergent additive. Prep Biochem Biotechnol, 44:392-417.

Srivastava PK, Kapoor M. 2015. Recombinant GH-26 endo-mannanase from Bacillus sp. CFR1601: Biochemical characterization and application in preparation of partially hydrolysed guar gum. LWT-Food Sci Technol, 64:809-816.

Gírio FM, Fonseca C, Carvalheiro F, et al. 2010. Hemicelluloses for fuel ethanol: A review. Bioresour Technol, 101:4775-4800.

Srivastava PK, Kapoor M. 2016. Production, properties, and applications of endo-β-mannanases. Biotechnol Adv, 35:1-19.

Barati R, Liang JT. 2014. A review of fracturing fluid systems used for hydraulic fracturing of oil and gas wells. J Appl Polym Sci, 131:318-323.

Mudgil D, Barak S, Khatkar BS. 2014. Guar gum: processing, properties and food applications-A review. Adv J Food Sci Technol, 51:409-418.

You J, Liu JF, Yang SZ, et al. 2016. Low-temperature-active and salt-tolerant β-mannanase from a newly isolated Enterobacter sp. strain N18. J Biosci Bioeng, 121:140-146.

McCutchen CM, Duffaud GD, Leduc P, et al. 1996. Characterization of extremely thermostable enzymatic breakers (α‐1,6‐galactosidase and β‐1,4‐mannanase) from the hyperthermophilic bacterium Thermotoga neapolitana 5068 for hydrolysis of guar gum. Biotechnol Bioeng, 52:332-9.

Dang JF, Gong WX, Zhang NX, et al. 2010. Research and application of biological enzyme breaker for fracturing fluid for low pressure gas reservoir in Hongtai. Oilfield Chem, 27:245-249.

Hu K, Li CX, Pan J, et al. 2014. Performance of a new thermostable mannanase in breaking guar-based fracturing fluids at high temperatures with little premature degradation. Appl Biochem Biotechnol, 172:1215-1226.

Wang CH, Luo HY, Niu CF, et al. 2015. Biochemical characterization of a thermophilic β-mannanase from Talaromyces leycettanus JCM12802 with high specific activity. Appl Microbiol Biotechnol, 99:1217-1228.

Yang H, Shi PJ, Lu HQ, et al. 2015. A thermophilic β-mannanase from Neosartorya fischeri P1 with broad pH stability and significant hydrolysis ability of various mannan polymers. Food Chem, 173:283-289.

Zhou JP, Zhang R, Gao YJ, et al. 2012. Novel low-temperature-active, salt-tolerant and proteases-resistant endo-1,4-β-mannanase from a new Sphingomonas strain. J Biosci Bioeng, 113:568-574.

Zakaria MM, Ashiuchi M, Yamamoto S, et al. 1998. Optimization for β-mannanase production of a psychrophilic bacterium, Flavobacterium sp. Biosci, Biotechnol, Biochem, 62:655-660.

Liu JF, Yang J, Yang SZ, et al. 2012. Effects of different amino acids in culture media on surfactin variants produced by Bacillus subtilis TD7. Appl Biochem Biotechnol, 166:2091-2100.

Songsiriritthigul C, Buranabanyat B, Haltrich D, et al. 2010. Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-β-mannosidase from Bacillus licheniformis in Escherichia coli. Microb Cell Fact, 9:1-13.

Pongsapipatana N, Damrongteerapap P, Chantorn S, et al. 2016. Molecular cloning of kman coding for mannanase from Klebsiella oxytoca KUB-CW2-3 and its hybrid mannanase characters. Enzyme Microb Technol, 89:39-51.

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.

Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685.

Pan CS, Xu SY, Zhou HJ, et al. 2007. Recent developments in methods and technology for analysis of biological samples by MALDI-TOF-MS. Anal Bioanal Chem, 387:193-204.

Rosengren A, Reddy SK, Sjöberg JS, et al. 2014. An Aspergillus nidulans β-mannanase with high transglycosylation capacity revealed through comparative studies within glycosidase family 5. Appl Microbiol Biotechnol, 98:10091-10104.

Egelhofer V, Büssow K, Luebbert C, et al. 2000. Improvements in protein identification by MALDI-TOF-MS peptide mapping. Anal Chem, 72:2741-2750.

You J, Liu JF, Yang SZ, et al. 2016. Activity and gel breaking performance of a low-temperature-tolerant complex enzyme MEMA10. Oilfield Chem, 33:612-618.

Morris JB. 2010. Morphological and reproductive characterization of guar (Cyamopsis tetragonoloba) genetic resources regenerated in Georgia, USA. Genet Resour Crop Evol, 57:985-993.

Mclean D, Agarwal V, Stack K, et al. 2011. Synthesis of guar gum-graft-poly (acrylamide-codiallyldimethylammonium chloride) and its application in the pulp and paper industry. Bioresources, 6:4168-4180.

Wan XF, Li YM, Wang XJ, et al. 2007. Synthesis of cationic guar gum-graft-polyacrylamide at low temperature and its flocculating properties. Eur Polym J, 43:3655-3661.

Katrolia P, Zhou P, Zhang P, et al. 2012. High level expression of a novel β-mannanase from Chaetomium sp. exhibiting efficient mannan hydrolysis. Carbohydr Polym, 87:480-490.

Kim dY, Ham SJ, Lee HJ, et al. 2011. A highly active endo-β-1,4-mannanase produced by Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bacterium in the gut of Eisenia fetida. Enzyme Microb Technol, 48:365-370.

Pan C, Zhou JG, Tian A, et al. 2011. High level expression of a truncated β-mannanase from alkaliphilic Bacillus sp. N16-5 in Kluyveromyces cicerisporus. Biotechnol Lett, 33:565-570.

Benech RO, Li X, Patton D, et al. 2007. Recombinant expression, characterization, and pulp prebleaching property of a Phanerochaete chrysosporium endo-β-1,4-mannanase. Enzyme Microb Technol, 41:740-747.

Zhang R, Zhou JP, Gao YJ, et al. 2015. Molecular and biochemical characterizations of a new low-temperature active mannanase. Folia Microbiol, 60:483-492.

Huang JL, Bao LX, Zou HY, et al. 2012. High-level production of a cold-active β-mannanase from Bacillus subtilis Bs5 and its molecular cloning and expression. Mol Genet Microbiol, 27:147-153.

Song JM, Nam KW, Kang SG, et al. 2008. Molecular cloning and characterization of a novel cold-active β-1,4-D-mannanase from the Antarctic springtail, Cryptopygus antarcticus. Comp Biochem Physiol, Part B: Biochem Mol Biol, 151:32-40.

Mendoza NS, Arai M, Kawaguchi T, Cubol FS, et al. 1994. Isolation of mannan-utilizing bacteria and the culture conditions for mannanase production. World J Microbiol Biotechnol, 10:51-54.

Jiang ZQ, Wei Y, Li DY, et al. 2006. High-level production, purification and characterization of a thermostable β-mannanase from the newly isolated Bacillus subtilis WY34. Carbohydr Polym, 66:88-96.

Qiao JY, Rao ZH, Dong B, et al. 2010. Expression of Bacillus subtilis MA139 β-mannanase in Pichia pastoris and the enzyme characterization. Appl Biochem Biotechnol, 160:1362-1370.

Gerday C, Aittaleb M, Bentahir M, et al. 2000. Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol, 18:103-107.

DOI: http://dx.doi.org/10.26789/AEB.2018.02.004


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