Characterization of Thermophilic β-Glucosidase of Rhizospheric Bacterial Strain (LSKB15) Isolated from Cholistan Desert, Pakistan

Amer Ahmed^1, , Faiz ul Hassan Nasim², Kashfa Batool², Aasia Bibi³ and Sarwat Rafi²

¹Department of Biochemistry and Biotechnology, Faculty of Science, The Islamia University of Bahawalpur, Bahawalpur, Pakistan

²Department of Chemistry, Biochemistry Section, Faculty of Science, The Islamia University of Bahawalpur, Bahawalpur, Pakistan

³Department of Biochemical Sciences, School of Molecular Biology and Medicine, La Sapienza University of Rome, Rome, Italy

Cite this paper:
Amer Ahmed, Faiz ul Hassan Nasim, Kashfa Batool, Aasia Bibi and Sarwat Rafi. Characterization of Thermophilic β-Glucosidase of Rhizospheric Bacterial Strain (LSKB15) Isolated from Cholistan Desert, Pakistan. American Journal of Microbiological Research. 2018; 6(4):173-180. doi: 10.12691/ajmr-6-4-5

Abstract

Fifty thermophilic bacterial strains isolated from rhizospheric soil of Cholistan desert, Pakistan, and designated as LSKB01-LSKB50 were screened for β-glucosidase gene (bgl) belonging to glycoside hydrolase family 1 (GH 1) using PCR technique. Subsequently, the same strains were screened for extracellular β-glucosidase production using esculin as substrate. All fifty strains were shown to be amplified for conserved region of bgl gene and to secrete extracellular β-glucosidase. One strain (LSKB15) secreted relative high amount of this enzyme as indicating by size of ferric-esculetin precipitate. This strain was further cultivated on cellulose containing media and β-glucosidase was purified by ammonium sulfate, dialysis and gel filtration chromatography. The purified enzyme showed an optimal temperature of 60°C and an optimal pH of 7. It also showed excellent temperature and pH stability retaining > 90% activity after incubation for 2 h at pH 5-8 and 40-60°C. Finally, the purified enzyme was run on Native-PAGE and subsequently incubated in phosphate buffer containing 5 mM of 4-methylumbelliferyl-β-D-glucoside (4-MUG) for 15 min at 50°C and visualized by UV light as white band. We concluded that thermophilic LSKB15 β- glucosidase may work with other cellulase to degrade available cellulose synthesized by plant and the properties exhibited by it such as high temperature and pH stability pointed out its potential industrial importance.

Keywords:
Screening cellulose cellulase β-glucosidase thermophile PCR biofuel

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]	Bhat M, Bhat S. Cellulose degrading enzymes and their potential industrial applications. Biotechnology Advances. 1997; 15(3): 583-620.
[2]	Zhang XZ, Zhang YHP. Cellulases: characteristics, sources, production, and applications. Bioprocessing Technologies in Biorefinery for Sustainable Production of Fuels, Chemicals, and Polymers. 2013; 1:131-46.
[3]	Ahmed A, Nasim FH, Batool K, Bibi A. Microbial ß-Glucosidase: Sources, Production and Applications. Journal of Applied & Environmental Microbiology. 2017; 5(1): 31-46.
[4]	Anwar Z, Gulfraz M, Irshad M. Agro-industrial lignocellulosic biomass a key to unlock the future bio-energy: a brief review. Journal of Radiation Research and Applied Sciences. 2014; 7(2): 163-73.
[5]	Tiwari P, Misra B, Sangwan NS. β-Glucosidases from the fungus Trichoderma: an efficient cellulase machinery in biotechnological applications. BioMed Research International. 2013; 2013.
[6]	Ahmed A, Aslam M, Ashraf M, Nasim FH, Batool K, and Bibi A, “Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications.” Journal of Applied & Environmental Microbiology, 2017 (2): 57-73.
[7]	Warzecha H, Gerasimenko I, Kutchan TM, Stöckigt J. Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis. Phytochemistry. 2000; 54(7): 657-66.
[8]	Fowler T, Brown RD, Jr. The bgl1 gene encoding extracellular beta-glucosidase from Trichoderma reesei is required for rapid induction of the cellulase complex. Molecular Microbiology. 1992; 6(21): 3225-35.
[9]	Kiran EU, Trzcinski AP, Liu Y. Enhancing the hydrolysis and methane production potential of mixed food waste by an effective enzymatic pretreatment. Bioresource Technology. 2015; 183: 47-52.
[10]	Zahoor S, Javed MM, Aftab MN, Ikram-ul-Haq. Cloning and expression of β-glucosidase gene from Bacillus licheniformis into E. coli BL 21 (DE3). Biologia. 2011; 66(2): 213-20.
[11]	Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A. Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresource Technology. 2013; 127: 500-7.
[12]	Seeberger PH, Werz DB. Synthesis and medical applications of oligosaccharides. Nature. 2007; 446(7139): 1046-51.
[13]	Bhatia Y, Mishra S, Bisaria V. Microbial β-glucosidases: cloning, properties, and applications. Critical Reviews in Biotechnology. 2002; 22(4): 375-407.
[14]	Arshad M, Ashraf MY, Ahmad M, Zaman F. Morpho-genetic variability potential of Cenchrus ciliaris L. from Cholistan desert, Pakistan. Pakistan Journal of Botany. 2007; 39(5): 1481-8.
[15]	Arshad M, Ashraf M, Arif N. Morphological variability of Prosopis cineraria (L.) Druce, from the Cholistan desert, Pakistan. Genetic Resources and Crop Evolution. 2006; 53(8): 1589-96.
[16]	Li H, Xu X, Chen H, Zhang Y, Xu J, Wang J, et al. Molecular analyses of the functional microbial community in composting by PCR-DGGE targeting the genes of the beta-glucosidase. Bioresource Technology. 2013; 134: 51-58.
[17]	Cuskey SM, Schamhart DHJ, Chase T, Jr., Montenecourt BS, Eveleigh DE. Screening for. Beta-glucosidase mutants of Trichoderma reesei with resistance to end-product inhibition. Development in Industrial Microbiology; (United States); 1980; 21: 471-480.
[18]	Matteotti C, Thonart P, Francis F, Haubruge E, Destain J, Brasseur C, et al. New glucosidase activities identified by functional screening of a genomic DNA library from the gut microbiota of the termite Reticulitermes santonensis. Microbiological Research. 2011; 166(8): 629-42.
[19]	Pandey S, Sree A, Dash S, Sethi D. A novel method for screening beta-glucosidase inhibitors. BMC Microbiology. 2013; 13(1): 55.
[20]	Karnchanatat A, Petsom A, Sangvanich P, Piaphukiew J, Whalley AJ, Reynolds CD. Purification and biochemical characterization of an extracellular β-glucosidase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.: Fr.) Rehm. FEMS Microbiology Letters. 2007; 270(1): 162-70.
[21]	Bhatti HN, Batool S, Afzal N. Production and Characterization of a Novel β-Glucosidase from Fusarium solani. International Journal of Agriculture & Biology. 2013; 15(1). 140-144
[22]	Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 1976; 72(1-2): 248-254.
[23]	Veena V, Poornima P, Parvatham R, Kalaiselvi K. Isolation and characterization of β-glucosidase producing bacteria from different sources. African Journal of Biotechnology. 2011; 10(66): 14891-906.
[24]	Hernndez-Guzmn A, Flores-Martnez A, Ponce-Noyola P, Villagmez-Castro JC. Purification and characterization of an extracellular β-glucosidase from Sporothrix schenckii. FEBS Open Bio. 2016; 6(11): 1067-77.
[25]	Verma A, Parinati R, Nishad S, Shalini S. Extracellular production, partial purification and biochemical characterization of a β-glucosidase from Bacillus subtilis strain Ps identified in sugarcane bagasse. World Journal of Agricultural Sciences. 2014; 10(6): 269-78.
[26]	An D-S, Cui C-H, Lee H-G, Wang L, Kim SC, Lee S-T, et al. Identification and characterization of a novel Terrabacter ginsenosidimutans sp. nov. β-glucosidase that transforms ginsenoside Rb1 into the rare gypenosides XVII and LXXV. Applied and Environmental Microbiology. 2010; 76(17): 5827-36.
[27]	Du J, Cui C-H, Park SC, Kim J-K, Yu H-S, Jin F-X, et al. Identification and Characterization of a Ginsenoside-Transforming β-Glucosidase from Pseudonocardia sp. Gsoil 1536 and Its Application for Enhanced Production of Minor Ginsenoside Rg 2 (S). PloS one. 2014; 9(6): e96914.
[28]	Shipkowski S, Brenchley JE. Characterization of an unusual cold-active β-glucosidase belonging to family 3 of the glycoside hydrolases from the psychrophilic isolate Paenibacillus sp. strain C7. Applied and Environmental Microbiology. 2005; 71(8): 4225-32.
[29]	Crespim E, Zanphorlin LM, de Souza FH, Diogo JA, Gazolla AC, Machado CB, et al. A novel cold-adapted and glucose-tolerant GH1 β-glucosidase from Exiguobacterium antarcticum B7. International Journal of Biological Macromolecules. 2016; 82: 375-80.
[30]	Agrawal R, Satlewal A, Verma AK. Development of a β-glucosidase hyperproducing mutant by combined chemical and UV mutagenesis. 3 Biotech. 2013; 3(5): 381-8.
[31]	Kengen SW, Luesink EJ, STAMS AJ, ZEHNDER AJ. Purification and characterization of an extremely thermostable β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. European Journal of Biochemistry. 1993; 213(1): 305-12.
[32]	Hong M-R, Kim Y-S, Park C-S, Lee J-K, Kim Y-S, Oh D-K. Characterization of a recombinant β-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus. Journal of Bioscience and Bioengineering. 2009; 108(1): 36-40.
[33]	Chen P, Fu X, Ng TB, Ye X-Y. Expression of a secretory β-glucosidase from Trichoderma reesei in Pichia pastoris and its characterization. Biotechnology Letters. 2011; 33(12): 2475-9.
[34]	Zahoor S, Javed M, Aftab M. Cloning and expression of β-glucosidase gene from Bacillus licheniformis into E. coli BL 21 (DE3). Biologia. 2011; 66(2): 213-20.
[35]	Yang F, Yang X, Li Z, Du C, Wang J, Li S. Overexpression and characterization of a glucose-tolerant β-glucosidase from T. aotearoense with high specific activity for cellobiose. Applied Microbiology and Biotechnology. 2015; 99(21): 8903-15.
[36]	Zhao L, Pang Q, Xie J, Pei J, Wang F, Fan S. Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovorans cellulose binding domain and its application in hydrolysis of microcrystalline cellulose. BMC Biotechnology. 2013; 13(1): 1.
[37]	Naz S, Ikram N, Rajoka MI, Sadaf S, Akhtar MW. Enhanced production and characterization of a beta-glucosidase from Bacillus halodurans expressed in Escherichia coli. Biochemistry. 2010; 75(4): 513-25.
[38]	Irchhaiya R, Kumar A, Yadav A, Gupta N, Kumar S, Gupta N, et al. Metabolites in plants and its classification. World Journal of Pharmacy and Pharmaceutical Sciences. 2015; 4(1): 287-305.
[39]	Biver S, Stroobants A, Portetelle D, Vandenbol M. Two promising alkaline beta-glucosidases isolated by functional metagenomics from agricultural soil, including one showing high tolerance towards harsh detergents, oxidants and glucose. Biotechnology. 2014; 41(3): 479-88.
[40]	Jiang C, Li S-X, Luo F-F, Jin K, Wang Q, Hao Z-Y, et al. Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning. Bioresource Technology. 2011; 102(3): 3272-8.
[41]	Liu D, Zhang R, Yang X, Zhang Z, Song S, Miao Y, et al. Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33. Microbial Cell Factories. 2012; 11(1): 1.
[42]	Choi J-Y, Park A-R, Kim YJ, Kim J-J, Cha C-J, Yoon J-J. Purification and characterization of an extracellular beta-glucosidase produced by Phoma sp. KCTC11825BP isolated from rotten mandarin peel. Journal of Microbiology and Biotechnology. 2011; 21(5): 503-8.
[43]	Dikshit R, Tallapragada P. Partial Purification and Characterization of β-glucosidase from Monascus sanguineus. Brazilian Archives of Biology and Technology. 2015; 58(2): 185-91.
[44]	Mallerman J, Papinutti L, Levin L. Characterization of β-Glucosidase Produced by the White Rot Fungus Flammulina velutipes. Journal of Microbiology & Biotechnology. 2015; 25(1): 57-65.
[45]	Daroit DJ, Simonetti A, Hertz PF, Brandelli A. Purification and characterization of an extracellular beta-glucosidase from Monascus purpureus. Journal of Microbiology and Biotechnology. 2008; 18(5): 933-41.
[46]	Chen H-L, Chen Y-C, Lu M-YJ, Chang J-J, Wang H-TC, Ke H-M, et al. A highly efficient β-glucosidase from the buffalo rumen fungus Neocallimastix patriciarum W5. Biotechnology for Biofuels. 2012; 5(1): 1-10.
[47]	Mahapatra S, Vickram AS, Sridharan TB, Parameswari R, Pathy MR. Screening, production, optimization and characterization of β-glucosidase using microbes from shellfish waste. 3 Biotech. 2016; 6(2): 213.
[48]	Li Y, Liu N, Yang H, Zhao F, Yu Y, Tian Y, et al. Cloning and characterization of a new β-Glucosidase from a metagenomic library of Rumen of cattle feeding with Miscanthus sinensis. BMC Biotechnology. 2014; 14(1): 1.
[49]	Uchiyama T, Yaoi K, Miyazaki K. Glucose-tolerant β-glucosidase retrieved from a Kusaya gravy metagenome. Frontiers in Microbiology. 2015; 6: 548.
[50]	Zhang Y, Yuan L, Chen Z, Fu L, Lu J, Meng Q, et al. Purification and characterization of beta-glucosidase from a newly isolated strain Tolypocladium cylindrosporum Syzx4. Chemical Research in Chinese Universities. 2011; 27: 557-61.
[51]	Bai H, Wang H, Sun J, Irfan M, Han M, Huang Y, Han X, Yang Q: Production, purification and characterization of novel betaglucosidase from newly isolated Penicillium simplicissimum H-11in submerged fermentation. EXCLI journal 2013, 12: 528.
[52]	Mai Z, Yang J, Tian X, Li J, Zhang S. Gene cloning and characterization of a novel salt-tolerant and glucose-enhanced β-glucosidase from a marine streptomycete. Applied Biochemistry and biotechnology. 2013; 169(5): 1512-22.
[53]	Sørensen A, Ahring BK, Lübeck M, Ubhayasekera W, Bruno KS, Culley DE, et al. Identifying and characterizing the most significant β-glucosidase of the novel species Aspergillus saccharolyticus. Canadian Journal of Microbiology. 2012; 58(9): 1035-46.
[54]	Riou C, Salmon J-M, Vallier M-J, Günata Z, Barre P. Purification, characterization, and substrate specificity of a novel highly glucose-tolerant β-glucosidase fromAspergillus oryzae. Applied and Environmental Microbiology. 1998; 64(10): 3607-14.
[55]	Yoon J-J, Kim K-Y, Cha C-J. Purification and characterization of thermostable β-glucosidase from the brown-rot basidiomycete Fomitopsis palustris grown on microcrystalline cellulose. The Journal of Microbiology. 2008; 46(1): 51-5.
[56]	Chan CS, Sin LL, Chan K-G, Shamsir MS, Manan FA, Sani RK, et al. Characterization of a glucose-tolerant β-glucosidase from Anoxybacillus sp. DT3-1. Biotechnology for Biofuels. 2016; 9(1): 174.
[57]	Saibi W, Amouri B, Gargouri A. Purification and biochemical characterization of a transglucosilating β-glucosidase of Stachybotrys strain. Applied Microbiology and Biotechnology. 2007; 77(2): 293-300.
[58]	Chen S, Hong Y, Shao Z, Liu Z. A cold-active β-glucosidase (Bgl1C) from a sea bacteria Exiguobacterium oxidotolerans A011. World Journal of Microbiology and Biotechnology. 2010; 26(8): 1427-35.