Highly thermostable GH39 β-xylosidase from a Geobacillus sp. strain WSUCF1

BMC Biotechnology

Aditya Bhalla²  Kenneth M Bischoff³  Rajesh K Sani¹ 

[1] Department of Chemical and Biological Engineering, South Dakota School of Mines and Technology, Rapid City 57701, SD, USA

[2] Present address: Great Lakes Bioenergy Research Center, Michigan State University, East Lansing 48824, MI, USA

[3] Renewable Product Technology Research Unit, Agricultural Research Service, National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Peoria 61604, IL, USA

关键词: Thermostable;    β-xylosidase;    Biofuels;    Lignocellulose;   

Others  :  1121352
DOI  :  10.1186/s12896-014-0106-8

 received in 2014-08-22, accepted in 2014-12-01,  发布年份 2014

【 摘 要 】

Background

Complete enzymatic hydrolysis of xylan to xylose requires the action of endoxylanase and β-xylosidase. β-xylosidases play an important part in hydrolyzing xylo-oligosaccharides to xylose. Thermostable β-xylosidases have been a focus of attention as industrially important enzymes due to their long shelf life and role in the relief of end-product inhibition of xylanases caused by xylo-oligosaccharides. Therefore, a highly thermostable β-xylosidase with high specific activity has significant potential in lignocellulose bioconversion.

Results

A gene encoding a highly thermostable GH39 β-xylosidase was cloned from Geobacillus sp. strain WSUCF1 and expressed in Escherichia coli. Recombinant β-xylosidase was active over a wide range of temperatures and pH with optimum temperature of 70°C and pH 6.5. It exhibited very high thermostability, retaining 50% activity at 70°C after 9 days. WSUCF1 β-xylosidase is more thermostable than β-xylosidases reported from other thermophiles (growth temperature ≤ 70°C). Specific activity was 133 U/mg when incubated with p-nitrophenyl xylopyranoside, with K_m and V_max values of 2.38 mM and 147 U/mg, respectively. SDS-PAGE analysis indicated that the recombinant enzyme had a mass of 58 kDa, but omitting heating prior to electrophoresis increased the apparent mass to 230 kDa, suggesting the enzyme exists as a tetramer. Enzyme exhibited high tolerance to xylose, retained approximately 70% of relative activity at 210 mM xylose concentration. Thin layer chromatography showed that the enzyme had potential to convert xylo-oligomers (xylobiose, triose, tetraose, and pentaose) into fermentable xylose. WSUCF1 β-xylosidase along with WSUCF1 endo-xylanase synergistically converted the xylan into fermentable xylose with more than 90% conversion.

Conclusions

Properties of the WSUCF1 β-xylosidase i.e. high tolerance to elevated temperatures, high specific activity, conversion of xylo-oligomers to xylose, and resistance to inhibition from xylose, make this enzyme potentially suitable for various biotechnological applications.

【 授权许可】

   
2014 Bhalla et al.; licencee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Juturu V, Wu JC: Microbial xylanases: engineering, production and industrial applications. Biotechnol Adv 2012, 30:1219-1227.
• [2]Yeoman CJ, Han Y, Dodd D, Schroeder CM, Mackie RI, Cann IK: Thermostable enzymes as biocatalysts in the biofuel industry. Adv Appl Microbiol 2010, 70:1-55.
• [3]Gao D, Uppugundla N, Chundawat SP, Yu X, Hermanson S, Gowda K, Brumm P, Mead D, Balan V, Dale BE: Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides. Biotechnol Biofuels 2011, 4:5. BioMed Central Full Text
• [4]Kumar R, Wyman C: Enzymatic hydrolysis of cellulosic biomass through enhanced removal of oligomers. 2010.
• [5]Shao W, Xue Y, Wu A, Kataeva I, Pei J, Wu H, Wiegel J: Characterization of a novel beta-xylosidase, XylC, from Thermoanaerobacterium saccharolyticum JW/SL-YS485. Appl Environ Microbiol 2011, 77:719-726.
• [6]Yan QJ, Wang L, Jiang ZQ, Yang SQ, Zhu HF, Li LT: A xylose-tolerant beta-xylosidase from Paecilomyces thermophila: characterization and its co-action with the endogenous xylanase. Bioresour Technol 2008, 99:5402-5410.
• [7]Anand A, Kumar V, Satyanarayana T: Characteristics of thermostable endoxylanase and β-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1 and its applicability in generating xylooligosaccharides and xylose from agro-residues. Extremophiles 2013, 17:357-366.
• [8]Bravman T, Mechaly A, Shulami S, Belakhov V, Baasov T, Shoham G, Shoham Y: Glutamic acid 160 is the acid–base catalyst of beta-xylosidase from Bacillus stearothermophilus T-6: a family 39 glycoside hydrolase. FEBS Lett 2001, 495:115-119.
• [9]Huang Z, Liu X, Zhang S, Liu Z: GH52 xylosidase from Geobacillus stearothermophilus: characterization and introduction of xylanase activity by site-directed mutagenesis of Tyr509. J Ind Microbiol Biotechnol 2014, 41:65-74.
• [10]Nanmori T, Watanabe T, Shinke R, Kohno A, Kawamura Y: Purification and properties of thermostable xylanase and beta-xylosidase produced by a newly isolated Bacillus stearothermophilus strain. J Bacteriol 1990, 172:6669-6672.
• [11]Shi H, Li X, Gu H, Zhang Y, Huang Y, Wang L, Wang F: Biochemical properties of a novel thermostable and highly xylose-tolerant β-xylosidase/α-arabinosidase from Thermotoga thermarum. Biotechnol Biofuels 2013, 6:27. BioMed Central Full Text
• [12]Bhalla A, Bansal N, Kumar S, Bischoff KM, Sani RK: Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresour Technol 2013, 128:751-759.
• [13]Czjzek M, Ben David A, Bravman T, Shoham G, Henrissat B, Shoham Y: Enzyme-substrate complex structures of a GH39 beta-xylosidase from Geobacillus stearothermophilus. J Mol Biol 2005, 353:838-846.
• [14]Rastogi G, Bhalla A, Adhikari A, Bischoff KM, Hughes SR, Christopher LP, Sani RK: Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains. Bioresour Technol 2010, 101:8798-8806.
• [15]Bhalla A, Kainth AS, Sani RK: Draft genome sequence of lignocellulose-degrading thermophilic bacterium Geobacillus sp. strain WSUCF1. Genome Announc 2013, 1:4.
• [16]Bhalla A, Bischoff KM, Uppugundla N, Balan V, Sani RK: Novel thermostable endo-xylanase cloned and expressed from bacterium Geobacillus sp. WSUCF1. Bioresour Technol 2014, 165:314-318.
• [17]Viikari L, Alapuranen M, Puranen T, Vehmaanpera J, Siika-Aho M: Thermostable enzymes in lignocellulose hydrolysis. Adv Biochem Eng Biotechnol 2007, 108:121-145.
• [18]Contreras LM, Gómez J, Prieto J, Clemente-Jiménez JM, Las Heras-Vázquez FJ, Rodríguez-Vico F, Blanco FJ, Neira JL: The family 52 beta-xylosidase from Geobacillus stearothermophilus is a dimer: structural and biophysical characterization of a glycoside hydrolase. Biochim Biophys Acta 2008, 1784:1924-1934.
• [19]Quintero D, Velasco Z, Hurtado-Gómez E, Neira JL, Contreras LM: Isolation and characterization of a thermostable beta-xylosidase in the thermophilic bacterium Geobacillus pallidus. Biochim Biophys Acta 2007, 1774:510-518.
• [20]Baba T, Shinke R, Nanmori T: Identification and characterization of clustered genes for thermostable xylan-degrading enzymes, beta-xylosidase and xylanase, of Bacillus stearothermophilus 21. Appl Environ Microbiol 1994, 60:2252-2258.
• [21]Ratnadewi AA, Fanani M, Kurniasih SD, Sakka M, Wasito EB, Sakka K, Nurachman Z, Puspaningsih NN: β-D-xylosidase from Geobacillus thermoleovorans IT-08: biochemical characterization and bioinformatics of the enzyme. Appl Biochem Biotechnol 2013, 170:1950-1964.
• [22]Wagschal K, Heng C, Lee CC, Robertson GH, Orts WJ, Wong DW: Purification and characterization of a glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08. Appl Biochem Biotechnol 2009, 155:304-313.
• [23]Lama L, Calandrelli V, Gambacorta A, Nicolaus B: Purification and characterization of thermostable xylanase and beta-xylosidase by the thermophilic bacterium Bacillus thermantarcticus. Res Microbiol 2004, 155:283-289.
• [24]Eneyskaya EV, Ivanen DR, Bobrov KS, Isaeva-Ivanova LS, Shabalin KA, Savel'ev AN, Golubev AM, Kulminskaya AA: Biochemical and kinetic analysis of the GH3 family beta-xylosidase from Aspergillus awamori X-100. Arch Biochem Biophys 2007, 457:225-234.
• [25]Yang JK, Yoon HJ, Ahn HJ, Lee BI, Pedelacq JD, Liong EC, Berendzen J, Laivenieks M, Vieille C, Zeikus GJ, Vocadlo DJ, Withers SG, Suh SW: Crystal structure of beta-D-xylosidase from Thermoanaerobacterium saccharolyticum, a family 39 glycoside hydrolase. J Mol Biol 2004, 335:155-165.
• [26]Shallom D, Leon M, Bravman T, Ben-David A, Zaide G, Belakhov V, Shoham G, Schomburg D, Baasov T, Shoham Y: Biochemical characterization and identification of the catalytic residues of a family 43 β-D-xylosidase from Geobacillus stearothermophilus T-6. Biochemistry 2005, 44:387-397.
• [27]Turner P, Mamo G, Karlsson EN: Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb Cell Fact 2007, 6:9. BioMed Central Full Text
• [28]Singh SK, Heng C, Braker JD, Chan VJ, Lee CC, Jordan DB, Yuan L, Wagschal K: Directed evolution of GH43 β-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis. J Ind Microbiol Biotechnol 2014, 41:489-498.
• [29]Zhang ZG, Yi ZL, Pei XQ, Wu ZL: Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis. Bioresour Technol 2010, 101:9272-9278.
• [30]Klein-Marcuschamer D, Oleskowicz-Popiel P, Simmons BA, Blanch HW: The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol Bioeng 2012, 109:1083-1087.
• [31]Tu M, Zhang X, Paice M, MacFarlane P, Saddler JN: The potential of enzyme recycling during the hydrolysis of a mixed softwood feedstock. Bioresour Technol 2009, 100:6407-6415.
• [32]Weiss N, Börjesson J, Pedersen LS, Meyer AS: Enzymatic lignocellulose hydrolysis: improved cellulase productivity by insoluble solids recycling. Biotechnol Biofuels 2013, 6:5. BioMed Central Full Text
• [33]Kristensen JB, Felby C, Jørgensen H: Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol Biofuels 2009, 2:11. BioMed Central Full Text
• [34]Zanoelo FF, PolizeliMdMde L, Terenzi HF, Jorge JA: Purification and biochemical properties of a thermostable xylose-tolerant beta- D-xylosidase from Scytalidium thermophilum. J Ind Microbiol Biotechnol 2004, 31:170-176.
• [35]Fujii T, Yu G, Matsushika A, Kurita A, Yano S, Murakami K, Sawayama S: Ethanol production from xylo-oligosaccharides by xylose-fermenting Saccharomyces cerevisiae expressing β-xylosidase. Biosci Biotechnol Biochem 2011, 75:1140-1146.
• [36]Kont R, Kurašin M, Teugjas H, Väljamäe P: Strong cellulase inhibitors from the hydrothermal pretreatment of wheat straw. Biotechnol Biofuels 2013, 6:135. BioMed Central Full Text
• [37]Zhang J, Viikari L: Xylo-oligosaccharides are competitive inhibitors of cellobiohydrolase I from Thermoascus aurantiacus. Bioresour Technol 2012, 117:286-291.
• [38]Qing Q, Yang B, Wyman CE: Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes. Bioresour Technol 2010, 101:9624-9630.
• [39]Herrmann MC, Vrsanska M, Jurickova M, Hirsch J, Biely P, Kubicek CP: The β-D-xylosidase of Trichoderma reesei is a multifunctional b-D-xylan xylohydrolase. Biochem J 1997, 321:375-381.
• [40]Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685.