Characterization of a New Glucose-Tolerant GH1 β-Glycosidase from Aspergillus fumigatus with Transglycosylation Activity
Abstract
:1. Introduction
2. Results and Discussion
2.1. AfBgl1.3 Structural Prediction
2.2. Expression of Recombinant β-Glycosidase, AfBgl1.3, in P. pastoris and Its Characterization
2.3. Substrate Specificity
2.4. Effect of pH and Temperature on AfBgl1 Activity and Stability
2.5. AfBgl1.3 Kinetic Parameters
2.6. Effects of Additives on AfBgl1.3 Activity
2.7. AfBgl1.3 Transglycosylation Activity
2.8. Effect of AfBgl1.3 on Saccharification
3. Materials and Methods
3.1. Strains, Culture Conditions, Vector and Materials
3.2. Sequence and Structural Analysis
3.3. RNA Extraction, cDNA Synthesis, and Gene Amplification
3.4. Cloning, P. pastoris Transformation, and Screening for Recombinant Transformants
3.5. Heterologous AfBgl1.3 Expression in P. pastoris and Protein Purification
3.6. Analysis by Circular Dichroism (CD) and Intrinsic Tryptophan Fluorescence Emission (ITFE)
3.7. AfBgl1.3 Activity Assay
3.8. AfBgl1.3 pH and Temperature Profiles
3.9. Influence of Additives on AfBgl1.3 Activity
3.10. Effects of Glucose on AfBgl1.3 Activity
3.11. Kinetic Assays
3.12. Determination of AfBgl1.3 Transglycosylation Activity by Thin Layer Chromatography (TLC)
3.13. Determination of AfBgl1.3 Transglycosylation Products by High Performance Liquid Chromatography (HPLC)
3.14. Activity with Celluclast® 1.5L by AfBgl1.3 Addition
3.15. Combined Assays
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Sluiter, J.B.; Ruiz, R.O.; Scarlata, C.J.; Sluiter, A.D.; Templeton, D.W. Compositional Analysis of Lignocellulosic Feedstocks. 1. Review and Description of Methods. J. Agric. Food Chem. 2010, 58, 9043–9053. [Google Scholar] [CrossRef] [PubMed]
- Gutiérrez-Antonio, C.; Romero-Izquierdo, A.G.; Gómez-Castro, F.I.; Hernández, S. Production Processes from Lignocellulosic Feedstock. In Production Processes of Renewable Aviation Fuel; Elsevier: Amsterdam, The Netherlands, 2021; pp. 129–169. [Google Scholar]
- Zoghlami, A.; Paës, G. Lignocellulosic Biomass: Understanding Recalcitrance and Predicting Hydrolysis. Front. Chem. 2019, 7, 874. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Aguiar, A.; Milessi, T.S.; Mulinari, D.R.; Lopes, M.S.; da Costa, S.M.; Candido, R.G. Sugarcane Straw as a Potential Second Generation Feedstock for Biorefinery and White Biotechnology Applications. Biomass Bioenergy 2021, 144, 105896. [Google Scholar] [CrossRef]
- Ashokkumar, V.; Venkatkarthick, R.; Jayashree, S.; Chuetor, S.; Dharmaraj, S.; Kumar, G.; Chen, W.-H.; Ngamcharussrivichai, C. Recent Advances in Lignocellulosic Biomass for Biofuels and Value-Added Bioproducts—A Critical Review. Bioresour. Technol. 2022, 344, 126195. [Google Scholar] [CrossRef]
- dos Santos, L.V.; de Barros Grassi, M.C.; Gallardo, J.C.M.; Pirolla, R.A.S.; Calderón, L.L.; de Carvalho-Netto, O.V.; Parreiras, L.S.; Camargo, E.L.O.; Drezza, A.L.; Missawa, S.K.; et al. Second-Generation Ethanol: The Need Is Becoming a Reality. Ind. Biotechnol. 2016, 12, 40–57. [Google Scholar] [CrossRef]
- Nascimento, C.V.; Souza, F.H.M.; Masui, D.C.; Leone, F.A.; Peralta, R.M.; Jorge, J.A.; Furriel, R.P.M. Purification and Biochemical Properties of a Glucose-Stimulated β-D-Glucosidase Produced by Humicola grisea Var. Thermoidea Grown on Sugarcane Bagasse. J. Microbiol. 2010, 48, 53–62. [Google Scholar] [CrossRef]
- Lopes, A.M.; Ferreira Filho, E.X.; Moreira, L.R.S. An Update on Enzymatic Cocktails for Lignocellulose Breakdown. J. Appl. Microbiol. 2018, 125, 632–645. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Singh, G.; Verma, A.K.; Kumar, V. Catalytic Properties, Functional Attributes and Industrial Applications of β-Glucosidases. 3 Biotech 2016, 6, 3. [Google Scholar] [CrossRef] [Green Version]
- Liu, G.; Qu, Y. Integrated Engineering of Enzymes and Microorganisms for Improving the Efficiency of Industrial Lignocellulose Deconstruction. Eng. Microbiol. 2021, 1, 100005. [Google Scholar] [CrossRef]
- de Carvalho, L.M.; Borelli, G.; Camargo, A.P.; de Assis, M.A.; de Ferraz, S.M.F.; Fiamenghi, M.B.; José, J.; Mofatto, L.S.; Nagamatsu, S.T.; Persinoti, G.F.; et al. Bioinformatics Applied to Biotechnology: A Review towards Bioenergy Research. Biomass Bioenergy 2019, 123, 195–224. [Google Scholar] [CrossRef]
- Sørensen, A.; Lübeck, M.; Lübeck, P.; Ahring, B. Fungal Beta-Glucosidases: A Bottleneck in Industrial Use of Lignocellulosic Materials. Biomolecules 2013, 3, 612–631. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Koppram, R.; Tomás-Pejó, E.; Xiros, C.; Olsson, L. Lignocellulosic Ethanol Production at High-Gravity: Challenges and Perspectives. Trends Biotechnol. 2014, 32, 46–53. [Google Scholar] [CrossRef] [PubMed]
- Bals, B.D.; Gunawan, C.; Moore, J.; Teymouri, F.; Dale, B.E. Enzymatic Hydrolysis of Pelletized AFEXTM-Treated Corn Stover at High Solid Loadings. Biotechnol. Bioeng. 2014, 111, 264–271. [Google Scholar] [CrossRef] [PubMed]
- Duque, A.; Manzanares, P.; Ballesteros, I.; Negro, M.J.; Oliva, J.M.; González, A.; Ballesteros, M. Sugar Production from Barley Straw Biomass Pretreated by Combined Alkali and Enzymatic Extrusion. Bioresour. Technol. 2014, 158, 262–268. [Google Scholar] [CrossRef] [PubMed]
- Gao, Y.; Xu, J.; Yuan, Z.; Zhang, Y.; Liu, Y.; Liang, C. Optimization of Fed-Batch Enzymatic Hydrolysis from Alkali-Pretreated Sugarcane Bagasse for High-Concentration Sugar Production. Bioresour. Technol. 2014, 167, 41–45. [Google Scholar] [CrossRef] [PubMed]
- Olsen, S.N.; Borch, K.; Cruys-Bagger, N.; Westh, P. The Role of Product Inhibition as a Yield-Determining Factor in Enzymatic High-Solid Hydrolysis of Pretreated Corn Stover. Appl. Biochem. Biotechnol. 2014, 174, 146–155. [Google Scholar] [CrossRef] [PubMed]
- Teugjas, H.; Väljamäe, P. Selecting β-Glucosidases to Support Cellulases in Cellulose Saccharification. Biotechnol. Biofuels 2013, 6, 105. [Google Scholar] [CrossRef] [Green Version]
- Salgado, J.C.S.; Meleiro, L.P.; Carli, S.; Ward, R.J. Glucose Tolerant and Glucose Stimulated β-Glucosidases—A Review. Bioresour. Technol. 2018, 267, 704–713. [Google Scholar] [CrossRef]
- Bhatia, Y.; Mishra, S.; Bisaria, V.S. Microbial β-Glucosidases: Cloning, Properties, and Applications. Crit. Rev. Biotechnol. 2002, 22, 375–407. [Google Scholar] [CrossRef]
- Meleiro, L.P.; Salgado, J.C.S.; Maldonado, R.F.; Alponti, J.S.; Zimbardi, A.L.R.L.; Jorge, J.A.; Ward, R.J.; Furriel, R.P.M. A Neurospora Crassa Ÿ-Glucosidase with Potential for Lignocellulose Hydrolysis Shows Strong Glucose Tolerance and Stimulation by Glucose and Xylose. J. Mol. Catal. B Enzym. 2015, 122, 131–140. [Google Scholar] [CrossRef]
- Yang, Y.; Zhang, X.; Yin, Q.; Fang, W.; Fang, Z.; Wang, X.; Zhang, X.; Xiao, Y. A Mechanism of Glucose Tolerance and Stimulation of GH1 β-Glucosidases. Sci. Rep. 2015, 5, 17296. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Böttcher, D.; Bornscheuer, U.T. Protein Engineering of Microbial Enzymes. Curr. Opin. Microbiol. 2010, 13, 274–282. [Google Scholar] [CrossRef] [PubMed]
- de Gouvêa, P.F.; Bernardi, A.V.; Gerolamo, L.E.; de Souza Santos, E.; Riaño-Pachón, D.M.; Uyemura, S.A.; Dinamarco, T.M. Transcriptome and Secretome Analysis of Aspergillus fumigatus in the Presence of Sugarcane Bagasse. BMC Genom. 2018, 19, 232. [Google Scholar] [CrossRef] [PubMed]
- Wild, R.; Kowal, J.; Eyring, J.; Ngwa, E.M.; Aebi, M.; Locher, K.P. Structure of the Yeast Oligosaccharyltransferase Complex Gives Insight into Eukaryotic N-Glycosylation. Science 2018, 359, 545–550. [Google Scholar] [CrossRef] [Green Version]
- Jeng, W.-Y.; Wang, N.-C.; Lin, M.-H.; Lin, C.-T.; Liaw, Y.-C.; Chang, W.-J.; Liu, C.-I.; Liang, P.-H.; Wang, A.H.-J. Structural and Functional Analysis of Three β-Glucosidases from Bacterium Clostridium cellulovorans, Fungus Trichoderma reesei and Termite Neotermes koshunensis. J. Struct. Biol. 2011, 173, 46–56. [Google Scholar] [CrossRef]
- Florindo, R.N.; Souza, V.P.; Mutti, H.S.; Camilo, C.; Manzine, L.R.; Marana, S.R.; Polikarpov, I.; Nascimento, A.S. Structural Insights into β-Glucosidase Transglycosylation Based on Biochemical, Structural and Computational Analysis of Two GH1 Enzymes from Trichoderma harzianum. New Biotechnol. 2018, 40, 218–227. [Google Scholar] [CrossRef]
- Liew, K.J.; Lim, L.; Woo, H.Y.; Chan, K.-G.; Shamsir, M.S.; Goh, K.M. Purification and Characterization of a Novel GH1 Beta-Glucosidase from Jeotgalibacillus malaysiensis. Int. J. Biol. Macromol. 2018, 115, 1094–1102. [Google Scholar] [CrossRef]
- Baiya, S.; Pengthaisong, S.; Kitjaruwankul, S.; Ketudat Cairns, J.R. Structural Analysis of Rice Os4BGlu18 Monolignol β-Glucosidase. PLoS ONE 2021, 16, e0241325. [Google Scholar] [CrossRef]
- Mohamad Sobri, M.F.; Abd-Aziz, S.; Abu Bakar, F.D.; Ramli, N. In-Silico Characterization of Glycosyl Hydrolase Family 1 β-Glucosidase from Trichoderma asperellum UPM1. Int. J. Mol. Sci. 2020, 21, 4035. [Google Scholar] [CrossRef]
- Micsonai, A.; Wien, F.; Bulyáki, É.; Kun, J.; Moussong, É.; Lee, Y.-H.; Goto, Y.; Réfrégiers, M.; Kardos, J. BeStSel: A Web Server for Accurate Protein Secondary Structure Prediction and Fold Recognition from the Circular Dichroism Spectra. Nucleic Acids Res. 2018, 46, W315–W322. [Google Scholar] [CrossRef]
- Kabsch, W.; Sander, C. Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-Bonded and Geometrical Features. Biopolymers 1983, 22, 2577–2637. [Google Scholar] [CrossRef] [PubMed]
- Kelley, L.A.; Mezulis, S.; Yates, C.M.; Wass, M.N.; Sternberg, M.J.E. The Phyre2 Web Portal for Protein Modeling, Prediction and Analysis. Nat. Protoc. 2015, 10, 845–858. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ireland, S.M.; Sula, A.; Wallace, B.A. Thermal Melt Circular Dichroism Spectroscopic Studies for Identifying Stabilising Amphipathic Molecules for the Voltage-gated Sodium Channel NavMs. Biopolymers 2018, 109, e23067. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Adlakha, N.; Sawant, S.; Anil, A.; Lali, A.; Yazdani, S.S. Specific Fusion of β-1,4-Endoglucanase and β-1,4-Glucosidase Enhances Cellulolytic Activity and Helps in Channeling of Intermediates. Appl. Environ. Microbiol. 2012, 78, 7447–7454. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Santos, C.A.; Zanphorlin, L.M.; Crucello, A.; Tonoli, C.C.C.; Ruller, R.; Horta, M.A.C.; Murakami, M.T.; de Souza, A.P. Crystal Structure and Biochemical Characterization of the Recombinant ThBgl, a GH1 β-Glucosidase Overexpressed in Trichoderma harzianum under Biomass Degradation Conditions. Biotechnol. Biofuels 2016, 9, 71. [Google Scholar] [CrossRef] [Green Version]
- Lima, R.A.T.; de Oliveira, G.; Souza, A.A.; Lopes, F.A.C.; Santana, R.H.; Istvan, P.; Quirino, B.F.; Barbosa, J.; de Freitas, S.; Garay, A.V.; et al. Functional and Structural Characterization of a Novel GH3 β-Glucosidase from the Gut Metagenome of the Brazilian Cerrado Termite Syntermes wheeleri. Int. J. Biol. Macromol. 2020, 165, 822–834. [Google Scholar] [CrossRef]
- Bashirova, A.; Pramanik, S.; Volkov, P.; Rozhkova, A.; Nemashkalov, V.; Zorov, I.; Gusakov, A.; Sinitsyn, A.; Schwaneberg, U.; Davari, M. Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability. Int. J. Mol. Sci. 2019, 20, 1602. [Google Scholar] [CrossRef] [Green Version]
- 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 from Aspergillus oryzae. Appl. Environ. Microbiol. 1998, 64, 3607–3614. [Google Scholar] [CrossRef] [Green Version]
- Kudo, K.; Watanabe, A.; Ujiie, S.; Shintani, T.; Gomi, K. Purification and Enzymatic Characterization of Secretory Glycoside Hydrolase Family 3 (GH3) Aryl β-Glucosidases Screened from Aspergillus oryzae Genome. J. Biosci. Bioeng. 2015, 120, 614–623. [Google Scholar] [CrossRef]
- Karami, F.; Ghorbani, M.; Sadeghi Mahoonak, A.; Khodarahmi, R. Fast, Inexpensive Purification of β-Glucosidase from Aspergillus niger and Improved Catalytic/Physicochemical Properties upon the Enzyme Immobilization: Possible Broad Prospects for Industrial Applications. LWT 2020, 118, 108770. [Google Scholar] [CrossRef]
- Monteiro, L.M.O.; Vici, A.C.; Pinheiro, M.P.; Heinen, P.R.; de Oliveira, A.H.C.; Ward, R.J.; Prade, R.A.; Buckeridge, M.S.; de Moraes Polizeli, M.d.L.T. A Highly Glucose Tolerant SS-Glucosidase from Malbranchea pulchella (MpBg3) Enables Cellulose Saccharification. Sci. Rep. 2020, 10, 6998. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- da Costa, S.G.; Pereira, O.L.; Teixeira-Ferreira, A.; Valente, R.H.; de Rezende, S.T.; Guimarães, V.M.; Genta, F.A. Penicillium citrinum UFV1 β-Glucosidases: Purification, Characterization, and Application for Biomass Saccharification. Biotechnol. Biofuels 2018, 11, 226. [Google Scholar] [CrossRef]
- Silva, L.D.M.; Gomes, T.C.; Ullah, S.F.; Ticona, A.R.; Hamann, P.R.; Noronha, E.F. Biochemical Properties of Carbohydrate-Active Enzymes Synthesized by Penicillium chrysogenum Using Corn Straw as Carbon Source. Waste Biomass Valorization 2020, 11, 2455–2466. [Google Scholar] [CrossRef]
- Xia, W.; Xu, X.; Qian, L.; Shi, P.; Bai, Y.; Luo, H.; Ma, R.; Yao, B. Engineering a Highly Active Thermophilic β-Glucosidase to Enhance Its PH Stability and Saccharification Performance. Biotechnol. Biofuels 2016, 9, 147. [Google Scholar] [CrossRef] [Green Version]
- Hasunuma, T.; Kondo, A. Consolidated Bioprocessing and Simultaneous Saccharification and Fermentation of Lignocellulose to Ethanol with Thermotolerant Yeast Strains. Process Biochem. 2012, 47, 1287–1294. [Google Scholar] [CrossRef]
- Pratto, B.; dos Santos-Rocha, M.S.R.; Longati, A.A.; de Sousa Júnior, R.; Cruz, A.J.G. Experimental Optimization and Techno-Economic Analysis of Bioethanol Production by Simultaneous Saccharification and Fermentation Process Using Sugarcane Straw. Bioresour. Technol. 2020, 297, 122494. [Google Scholar] [CrossRef]
- Fusco, F.A.; Fiorentino, G.; Pedone, E.; Contursi, P.; Bartolucci, S.; Limauro, D. Biochemical Characterization of a Novel Thermostable β-Glucosidase from Dictyoglomus turgidum. Int. J. Biol. Macromol. 2018, 113, 783–791. [Google Scholar] [CrossRef] [PubMed]
- Yin, Y.R.; Sang, P.; Xiao, M.; Xian, W.D.; Dong, Z.Y.; Liu, L.; Yang, L.Q.; Li, W.J. Expression and Characterization of a Cold-Adapted, Salt- and Glucose-Tolerant GH1 β-Glucosidase Obtained from Thermobifida halotolerans and Its Use in Sugarcane Bagasse Hydrolysis. Biomass Convers. Biorefin. 2021, 11, 1245–1253. [Google Scholar] [CrossRef]
- Chen, P.; Fu, X.; Ng, T.B.; Ye, X.Y. Expression of a Secretory β-Glucosidase from Trichoderma reesei in Pichia pastoris and Its Characterization. Biotechnol. Lett. 2011, 33, 2475–2479. [Google Scholar] [CrossRef] [PubMed]
- de Almeida, P.Z.; de Oliveira, T.B.; de Lucas, R.C.; Salgado, J.C.S.; Pérez, M.M.; Galan, B.; García, J.L.; de Moraes, M.D.L.T. Heterologous Production and Biochemical Characterization of a New Highly Glucose Tolerant GH1 β-Glucosidase from Anoxybacillus thermarum. Process Biochem. 2020, 99, 1–8. [Google Scholar] [CrossRef]
- Saleh Zada, N.; Belduz, A.O.; Güler, H.I.; Khan, A.; Sahinkaya, M.; Kaçıran, A.; Ay, H.; Badshah, M.; Shah, A.A.; Khan, S. Cloning, Expression, Biochemical Characterization, and Molecular Docking Studies of a Novel Glucose Tolerant β-Glucosidase from Saccharomonospora sp. NB11. Enzyme Microb. Technol. 2021, 148, 109799. [Google Scholar] [CrossRef] [PubMed]
- Jiang, Z.; Long, L.; Liang, M.; Li, H.; Chen, Y.; Zheng, M.; Ni, H.; Li, Q.; Zhu, Y. Characterization of a Glucose-Stimulated β-Glucosidase from Microbulbifer sp. ALW1. Microbiol. Res. 2021, 251, 126840. [Google Scholar] [CrossRef] [PubMed]
- Yin, Y.-R.; Sang, P.; Yang, F.-L.; Li, T.; Yang, R.-F.; Liu, H.-Y.; Luo, Z.-L.; Li, W.-J.; Yang, L.-Q. Characterization of a Cu2+, SDS, Alcohol and Glucose Tolerant GH1 β-Glucosidase from Bacillus sp. CGMCC 1.16541. Antonie Van Leeuwenhoek 2020, 113, 1467–1477. [Google Scholar] [CrossRef] [PubMed]
- Yang, S.; Hua, C.; Yan, Q.; Li, Y.; Jiang, Z. Biochemical Properties of a Novel Glycoside Hydrolase Family 1 β-Glucosidase (PtBglu1) from Paecilomyces thermophila Expressed in Pichia pastoris. Carbohydr. Polym. 2013, 92, 784–791. [Google Scholar] [CrossRef] [PubMed]
- Nam, K.H.; Sung, M.W.; Hwang, K.Y. Structural Insights into the Substrate Recognition Properties of β-Glucosidase. Biochem. Biophys. Res. Commun. 2010, 391, 1131–1135. [Google Scholar] [CrossRef]
- Chen, A.; Wang, D.; Ji, R.; Li, J.; Gu, S.; Tang, R.; Ji, C. Structural and Catalytic Characterization of TsBGL, a β-Glucosidase From Thermofilum sp. Ex4484_79. Front. Microbiol. 2021, 12, 723678. [Google Scholar] [CrossRef]
- Sun, J.; Wang, W.; Ying, Y.; Hao, J. A Novel Glucose-Tolerant GH1 β-Glucosidase and Improvement of Its Glucose Tolerance Using Site-Directed Mutation. Appl. Biochem. Biotechnol. 2020, 192, 999–1015. [Google Scholar] [CrossRef]
- Bonfá, E.C.; de Souza Moretti, M.M.; Gomes, E.; Bonilla-Rodriguez, G.O. Biochemical Characterization of an Isolated 50 kDa Beta-Glucosidase from the Thermophilic Fungus Myceliophthora thermophila M.7.7. Biocatal. Agric. Biotechnol. 2018, 13, 311–318. [Google Scholar] [CrossRef] [Green Version]
- Bauermeister, A.; Amador, I.R.; Pretti, C.P.; Giese, E.C.; Oliveira, A.L.M.; Alves da Cunha, M.A.; Rezende, M.I.; Dekker, R.F.H.; Barbosa, A.M. β-(1→3)-Glucanolytic Yeasts from Brazilian Grape Microbiota: Production and Characterization of β-Glucanolytic Enzymes by Aureobasidium pullulans 1WA1 Cultivated on Fungal Mycelium. J. Agric. Food Chem. 2015, 63, 269–278. [Google Scholar] [CrossRef]
- Watanabe, T.; Sato, T.; Yoshioka, S.; Koshijima, T.; Kuwahara, M. Purificication and Properties of Aspergillus niger Beta-Glucosidase. Eur. J. Biochem. 1992, 209, 651–659. [Google Scholar] [CrossRef]
- Workman, W.E.; Day, D.F. Purification and Properties of β-Glucosidase from Aspergillus terreus. Appl. Environ. Microbiol. 1982, 44, 1289–1295. [Google Scholar] [CrossRef] [Green Version]
- Lin, C.; Shen, Z.; Qin, W. Characterization of Xylanase and Cellulase Produced by a Newly Isolated Aspergillus fumigatus N2 and Its Efficient Saccharification of Barley Straw. Appl. Biochem. Biotechnol. 2017, 182, 559–569. [Google Scholar] [CrossRef] [PubMed]
- Milles, L.F.; Unterauer, E.M.; Nicolaus, T.; Gaub, H.E. Calcium Stabilizes the Strongest Protein Fold. Nat. Commun. 2018, 9, 4764. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fang, Z. Cloning and Characterization of a β-Glucosidase from Marine Microbial Metagenome with Excellent Glucose Tolerance. J. Microbiol. Biotechnol. 2010, 20, 1351–1358. [Google Scholar] [CrossRef] [PubMed]
- di Lauro, B.; Rossi, M.; Moracci, M. Characterization of a β-Glycosidase from the Thermoacidophilic Bacterium Alicyclobacillus acidocaldarius. Extremophiles 2006, 10, 301–310. [Google Scholar] [CrossRef]
- Bernardi, A.V.; Gerolamo, L.E.; Uyemura, S.A.; Dinamarco, T.M. A Thermophilic, PH-Tolerant, and Highly Active GH10 Xylanase from Aspergillus fumigatus Boosted Pre-Treated Sugarcane Bagasse Saccharification by Cellulases. Ind. Crops Prod. 2021, 170, 113697. [Google Scholar] [CrossRef]
- Shah, M.A.; Mishra, S.; Chaudhuri, T.K. Structural Stability and Unfolding Transition of β-Glucosidases: A Comparative Investigation on Isozymes from a Thermo-Tolerant Yeast. Eur. Biophys. J. 2011, 40, 877–889. [Google Scholar] [CrossRef]
- Ostermeier, L.; Oliva, R.; Winter, R. The Multifaceted Effects of DMSO and High Hydrostatic Pressure on the Kinetic Constants of Hydrolysis Reactions Catalyzed by α-Chymotrypsin. Phys. Chem. Chem. Phys. 2020, 22, 16325–16333. [Google Scholar] [CrossRef]
- Rajasree, K.P.; Mathew, G.M.; Pandey, A.; Sukumaran, R.K. Highly Glucose Tolerant β-Glucosidase from Aspergillus unguis: NII 08123 for Enhanced Hydrolysis of Biomass. J. Ind. Microbiol. Biotechnol. 2013, 40, 967–975. [Google Scholar] [CrossRef]
- Tiwari, R.; Singh, P.K.; Singh, S.; Nain, P.K.S.; Nain, L.; Shukla, P. Bioprospecting of Novel Thermostable β-Glucosidase from Bacillus subtilis RA10 and Its Application in Biomass Hydrolysis. Biotechnol. Biofuels 2017, 10, 246. [Google Scholar] [CrossRef] [Green Version]
- Belancic, A.; Gunata, Z.; Vallier, M.-J.; Agosin, E. β-Glucosidase from the Grape Native Yeast Debaryomyces vanrijiae: Purification, Characterization, and Its Effect on Monoterpene Content of a Muscat Grape Juice. J. Agric. Food Chem. 2003, 51, 1453–1459. [Google Scholar] [CrossRef] [PubMed]
- Pérez-Pons, J.A.; Rebordosa, X.; Querol, E. Properties of a Novel Glucose-Enhanced β-Glucosidase Purified from Streptomyces Sp. (ATCC 11238). Biochim. Biophys. Acta (BBA) Protein Struct. Mol. Enzymol. 1995, 1251, 145–153. [Google Scholar] [CrossRef]
- Matsuzawa, T.; Yaoi, K. Screening, Identification, and Characterization of a Novel Saccharide-Stimulated β-Glycosidase from a Soil Metagenomic Library. Appl. Microbiol. Biotechnol. 2017, 101, 633–646. [Google Scholar] [CrossRef] [PubMed]
- Lu, J.; Du, L.; Wei, Y.; Hu, Y.; Huang, R. Expression and Characterization of a Novel Highly Glucose-Tolerant Beta-Glucosidase from a Soil Metagenome. Acta Biochim. Biophys. Sin. 2013, 45, 664–673. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Guo, B.; Amano, Y.; Nozaki, K. Improvements in Glucose Sensitivity and Stability of Trichoderma reesei β-Glucosidase Using Site-Directed Mutagenesis. PLoS ONE 2016, 11, e0147301. [Google Scholar] [CrossRef] [Green Version]
- de Giuseppe, P.O.; Souza, T.D.A.; Souza, F.H.M.; Zanphorlin, L.M.; Machado, C.B.; Ward, R.J.; Jorge, J.A.; Furriel, R.P.M.; Murakami, M.T. Structural Basis for Glucose Tolerance in GH1 β-Glucosidases. Acta Crystallogr. D Biol. Crystallogr. 2014, 70, 1631–1639. [Google Scholar] [CrossRef]
- Uchiyama, T.; Miyazaki, K.; Yaoi, K. Characterization of a Novel β-Glucosidase from a Compost Microbial Metagenome with Strong Transglycosylation Activity. J. Biol. Chem. 2013, 288, 18325–18334. [Google Scholar] [CrossRef] [Green Version]
- Sternberg, D.; Mandels, G.R. Induction of Cellulolytic Enzymes in Trichoderma reesei by Sophorose. J. Bacteriol. 1979, 139, 761–769. [Google Scholar] [CrossRef] [Green Version]
- Singhania, R.R.; Patel, A.K.; Pandey, A.; Ganansounou, E. Genetic Modification: A Tool for Enhancing Beta-Glucosidase Production for Biofuel Application. Bioresour. Technol. 2017, 245, 1352–1361. [Google Scholar] [CrossRef]
- Ubiparip, Z.; Moreno, D.S.; Beerens, K.; Desmet, T. Engineering of Cellobiose Phosphorylase for the Defined Synthesis of Cellotriose. Appl. Microbiol. Biotechnol. 2020, 104, 8327–8337. [Google Scholar] [CrossRef]
- Vianna Bernardi, A.; Kimie Yonamine, D.; Akira Uyemura, S.; Magnani Dinamarco, T. A Thermostable Aspergillus fumigatus GH7 Endoglucanase Over-Expressed in Pichia pastoris Stimulates Lignocellulosic Biomass Hydrolysis. Int. J. Mol. Sci. 2019, 20, 2261. [Google Scholar] [CrossRef] [Green Version]
- Bernardi, A.V.; Gerolamo, L.E.; de Gouvêa, P.F.; Yonamine, D.K.; Pereira, L.M.S.; de Oliveira, A.H.C.; Uyemura, S.A.; Dinamarco, T.M. LPMO AfAA9_B and Cellobiohydrolase AfCel6A from A. fumigatus Boost Enzymatic Saccharification Activity of Cellulase Cocktail. Int. J. Mol. Sci. 2020, 22, 276. [Google Scholar] [CrossRef] [PubMed]
- Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; et al. Highly Accurate Protein Structure Prediction with AlphaFold. Nature 2021, 596, 583–589. [Google Scholar] [CrossRef] [PubMed]
- Mirdita, M.; Schütze, K.; Moriwaki, Y.; Heo, L.; Ovchinnikov, S.; Steinegger, M. ColabFold: Making Protein Folding Accessible to All. Nat. Methods 2022, 19, 679–682. [Google Scholar] [CrossRef] [PubMed]
- Heo, L.; Park, H.; Seok, C. GalaxyRefine: Protein Structure Refinement Driven by Side-Chain Repacking. Nucleic Acids Res. 2013, 41, W384–W388. [Google Scholar] [CrossRef] [Green Version]
- Laskowski, R.A.; Rullmann, J.A.C.; MacArthur, M.W.; Kaptein, R.; Thornton, J.M. AQUA and PROCHECK-NMR: Programs for Checking the Quality of Protein Structures Solved by NMR. J. Biomol. NMR 1996, 8, 477–486. [Google Scholar] [CrossRef]
- Laskowski, R.A.; MacArthur, M.W.; Moss, D.S.; Thornton, J.M. PROCHECK: A Program to Check the Stereochemical Quality of Protein Structures. J. Appl. Crystallogr. 1993, 26, 283–291. [Google Scholar] [CrossRef]
- Quan, J.; Tian, J. Circular Polymerase Extension Cloning for High-Throughput Cloning of Complex and Combinatorial DNA Libraries. Nat. Protoc. 2011, 6, 242–251. [Google Scholar] [CrossRef]
- Greenberg, D.M. The Colorimetric Determination of the Serum Proteins. J. Biol. Chem. 1929, 82, 545–550. [Google Scholar] [CrossRef]
- Savitzky, A.; Golay, M.J.E. Smoothing and Differentiation of Data by Simplified Least Squares Procedures. Anal. Chem. 1964, 36, 1627–1639. [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]
- Fontana, J.D.; Gebara, M.; Blumel, M.; Schneider, H.; MacKenzie, C.R.; Johnson, K.G. α-4-O-Methyl-d-Glucuronidase Component of Xylanolytic Complexes. In Methods in Enzymology; Academic Press: Cambridge, MA, USA, 1988; pp. 560–571. [Google Scholar]
- Yepes, C.; Estévez, J.; Arroyo, M.; Ladero, M. Immobilization of an Industrial β-Glucosidase from Aspergillus fumigatus and Its Use for Cellobiose Hydrolysis. Processes 2022, 10, 1225. [Google Scholar] [CrossRef]
Temperature (°C) | α-Helices (%) | β-Sheets Antiparallel (%) | β-Sheets Parallel (%) | Loops (%) | Others and Disorderly (%) |
---|---|---|---|---|---|
25 | 36.62 | 3.97 | 13.84 | 11.04 | 34.53 |
30 | 38.9 | 0.43 | 13.17 | 11.83 | 35.67 |
40 | 34.72 | 5.99 | 13.23 | 11.84 | 34.22 |
50 | 31.27 | 9.28 | 12.57 | 11.31 | 35.56 |
60 | 27.23 | 17.43 | 7.54 | 10.14 | 37.66 |
70 | 21.16 | 19.09 | 10.17 | 11.14 | 38.44 |
80 | 22.83 | 16.75 | 7.53 | 11.47 | 41.37 |
Dictyoglomus turgidum | T. halotolerans YIM 90462T | Trichoderma reesei | Anoxybacillus thermarum | Saccharomonospora sp. NB11 | Microbulbifer sp. ALW1 | Bacillus sp. CGMCC 1.16541 | Jeotgalibacillus malaysiensis | Paecilomyces thermophila | Aspergillus fumigatus Af293 | Source Organism |
---|---|---|---|---|---|---|---|---|---|---|
DturβGlu | ThBGL1A | bgl1 | BgAt | BglNB11 | MaGlu1A | BsBgl1A | BglD5 | PtBglu1 | AfBgl1.3 | Protein |
E. coli BL21(DE3)-RIL | E. coli BL21 | Pichia pastoris GS115 | E. coli BL21 (DE3) | E. coli BL21 (DE3) | E. coli BL21 (DE3) | E. coli BL21 (DE3) | E. coli BL21 (DE3) | Pichia pastoris GS115 | Pichia pastoris X-33 | Expression system |
80 °C | 40–55 °C | 70 °C | 65 °C | 40 °C | 40 °C | 45 °C | 65 °C | 55 °C | 35–45 °C | Optimum Temperature |
5.4 | 5.6–6.6 | 5 | 7 | 7 | 4.5 | 5.6–7.6 | 7 | 6 | 5.4–6.4 | Optimum pH |
After 2 h of pre-incubation at 70 °C and 80 °C, the residual activity was 70% and 50%, respectively | 100% of its residual activity at 40 °C after 2 h; | Residual activity >90% at 60 °C for 60 min | Residual activity about 100% after 24 h at 50 °C; | 40% of residual activity at 40 °C for 2 h and 70% of residual activity after 2 h in the temperature range of 25−35 °C | 34.0% of residual activity after 0.5 h at 40 °C | Residual activity about 100% after 120 min at 45 °C | t1/2 65 °C = 35 min (with calcium); t1/2 65 °C = 70 min (without calcium) | Residual activity about 88% activity after 30 min at 55 °C | Residual activity about 60% after 15 h at 40 °C and 90% residual activity after 24 h at 30 °C | Thermostability |
Residual activity remained close to 90% after 1 h of pre-incubation in the pH range 5.0–8.0 | Residual activity >60% after 12 h and 24 h pre-incubation at pH 5–8 | Residual activity >90% in the pH range 4–7 | Enzyme activity induction in the pH range 4.0–6.5 after 15min of pre-incubation | Residual activity >50% after 8 h pre-incubation in the pH range 6–10 | - | Residual activity about 100% in the pH range (4–9) after 24 and 48 h of pre-incubation | - | Residual activity >80% in the pH range (5.0–11.0) | Residual activity >70% after 24 and 48 h of incubation in the pH range 5–8 | pH stability |
pNPG Salicin | pNPG Cellobiose | - | pNPG | pNPG | pNPG Cellobiose | pNPG Cellobiose | pNPG | pNPG Salicin Cellobiose | pNPG Salicin Cellobiose | Substrate |
- | 52.6 U mg−1 33.8 U mg−1 | - | 7614 U mg−1 | 5735.8 U mg−1 | 4.52 U mg−1 151.52 U mg−1 | 36 ± 0.6 U mg−1 78 ± 2 U mg−1 | 39.48 ± 0.63 U mg−1 | 328.8 ± 7.5 U mg−1 66.0 ± 0.4 U mg−1 306.3 ± 6.3 U mg−1 | 656.0 ± 17.5 U mgˉ1 706.5 ± 23.8 U mgˉ1 132.6 ± 7.1 U mgˉ1 | Vmax |
0.84 mM 8.12 mM | 21.96 mM 3.06 mM | - | 0.360 mM | 0.4037 mM | 2.71 mM 24.44 mM | 9 ± 0.2 mM 0.11 ± 0.02 mM | 0.50 ± 0.02 | 0.55 ± 0.03 mM 6.85 ± 0.06 mM 1.0 ± 0.06 mM | 7.6 ± 0.8 mM 17.6 ± 1.9 mM 15.4 ± 2.5 mM | KM |
8710 s−1 659 s−1 | 41.8 s−1 30 s−1 | - | 0.63 × 104 s−1 | 5042.16 s−1 | - | 31.3 ± 0.5 s−1 67.8 ± 1.7 s−1 | 33.93 ± 0.54 s−1 | 5.5 s−1 1.1 s−1 5.1 s−1 | 595.8 s−1 621.7 s−1 120.4 s−1 | kcat |
1 × 104 mM−1 s −1 81 mM−1 s −1 | 1.9 s−1 mM−1 9.8 s−1 mM−1 | - | 1.74 × 104 s−1 mM−1 | 12,487.71 s−1 mM−1 | - | 3.5 ± 0.1 s−1/mg/mL 616 ± 2 s−1/mg/mL | - | 9.96 mM−1 s−1 0.16 mM−1 s−1 5.10 mM−1 s−1 | 7.8 × 104 Mˉ1 sˉ1 3.5 × 104 Mˉ1 sˉ1 7.8 × 103 Mˉ1 sˉ1 | kcat/KM |
[48] | [49] | [50] | [51] | [52] | [53] | [54] | [28] | [55] | This work | Reference |
Substrate | Vmax (U mg−1) | KM (mM) | kcat (s−1) | kcat/KM (M−1s−1) |
---|---|---|---|---|
pNPG | 656.0 ± 17.5 | 7.6 ± 0.8 | 595.8 | 7.8 × 104 |
Salicin | 706.5 ± 23.8 | 17.6 ± 1.9 | 621.7 | 3.5 × 104 |
Cellobiose | 132.6 ± 7.1 | 15.4 ± 2.5 | 120.4 | 7.8 × 103 |
Additive | Relative Activity (%) | Additive | Relative Activity (%) |
---|---|---|---|
Control | 100.0 ± 3.0 | ZnSO4 | 0 |
(NH4)2SO4 | 108.7 ± 1.2 | EDTA | 94.5 ± 2.3 |
MnCl2 | 114.5 ± 1.2 | β-mercaptoethanol | 103.5 ± 3.8 |
KCl | 108.4 ± 2.1 | DMSO | 116.3 ± 3.9 |
MgSO4 | 91.4 ± 0.0 | DTT | 111.2 ± 2.3 |
CaCl2 | 83.8 ± 1.7 | Triton X-100 | 123.1 ± 2.6 |
CoCl2 | 39.9 ± 5.4 | Tween 20 | 118.2 ± 2.5 |
FeSO4 | 31.4 ± 1.1 | SDS | 0 |
AgNO3 | 0 | SLS | 0 |
CuSO4 | 0 | Ascorbic acid | 0 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 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 (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Pereira, L.M.S.; Bernardi, A.V.; Gerolamo, L.E.; Pedersoli, W.R.; Carraro, C.B.; Silva, R.d.N.; Uyemura, S.A.; Dinamarco, T.M. Characterization of a New Glucose-Tolerant GH1 β-Glycosidase from Aspergillus fumigatus with Transglycosylation Activity. Int. J. Mol. Sci. 2023, 24, 4489. https://doi.org/10.3390/ijms24054489
Pereira LMS, Bernardi AV, Gerolamo LE, Pedersoli WR, Carraro CB, Silva RdN, Uyemura SA, Dinamarco TM. Characterization of a New Glucose-Tolerant GH1 β-Glycosidase from Aspergillus fumigatus with Transglycosylation Activity. International Journal of Molecular Sciences. 2023; 24(5):4489. https://doi.org/10.3390/ijms24054489
Chicago/Turabian StylePereira, Lucas Matheus Soares, Aline Vianna Bernardi, Luis Eduardo Gerolamo, Wellington Ramos Pedersoli, Cláudia Batista Carraro, Roberto do Nascimento Silva, Sergio Akira Uyemura, and Taísa Magnani Dinamarco. 2023. "Characterization of a New Glucose-Tolerant GH1 β-Glycosidase from Aspergillus fumigatus with Transglycosylation Activity" International Journal of Molecular Sciences 24, no. 5: 4489. https://doi.org/10.3390/ijms24054489