Recent Advances in β-Glucosidase Sequence and Structure Engineering: A Brief Review
Abstract
:1. Introduction
2. BGL Engineering Strategies
2.1. Directed Evolution
2.1.1. Generation of Diverse Mutants
2.1.2. Mutant Screening
2.1.3. Machine Learning-Assisted Directed Evolution
2.2. Rational Design
2.2.1. Structural Analysis
2.2.2. Multiple Sequence Alignment (MSA)
2.2.3. Computational Approaches
2.2.4. Site-Directed Mutagenesis (SDM)
2.3. Semi-Rational Design
3. Engineering of BGL Functionalities
3.1. Enhancing Activity
3.2. Improving Product Tolerance
3.3. Improving Transglycosylation
3.4. Improving Thermostability
3.5. Improving Catalytic Performance in Unconventional Phase
3.6. Improving pH Stability
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Organism Source | Engineering Methods | High Throughput Screening Method | Improved Characteristics | Reference |
---|---|---|---|---|
Clostridium thermocellum (BglA) | Directed evolution Error-prone PCR | Assay medium screening method (0.02% Magenta GlcA) | Thermostability: ↑ Ti by 6.4 °C. | [18] |
Alteromonas sp. L82 (bgla) | Rational design Site-directed mutation | --- | Glucose tolerance: 40.7% of relative activity (glucose: 4 M). | [13] |
Metagenomic library of Turpan Depression (bgl1317) | Rational design Site-directed mutation | --- | Activity: ↑ by 80%. Glucose tolerance: IC50 from 0.8 to 1.5 M. | [19] |
P. oxalicum 16 (16BGL) | Directed evolution Error-prone PCR | Assay medium screening method 6-(β-d-glucopyranosyloxy)-7-hydroxy-2H -1-benzopyran-2-one | Activity: ↑ specific activity 70% at 40 °C. | [20] |
Thermotoga naphthophila RKU-10 (TN0602) | Rational design Site-directed mutation | --- | Transglycosylation: ↑ GOS productivity by 50%. | [9] |
Soil Macrogenome Library (Bgl15) | Directed evolution Error-prone PCR | Double assay medium screening method (0.1% hesperidin) | Glucose tolerance: IC50 from 0.04 to 2.1 M. Thermostability: T1/2 from 0.8 h to 180 h at 50 °C. | [21] |
Trichoderma harzianum (ThBgl) | Rational design Site-directed mutation | --- | Glucose tolerance: 300% of relative activity (glucose: 0.25 M). pH: stability at broad range (pH 4–9). | [22] |
Caldicellulosirutor Saccharolyticus (CsBglA) | Semi-rational design Site-directed mutation | Cell surface display Fluorescence detection medium screening method (pNPG) | Activity: ↑ Kcat/KM by 150% at 55 °C. | [23] |
GenBank FJ686869 (Bgl1D) | Directed evolution Error-prone PCR | Assay medium screening method (0.1% hesperidin) | Activity: ↑ Kcat/KM~23-fold. Thermostability: ↑ T1/2~10-fold. | [24] |
Paenibacillus polymyxa (Glu1C) | Rational design Site-directed mutation | --- | Thermostability: ↑ ~4-fold. Glucose tolerance: from 50% to 75% active at 1 M glucose. | [25] |
Talaromyces amestolkiae (BGL-1) | Rational design Site-directed mutation | --- | Transglycosylation: ↑ epigallocatechin gallate productivity by 48.8%. | [26] |
Trichoderma reesei | Directed evolution (UV light, N-methyl-N′-nitro-N-nitrosoguanidine) | Detection medium screening method (phosphoric acid-swollen cellulose) | Activity: ↑ ~5-fold. | [27] |
T. reesei (TrCel1b) | Rational design (Hydropathy index for enzyme activity) Site-directed mutation | --- | Transglycosylation: ↑disaccharides productivity by 3.5-fold. | [28] |
Bacillus sp. D1 (BglD1) | Semi-rational design Site-directed mutagenesis | --- | Transglycosylation: ↑ GOS productivity by 11.5%. | [29] |
Marine microbial metagenomic library (Bgl1A) | Semi-rational design Site-directed mutagenesis | --- | Glucose tolerance: ↑ IC50~1.4- to 2.4-fold. Thermostability: ↑ T1/2~4.3-fold. | [30] |
Lentinula edodes (LXYL-P1) | Rational design Site-directed mutagenesis | --- | Activity: ↑ ~3-fold. | [31] |
Penicillium piceum H16 | Rational design Site-directed mutation | --- | Thermostability: ↑ by 46.3%. | [32] |
Thermotoga Neapolitana (TnBgl1A) | Rational design Site-directed mutation | --- | Transglycosylation: ↑ by 7-fold. | [33] |
Neosartorya fischeri (NfBGL) | Rational design Site-directed mutation | --- | Activity: ↑ by 8%. | [34] |
A dairy run-off metagenome (BG3L) | Rational design Site-directed mutation | --- | Activity: ↑ ~2 or 3-fold. | [35] |
Metagenomic library of Turpan Depression (Bgl6-M3) | Semi-rational design Site-directed mutation | --- | Thermostability: ↑ T1/2~20-fold. Activity: ↑Kcat/KM~5.6-fold. Glucose tolerance: ↑ ΔIC50 of 200 mM. | [36] |
Organism | Strategy | Mutations | Molecular Effects | Improved Characteristics | References |
---|---|---|---|---|---|
Halothermothrix orenii (B8CYA8) | Rational design OEP | V169C, I246A | Lack of stable polar contacts; Reduction in side chain volume | Specific activity ↑ ~2-fold. | [81] |
Coniophora puteana (CpBgl) | Semi-rational design (HotSpot, Alanine scanning technique) SDM | Q20C, A240S | A combination of structural changes in the active pocket and protein–ligand interactions | ↑ By 65.75% and 58.58%, respectively. | [73] |
Chaetomella raphigera (D2-BGL) | Directed evolution Error-prone PCR | F256M/Y260D /D224G | F256 and Y260 on a short loop related to the high substrate affinity of the enzyme | ↑ ~2.7-fold. | [82] |
Metagenomic library of Turpan soil (Bgl1317) | Rational design SDM | A397R, L188A, A262S | Increase in the polarity of residues and hydrogen bonding contacts | ↑ By 80%. | [19] |
Talaromyces leycettanus JCM12802 | Rational design OEP | M36E, M36N, F66Y, E168Q | Increase in hydrophobic stacking interactions and hydrogen bonding networks of active centers | ↑ ~1.4–2.3-fold. | [83] |
P. oxalicum 16 (16BGL) | Directed evolution Error-prone PCR | M280T/V484L /D589E | Increase in the number of hydrogen bonds formed by the substrate to increase the binding free energy | ↑ By 22%. | [6] |
C. saccharolyticus | Directed evolution Error-prone PCR, Random drift mutagenesis | --- | Smaller residues near catalytic residues allow more flexibility in the active site or more access to the substrate | ↑ ~2-fold. | [47] |
P. oxalicum 16 (16BGL) | Directed evolution Error-prone PCR | G414S/D421V/ T441S | Tighter active site pockets | ↑ Specific activity 70% at 40 °C. | [20] |
Pyrococcus furiosus (CelB) | Directed evolution DNA shuffling | N415S | --- | ↑ Up to 3-fold. | [40] |
C. saccharolyticus (CsBglA) | Semi-rational design (SDM combined with random mutagenesis) | L64R/Y73F/ T221N/H324L | --- | ↑ Kcat/KM by 150% at 55 °C. | [23] |
Organism | Strategy | Mutations | Molecular Effects | Improved Characteristics | References |
---|---|---|---|---|---|
Metagenomic library of Turpan soil (Bgl1317) | Rational design SDM | L188A, A262S | Active site metastable interactions | IC50 from 0.8 to 1.5 M. | [19] |
Agrobacterium tumefaciens 5A (H0HC94) | Rational design SDM OEP | W127F, C174V, V176A, L178A, L178E, H229S | Increase in the hydrophobicity of the aglycone-binding sites and gatekeeper regions | ↑ ~2.2-fold | [87] |
Trichoderma Harzianum (ThBgl) | Rational design SDM | L167W/P172L | Replacement of gatekeeper residues to alter active site accessibility | 300% of relative activity (glucose: 0.25 M). | [22] |
T. Cel1A (Bgl II) | Rational design SDM OEP | L167W/P172L | Replacement of gatekeeper residues to narrow the entrance to the active pocket | IC50 = 650 mM. | [88] |
Humicola insolens (Bglhi) | Directed evolution Error-prone PCR | H307Y, D237V, N235S | Increasing trans-glycosylation Unbinding of unproductive substrates | --- | [89] |
A. tumefaciens 5A | --- | --- | Presence of separate glucose binding sites | --- | [90] |
Marine microbial Metagenome (SrBGL) | Rational design SDM | H228T | Interaction leading to glucose excretion by slingshot mechanism | ↑ Affinity score for cellobiose. | [91] |
H. orenii (B8CYA8) | Rational design SDM | V169C/E173L/ I246A | Increasing backbone kinetics of active channel residues and flexibility of active site pockets | 75% of specific activity in 1.0 M glucose. | [84] |
GenBank MK490918 (Bgl15) | Directed evolution Error-prone PCR Petri-dish-based double-layer high-throughput screening | S167V/W178L | Increasing transglycosylation activity | IC50 from 0.04 to 2.1 M. | [21] |
Marine bacteria (bgla) | Rational design OEP | F171W | Increase in volume of side chains near the active site | 40.7% of relative activity (glucose: 4 M). | [13] |
Hot-spring metagenome (BglM) | --- | --- | The narrow space between the remnants of the gatekeeper’s base | --- | [92] |
Organism | Strategy | Mutations | Molecular Effects | Improved Characteristics | References |
---|---|---|---|---|---|
T. amestolkiae (BGL-1) | Rational design SDM | E521G | Stimulating glycosyl donor departure. Absence of side chains to reduce steric hindrance | ↑ Epigallocatechin gallate productivity by 48.8%. | [26] |
T. naphthophila RKU-10 (Tn0602) | Rational design SDM | F226G/F414S | Reducing steric hindrance and removing interactions at the aglycone-binding sites | ↑GOS productivity ~1.3-fold. | [97] |
T. naphthophila RKU-10 (Tn0602) | Rational design SDM | F414S | Improving hydrophilicity of the lumen of the −1 subsite | ↑GOS productivity by 50%. | [9] |
Thermotoga maritima (TmBglA) | Rational design SDM | N222F/Y295F /F414S | Creating a more suitable environment for hexanol in the active center pocket to inhibit hydrolysis | Hexyl-β-glycoside productivity from 14.49 to 22.8 mM. | [96] |
A. niger (BGL1) | Directed evolution Error-prone PCR | Y305C | Reducing hydrolytic activity | Ki from 2.98 to 4.78 mM. | [98] |
T. neapolitana (TnBgl1A) | Rational design SDM | N220F, N220R, N220Y | Inhibiting hydrolysis | Transglycosylation/hydrolysis from 0.33 to 1.45–2.71. | [95] |
T. reesei (TrCel1b) | Rational design SDM HIFEA Strategy | I177S/I174S/ W173H | Inhibition of hydrophilicity of key amino acid residues in the catalytic sites | ↑ Disaccharides productivity by 3.5-fold. | [28] |
Method | Access | Description | Reference |
---|---|---|---|
Constraint network analysis (CAN) | --- | Local and global flexibility/stiffness properties of proteins calculated by the graph theory-based rigidity analysis of thermal unfolding simulation. | [105] |
MD simulation | e.g., GROMACS | Analysis of protein unfolding pathways at higher temperatures. | [71] |
B-Fitter | https://www.kofo.mpg.de/en/research/organic-synthesis, accessed on 23 April 2023 | Calculates and averages the B-factor values for all atoms in an amino acid. | [71] |
FoldUnfold | http://bioinfo.protres.ru/ogu/, accessed on 23 April 2023 | Uses the expected average number of contacts per residue calculated from the amino acid sequence as an indicator for whether a given region is folded or unfolded. | [106] |
PredyFlexy | https://www.dsimb.inserm.fr/dsimb_tools/predyflexy/, accessed on 23 April 2023 | Combines the B-factor with the state of motion of amino acid residues during molecular dynamics simulations. | [104] |
FIRST | -- | Representation of protein structure as a set of constraints on bond-angle interactions, identification of rigid and flexible regions of protein conformation by CAN. | [107] |
FlexPred | https://kiharalab.org/flexPred/, accessed on 23 April 2023 | Flexibility in predicting elastic residues using the SVM algorithm. | [104] |
Rosetta Design | Rosetta 3.13 software | Design of thermally stable proteins based on iterative sidechain optimization and backbone relaxation through optimizing packing and idealizing backbone conformation. | [100] |
FRESCO | --- | Combined with MD simulations to predict flexible regions of proteins that can incorporate stable disulfide bonds. | [108] |
HINGEprot | http://bioinfo3d.cs.tau.ac.il/HingeProt/, accessed on 23 April 2023 | Predicts the hinge region of a protein. | [104] |
PROSS | http://pross.weizmann.ac.il, accessed on 23 April 2023 | Calculation of ΔΔG and thus analysis of potential stable mutation locations using Rosetta combination sequences. | [109] |
FireProtDB | https://loschmidt.chemi.muni.cz/fireprotdb/, accessed on 23 April 2023 | Numerical data, structural information for mutation experiments with a variety of proteins. | [110] |
Organism | Strategy | Mutations | Molecular Effects | Improved Characteristics | References |
---|---|---|---|---|---|
Penicillium funiculosum (PfBgl3A) | Rational design SDM | --- | --- | --- | [113] |
A. tumefaciens 5A (H0HC94) | Rational design SDM OEP | W127F, V176A, L178A, L178E | Enhancement of hydrophobic interactions | ↑ T1/2~2 or 3-fold. | [87] |
Metagenomic library of Turpan Depression (Bgl6) | Directed evolution Quikchange | V174C/A404V/ L441F | Enhancement of hydrophobic interactions within the enzyme | --- | [85] |
Thermomicrobium roseum (B9L147) | Rational design SDM OEP | V169C | -- | ↑ T1/2~2-fold. | [114] |
H. orenii | Rational design SDM | V169C/E173L/ I246A | Increase in hydrophobic interactions | T1/2 > 7 h at 70 °C. | [84] |
GenBank MK490918 (Bgl15) | Directed evolution Error-prone PCR Petri-dish-based double-layer high-throughput screening | S39T/L42N/ V167C/W178L/ A251L/E319A/ E326P/A396V/ L433F | Increasing hydrophobic interactions and formation of more additional hydrogen bonds | T1/2 from 0.8 h to 180 h at 50 °C. | [21] |
P. piceum H16 | Rational design Proline theory Computer-assisted virtual saturation mutation | S507F/Q512W/ S514W | Mutation of glycine by proline reducing conformational entropy Increased hydrophobic interactions | ↑ By 46.3%. | [32] |
C. thermocellum (BglA) | Directed evolution Error-prone PCR | A17S/K268N | Increasing hydrophobic interactions | ↑ Ti by 6.4 °C. | [18] |
GenBank FJ686869 (Bgl1D) | Directed evolution DNA shuffling | S28T/Y37H/ D44E/R91G/ L115N | Enhancing interaction with protein structure around water molecules and introduction of more hydrogen bonds | ↑ T1/2~10-fold. | [24] |
GenBank HV348683 (Ks5A7) | Directed evolution Error-prone PCR | T167I/V181F/ K186T/A187E/ A298G | Increasing hydrophobic interactions with the protein core | ↑ T1/2~8640-fold. | [115] |
Coniophora puteana (CpBgl) | Semi-rational design (HotSpot, Alanine scanning technique) SDM | Q20C, A240S | A combination of structural changes in the active pocket and protein–ligand interactions | ↑ T1/2~5-fold. | [73] |
MeBglD2 | Rational design Directed evolution | His8/Asn59/ Gly295 | Increasing hydrophobic interactions with the protein core | ↑ Tm by 9 °C. | [116] |
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Ouyang, B.; Wang, G.; Zhang, N.; Zuo, J.; Huang, Y.; Zhao, X. Recent Advances in β-Glucosidase Sequence and Structure Engineering: A Brief Review. Molecules 2023, 28, 4990. https://doi.org/10.3390/molecules28134990
Ouyang B, Wang G, Zhang N, Zuo J, Huang Y, Zhao X. Recent Advances in β-Glucosidase Sequence and Structure Engineering: A Brief Review. Molecules. 2023; 28(13):4990. https://doi.org/10.3390/molecules28134990
Chicago/Turabian StyleOuyang, Bei, Guoping Wang, Nian Zhang, Jiali Zuo, Yunhong Huang, and Xihua Zhao. 2023. "Recent Advances in β-Glucosidase Sequence and Structure Engineering: A Brief Review" Molecules 28, no. 13: 4990. https://doi.org/10.3390/molecules28134990