Characterization of the Glucose Tolerance of ꞵ-Glucosidase from Glycosyl Family 1 (H0HC94) and Family 3 (D0MD38)

Goswami, Shubhasish (2021) Characterization of the Glucose Tolerance of ꞵ-Glucosidase from Glycosyl Family 1 (H0HC94) and Family 3 (D0MD38). PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Lignocellulosic biomass is an attractive renewable energy source as it contains the world's most abundant biopolymer called cellulose. Cellulose is the polymer of glucose linked by β-1,4 linkage and is hydrolyzed into fermentable glucose by the action of enzymes called cellulases. Upon fermentation, the glucose is converted into ethanol and used as biofuel. There are mainly three classes of cellulases required for the hydrolysis of the biomass. Endoglucanases (EG) (EC randomly cleave the β-1,4 glycosidic linkages of cellulose; cellobiohydrolase (CBH) (EC attack cellulose chain ends to produce cellobiose and β-glucosidases (BG) (EC hydrolyze cellobiose into two molecules of glucose. β-Glucosidases are mostly predominant in glycoside hydrolase (GH) family 1 and family 3. β-Glucosidases activity is important in biomass saccharifications. By hydrolyzing cellobiose into fermentable glucose, these enzymes remove cellobiose-mediated inhibition of other cellulases, such as, EG and CBH required for biomass saccharification. Nevertheless, most of the β-glucosidases are prone to inhibition by their reaction product, glucose. Thus, enzymatic hydrolysis of biomass becomes inefficient, resulting in increased loading of β-glucosidases that ultimately increase biofuel cost. Therefore, there is a requirement for glucose tolerant β-glucosidases to enhance the rate of saccharifications of lignocellulosic biomass that would reduce biofuel cost. There are very few GH1 β-glucosidases reported to be glucose tolerant, and only two GH3 β-glucosidases are known for their moderate glucose tolerance. Both GH1 and GH3 β-glucosidases are known to undergo severe competitive inhibition by their reaction product glucose. This thesis focuses on the biochemical characterization of glucose tolerant GH1 and GH3 β-glucosidases and understanding the molecular basis of glucose tolerant β-glucosidases to biomass saccharifications for the generation of biofuel. The cloning and biochemical characterization of a putative GH1 β- glucosidase (Uniprot ID H0HC94) confirmed β-glucosidase activity. This work showed that H0HC94 is active over a wide range of pH and temperature and had the highest kcat/Km against the natural substrate, cellobiose, amongst the reported β-glucosidase from Agrobacterium tumefaciens 5A. The thermostability and activity of H0HC94 in the presence of 0.9 M of 1- Ethyl-3-methylimidazolium diethyl phosphate ([C₂C₁im][C₂C₂PO₄]), 1-Ethyl-3- methylimidazolium dimethyl phosphate ([C₂C₁im][C₁C₁PO₄]), and 1-Ethyl-3- methylimidazolium L-(+)-lactate ([C₂C₁im][MeCHOHCO₂]) is decreased by only 20 to 30 % compared to the activity in the absence of IL. The apparent inhibition constant, Ki, app, of glucose was 686 mM. Thus, H0HC94 is moderately glucose tolerant, though the inhibition constant was much higher than the other β-glucosidases in Agrobacterium tumefaciens 5A. An uncompetitive type of inhibition was observed in H0HC94 in contrast to competitive inhibition by most of the other β-glucosidases. The glucose tolerance (modulating non-conserved residues) of W127F, V176A, L178A, and L78E is similar to the WT. The glucose tolerance of the mutants C174V and H229S however, increased 2-fold compared to WT. Significantly, while the WT, W127F, V176A, L178A, and L78E undergone the uncompetitive mode of inhibition mutants C174V and H229S undergo competitive inhibition. To understand the possible role of conformational changes and glucose binding sites to enhanced glucose tolerance of the mutants C174V and H229S, various fluorescence-based biophysical techniques and molecular dynamic simulations were used. In WT, the enzyme conformational changes in the presence of glucose and upon binding of glucose at the glucose binding site may explain the moderately high glucose tolerance. In C17V and H229S, the conformational changes due to these mutants led to creation of more glucose binding sites and resulted in a two-fold increase in glucose tolerance over the WT. Thus, the higher glucose tolerance of C174V and H229S than the HOHC94 wild-type is due to the presence of glucose-binding sites facilitated by conformational changes. While there are reports of some GH1 glucose tolerant ꞵ-glucosidases but there is only two moderately tolerated GH3 ꞵ-glucosidases are reported. A highly glucose tolerant GH3 family ꞵ- glucosidases (D0MD38: Uniprot ID) from Rhodothermus marinus DSM 4252 has been biochemically characterized. Its glucose tolerance, Ki is found to be 4.4 M, highest among reported GH3 family ꞵ-glucosidases. It is competitively inhibited with glucose. An absence of transglycosylation was observed in the presence of a saturated concentration of glucose. Isothermal titration calorimetry (ITC) studies in the presence of glucose confirm the presence of two glucose binding sites might be one reason for high glucose tolerance. D0MD38 having Topt of 72°C, the half-life of 12 hours presents a potential GH3 ꞵ-glucosidases for industrial usage. Thus, the presence of glucose binding sites is one of the critical parameters to increase the glucose tolerance of these enzymes, and these studies show that glucose binding is one of the factors for glucose tolerance. This study advances the current knowledge of ꞵ-glucosidase. It shows that conformation changes in the presence of glucose are an essential characteristic of glucose tolerance. The importance of non-conserved residues, hydrophobicity, and sterics at the active site pocket entry is also an essential factor in glucose tolerance. The binding of glucose to the enzyme was shown for the first time and linked to enhanced glucose tolerance of such enzymes.

Item Type: Thesis (PhD)
Additional Information: Supervisor: Dr. Supratim Datta
Uncontrolled Keywords: Beta-Glucosidase; D0MD38; Glucose Tolerance; Glycosyl Family 1; Glycosyl Family 3; H0HC94
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Department of Biological Sciences
Depositing User: IISER Kolkata Librarian
Date Deposited: 25 Oct 2021 11:39
Last Modified: 02 Dec 2021 06:02

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