Characterization and engineering of thermostable endoglucanase enzymes from bacterial and fungal sources

Aich, Shritama (2020) Characterization and engineering of thermostable endoglucanase enzymes from bacterial and fungal sources. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Shritama Aich (14RS041)) 14RS041.pdf - Submitted Version Restricted to Repository staff only Download (9MB)

Abstract

Lignocellulosic biomass is one of the major forms of primary energy source on earth. Due to its recalcitrance, the biomass needs to be treated by several means by which it becomes accessible to the enzymes for hydrolysis. The enzymes from fungal and bacterial sources are a rate limiting step in this whole biomass hydrolysis process where catalytically efficient and stable enzymes make the hydrolysis of pretreated lignocellulosic biomass to simple units of glucose much more economical and efficient. The sugar units are then fermented and processed further for generation of biofuel and several chemicals. Biofuels produced from lignocellulosic biomass has many potential benefits over first-generation biofuel, including lower CO2 emissions, without any competition with food for human consumption. Thus, identifying efficient enzymes from the huge repertoire of cellulase enzyme and a detailed characterization of these enzymes would be the first step towards their biotechnological applications in biomass hydrolysis. Cellulose is the predominant part of the lignocellulosic biomass and the most common organic compound of primary energy source on earth and degradation of cellulose is important for global carbon recycling. The enzymes that hydrolyses cellulose into units of glucose are known as cellulases. Cellulases are mainly comprised of three enzyme classes which are endoglucanases, cellobiohydrolases and β-glucosidases. All the cellulases are involved in hydrolysis of glycosidic bonds and are grouped in the family of glycoside hydrolases of the Carbohydrate-active Enzymes’ (CAZy) database. These cellulase enzymes help in the enzymatic hydrolysis of cellulosic biomass for the formation of fermentable sugars. Endoglucanase are the enzymes that play a prominent role in initiating and sustaining the hydrolytic process of cellulose breakdown. It helps in random cleavage of cellulose chains into smaller units that can be further hydrolyzed by cellobiohydrolases from one end of the smaller cellulosic units to generate cellobiose. Cellobiose is further hydrolyzed into two units of glucose by β-glucosidase. These glucose units are further fermented and processed for generation of biofuel and chemicals for different industrial purposes. Other than biofuel, the cellulases have potential applications in industries like textile, laundry and detergent, paper, medicine, agriculture and food industries. Identifying homologs of endoglucanases gene from the environment and characterization and quantification of endoglucanase activity is important for industry and in the overall study of cellulose degradation. The thesis mostly emphasizes on identifying endoglucanases from fungal and bacterial sources as because in any cellulolytic enzyme activity, endoglucanase plays a prominent role in initiating and sustaining the hydrolytic process after pretreatment of the biomass. The endoglucanase enzymes characterized are from the glycoside hydrolase (GH) family 7 and 9 from the CAZy database. GH7 family cellulases are one of the most important cellulases with highest cellulolytic turnover and GH9 family cellulases are the ones with industrial importance because of its huge amount of secreted enzymes and because of their processive nature. The highly stable endoglucanase from the GH7 family of a fungal plant pathogen Bipolaris sorokiniana and endoglucanase from the GH9 family of the soil bacterium, Bacillus licheniformis are the two enzymes that will be mainly discussed in this thesis. GH7 family has both cellobiohydrolase and endoglucanase enzymes from fungal sources where as GH9 enzymes are primarily endoglucanase from bacterial sources. GH7 endoglucanases have an open substrate binding cleft whereas the cellobiohydrolases have further elongated loops which make the substrate binding site look like a closed tunnel. The protein structures are β-jellyroll folded frame work where the antiparallel β-sheets face each other and form a β-sandwich structure. The catalytic domain of GH9 endoglucanases on the contrary, have an (α/α)6 barrel fold which forms an open active site cleft and the cleft contains six sugar binding subsites. GH7 family enzymes operate by retention of anomeric stoichiometry where as GH9 enzymes hydrolysis causes inversion of the anomeric stoichiometry. The catalytic residues of GH7 enzymes maintain a consensus motif of -Glu-X-Asp-X-X-Glu and are positioned close to one another. The first Glu is the catalytic nucleophile and the second one is the general acid/base. In GH9, a conserved Glu acts as catalytic general acid and two other conserved Asp residues of which acts as catalytic general base. In the last part of the thesis I have discussed about the role of relatively newly discovered auxiliary active (AA) enzymes in efficient hydrolysis of cellulosic biomass and how incorporating these enzymes into a cellulase cocktail of endoglucanase, cellobiohydrolase and β-glucosidase might further help in efficient cellulose dissolution. Bipolaris sorokiniana is a filamentous fungus that causes spot blotch disease in cereals like wheat and has severe economic consequences. However, information on the identities and role of the cell wall-degrading enzymes (CWDE in B. sorokiniana is very limited. Several fungi produce CWDE like glycosyl hydrolases (GHs) that help them in host cell invasion. B. sorokiniana, has CWDE from GH families like GH3, GH6, GH7, GH45 and AA9. Quantitative real-time PCR analyses of these homologs revealed that the transcripts of the BsGH7-3 (3rd homolog of the GH 7 family in B. sorokiniana) were most abundant. BsGH7-3, the gene of BsGH7-3, was thus cloned into pPICZαC Pichia pastoris vector and expressed in X33 P. pastoris host to be characterized. BsGH7-3 enzyme showed a temperature optimum of 60 °C and a pHopt of 8.1. BsGH7-3 was identified to be an endoglucanase based on its broad substrate specificity and structural comparisons with other such endoglucanases. BsGH7-3 had a very long half-life and retains 100% activity even in the presence of 4 M NaCl, 4 M KCl and 20% (v/v) ionic liquids. The enzyme activity was stimulated up to fivefold in the presence of Mn⁺² and Fe⁺² without any deleterious effects on enzyme thermostability. However, the catalytic efficiency of the enzyme was lower than for some other endoglucanases of the GH7 family reported in the literature. To engineer a more active enzyme, conserved residues in the substrate-binding tunnel and on the surface of the protein that could play a role in charge-charge interaction and stabilize the structure was identified. The mutants D257W and Q225H in the substrate-binding tunnel and Y222R and Q401N on the protein surface showed a 2-fold increase in specific activity and a 1.5-fold increase in turnover number and were active over a broader range of pH. The mutants also showed a higher tolerance to NaCl and was also stable with 50 % activity retained even after 10 days of incubation at its optimum temperature. The rational design of the BsGH7-3 mutants helped in increasing the catalytic efficiency of the thermostable enzyme and may be useful in combination with other cellulases like cellobiohydrolase and β-glucosidase towards complete saccharification of cellulose into glucose. One of the critical steps in lignocellulosic deconstruction is the processive hydrolysis of crystalline cellulose by cellulases. The endoglucanase catalytic domains of glycoside hydrolase family 9 (GH9) are arranged with specific carbohydrate-binding domains to ensure stability and activity and GH9 is the second largest cellulase family which consists of many endoglucanases. An endoglucanase gene, gh9A from GH9 family was isolated from the soil bacterium Bacillus licheniformis and the endoglucanase enzyme, H1AD14 was characterized and its catalytic efficiency was determined. The role of the carbohydrate binding module (CBM) fused to the C-terminal end of the protein structure was discussed. The role of the CBM in the enzyme processivity and substrate hydrolysis had been shown. Saccharification of different pretreated forms of sugarcane bagasse by H1AD14 and H1AD14 without the CBM (H1AD14-CBM) had also been studied. The newly discovered AA enzymes has also been studied from AA family 10 of Thermobifida fusca to understand how it might enhance the rate of saccharification of cellulosic biomass when supplemented with cellulases. The AA10 enzymes are the copper dependent lytic polysaccharide monooxygenase which helps in enzymatic conversion of recalcitrant polysaccharides such as cellulose and makes it more accessible to the other cellulase enzymes for their hydrolysis. The codon optimized AA10 gene from T. fusca was cloned into pET21b vector and the enzyme Q47QG3 was purified to study its synergistic effect on the endoglucanases and how the enzymes help in enhanced saccharification of cellulosic biomass by commercially used cellulase cocktails and commercial endoglucanase. Having a characterized LPMO will help towards increasing the overall saccharification of the biomass. This thesis, that mainly focusses on identifying active and thermostable endoglucanases leads to a step forward towards generation of a highly efficient and stable cellulase cocktail where all the minimum components required for hydrolysis i.e auxiliary active enzymes, endoglucanase, cellobiohydrolase and β-glucosidase are compatible with each other and be catalytically efficient without any product inhibition which limits the rate of hydrolysis and product generation. Comparing the cellulase cocktail generated with highly active commercial cellulase cocktails to have an industrially relevant cellulase for hydrolysis of cellulosic biomass is the broader aspect of the studies done in this thesis and the thesis tries to contribute towards generating an efficient cellulase cocktail which is catalytically more efficient as compared to all the other commercially available cellulase cocktail used for cellulosic breakdown.

Actions (login required)

View Item