STUDIES ON ENZYMES OF HYDROLASE FAMILY DEPARTMENT OF BIOTECHNOLOGY INDIAN INSTITUTE OF TECHNOLOGY ROORKEE β-GLUCOSIDASE AND ACID PHOSPHATASE

Abstract

Proteins are highly complex most abundant biological macromolecules common to all life present on earth today and they are responsible for most of the complex functions that make life possible. All the forms of life i.e. plant, animal, fungi, bacteria, virus contain thousands of proteins for proper functioning. In modern classification proteins are classified in three major classes; storage proteins, structural proteins and biological active proteins and some of these proteins also play combined roles. The metabolic proteins mainly comprised of enzymes. Enzymes are awesome machines with a suitable level of complexity. They are central to every biochemical process and have extraordinary catalytic power. Among six different classes of enzymes, ‘Hydrolase’ is one of the most important group of enzymes which catalyze the hydrolysis of a chemical bond like C-C, C-O, C-N, ether, ester and halide bonds etc. Hydrolases are classified into several subclasses, based upon the bonds they act upon e.g. glycosyl hydrolase, esterase, phosphatase, lipase, DNA glycosylases etc. In the hydrolase family, glycosyl hydrolases and phosphatases are two important groups of enzymes as they play very crucial role in cell metabolism. We have studied two enzymes from each of these two groups. First enzyme is a β-glucosidase from family 1 glycosyl hydrolase. Glycosyl hydrolases or Glycoside hydrolases or Glycosidases (GHs; EC 3.2.1.x) catalyze the hydrolysis of O-, N- or S-linked glycosidic bonds between a carbohydrate and non-carbohydrate moiety or between two carbohydrates. The cleavage of these glycosidic bonds is crucial for the processes like hydrolysis of structural polysaccharides during penetration of pathogens, recycling of cell surface carbohydrates, defense against pathogens expansion of cell wall, energy uptake, starch metabolism, symbiosis and recycling of signaling molecules etc. On the basis of amino acid sequence and structural analogy, at present there are 133 Glysoside hydrolase families available on the CAZY web server (URL- http://www.cazy.org/). Among them, family 1 glycosyl hydrolase is most studied. Glycoside hydrolases family 1 contains different enzymes with some wellknown functions, such as β-glucosidase (EC 3.2.1.21), β-fucosidase (EC 3.2.1.38), β- galactosidase (EC 3.2.1.23), β-mannosidase (EC 3.2.1.25), exo-β-1, 4-glucanase (EC 3.2.1.74) etc. β-glucosidase characterized till date fall primarily in glycoside hydrolase families 1, 3 and 5 with family 1 β-glucosidases being more abundant in plants. β-glucosidase of this family may have high specificity for glucosides or in addition to this may hydrolyse fucosides and/or galactosides. ii Carbohydrates are the most abundant biomolecules on earth and also in plant cells. The controlled regulation of synthesis, breakdown and modification of these macromolecules in nature is one of the most fundamentally important processes. This carbohydrate metabolism is only possible due to some enzymes i.e. glycosyl hydrolases (hydrolyzes and/or rearrange the glycosidic bonds), glycosyl transferases (build glycosidic bonds), polysaccharide lyases (non-hydrolytic cleavage of glycosidic bonds) and polysaccharide lyases (hydrolyzes carbohydrate esters). The importance of these processes can be assessed by the fact that 1-2% of an organism’s total gene dedicates for the synthesis of glycosyl hydrolases and glycosyl transferases alone. The second enzyme, we have studied is a phosphatase from HAD superfamily. Like glycosyl hydrolase HAD superfamily (HADSF) hydrolases are also very important in their functional point of view. The HADSF is the major family found with almost 48,000 sequences and is present ubiquitously in the living cells. HADSF constitute different enzymes like dehalogenase, phosphatase, phosphonatase, β-phosphoglucomutase and ATPases. Phosphatase forming the majority in HADSF can vary in figure from 30 in prokaryotes to 200-300 in the eukaryotes. They help in phosphorous transfer reactions by transferring the phosphate group to an Asp as an active site residue. In the present work, a β-glucosidase enzyme from Putranjiva roxburghii plant (PRGH1) was characterized, cloned and expressed in both prokaryotic and eukaryotic system. A comparison study between the native enzyme and bacterial expressed recombinant enzyme has been carried out to understand the possible role of post translational modifications on the enzyme. Several mutation studies were done to locate the possible active site. Meticulous bioinformatics work was carried out to understand the substrate preference of this enzyme. The possible role of N-linked glycosylation in the stability and the activities of the enzyme were examined. We have used this enzyme in bioethanol production by expressing this enzyme in eukaryotic system. Along with this enzyme we have characterized another hydrolase enzyme from Staphylococcus lugdunensis. This enzyme belongs to HADSF and showed phosphatase activity. This thesis is divided into five chapters and covers the studies carried out on two hydrolases. The first one is a glycosyl hydraolase sourced from the Putranjiva roxburghii plant and the second one is a phosphatase from HAD family sourced from Staphylococcus lugdunensis, which is a human pathogen. iii Chapter 1 reviews the literature; describing the recent studies on family 1 Glycosyl hydrolase (GH1), role of N-linked glycosylation, application of β-glucosidase in bioethanol production and studies on HADSF. Chapter 2 describes the cloning, overexpression, purification and characterization of a glycosidase 1 enzyme. A 66 kDa, thermostable enzyme with β-fucosidase, β-glucosidase and β-galactosidase activities was purified from the seeds of Putranjiva roxburghii by employing concanavalin-A affinity chromatography. The deduced amino acid sequence showed considerable similarity with plant β-glucosidases. The enzyme hydrolyzes p-nitrophenyl-β-Dglucopyranoside (pNP-Fuc) with higher efficiency (Kcat/Km = 3.79 × 104 M-1s-1) as compared to pNP-Glc (Kcat/Km = 2.27 × 104 M-1s-1) and pNP-Gal (Kcat/Km = 1.15 × 104 M-1s-1). Both the native and recombinant protein has pH optima of 4.8. The thermostability of the recombinant enzyme is much lower than native enzyme. Mutational study showed that disruption of active site residues affect the activity of the enzyme. Oligomerization study showed that at higher concentration the enzyme form various species of oligomers having molecular mass around a decamer. Mixed substrate analysis showed that all the three activities (glucosidase, galactosidase and fucosidase) of the enzyme were performed by a single active site. This preference for the substrate has also been studied and proved using meticulous bioinformatics work. Chapter 3 describes the role of glycosylation for the stability and consequence effect on the activity of a very efficient thermostable β-glucosidase from Putranjiva roxburghii plant (PRGH1). We successfully produced deglycosylated PRGH1 by using PNGase F. We compared the activities of both forms of this enzyme under various conditions like different temperature, pH and organic solvents. At higher pH the deglycosylated PRGH1 showed a sharp decrease in activity. The temperature profile of both the glycosylated and deglycosylated enzyme clearly reflect that glycosylated form of this enzyme have greater stability at higher temperature. CD and intrinsic fluorescence studies of both glycosylated and deglycosylated enzyme showed that the conformation of the native protein changed to a certain extent after removal of the N-linked sugars. Proteolysis study along with the spectral studies suggests that the structure of native glycosylated PRGH1 is quite compact and rigid than the deglycosylated counterpart. Mutagenesis studied shows that out of seven potential glycosylation site three sites were glycosylated. Chapter 4 describes the expression of a plant β-glucosidase gene (PRGH1) in the S. cerevisiae Y294 and studies on the properties of the enzyme. The optimal pH and temperature of the enzyme activity was found to be 5.0 and 65 °C respectively indicating this enzyme is a iv thermostable enzyme with preference towards moderate acidic condition. The enzyme showed broad substrate specificity and able to hydrolyze cellobiose significantly. The enzyme showed resistance towards alcohols, suggesting the enzyme can be used in fermentation industry more efficiently. The recombinant S. cerevisiae Y294 harbouring prgh1 gene showed better growth profile, cellobiose consumption and ethanol production. In addition to this, complementing with commercial cellulose enzyme the recombinant S. cerevisiae Y294 was used in SSF experiment using CMC, rice straw, sugarcane bagesse as sole carbon source. The study demonstrated the feasibility of using the β-glucosidase gene to enhance the second generation cellulosic ethanol production. Chapter 5 describes the cloning, overexpression and characterization of a acid phosphatase from Haloacid dehalogenase superfamily. SHFD gene with ~840 bp has been cloned and overexpressed in E.coli. The enzyme with a molecular mass of ~32 kDa has been purified using Ni2+-NTA affinity chromatography. SHFD showed phosphatase activity with an optimum temperature of 25 °C. SHFD is an acid phosphatase with an optimum pH of 5.0. The kinetic parameters (Km= 0.32 mM, Vmax = 0.36 U/mg, kcat = 21.43 ± 0.85 s-1 and kcat/Km = 66.96 mM-1s-1) indicate that it is a very efficient enzyme. SHFD is a mixed α/β protein as predicted by the ESPript and CD spectrum. Multiple sequence alignment shows the conservation of nucleophilic Asp10, acid/base catalyst Asp12, phosphate binding Ser43, and many other catalytic residues like Arg45, Lys210, Asp233, Asn236 and Asp237. SHFD is a two domain protein, with a larger core domain comprising four conserved loops surrounding the active site. The core domain also has a modified Rossmann fold with six stranded β-sheets surrounded by six α-helices. It is responsible for binding, reorienting of the phosphate group along with the co-factor Mg2+ and also in preparing the Asp10 for the nucleophilic attack.