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Home > Glycosyl Hydrolases from Hyperthermophilic Archaeon Pyrobaculum Calidifontis: Cloning & Characterization

Glycosyl Hydrolases from Hyperthermophilic Archaeon Pyrobaculum Calidifontis: Cloning & Characterization

Thesis Info

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External Link

Author

Mehboob, Sumaira

Program

PhD

Institute

University of the Punjab

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2016

Thesis Completion Status

Completed

Subject

Biological Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/13131/1/thesis%20new%20title.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726341030

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4-α-glucanotransferases (4-α-GTases) play a vital role in starch modification for the production of resistant starches, cycloamyloses, prebiotics and thermoreversible starch gels. These products have a wide application in the development of starch based healthy foods. Synthesis of these products at industrial level requires highly thermostable enzymes. The present study describes the cloning and characterization of: 1) Pcal_0630, a β-glucosidase, 2) Pcal_0672, a glycosyl hydrolase and 3) Pcal_0768, a novel and highly thermostable 4-α-glucanotransferase from a hyperthermophilic aerobic archaeon Pyrobacculum calidifontis VA1. The genes encoding these enzymes were cloned in pET-21a(+) or pET-28a(+) and expressed in Escherichia coli. The blast search showed that Pcal_0630 belonged to family GH1 and exhibited a good sequence similarity with other uncharacterized enzymes from genus Pyrobaculum (upto 69%) while poor similarity was observed with the characterized thermophilic bacterial and archaeal enzymes like Pyrococcus furiosus (24%), Pyrococcus horikoshii (24%) and Halothermothrix orenii (22%). The gene sequence of Pcal_0630 is 1095 nucleotides in length which encoded a protein of 365 amino acids with a calculated molecular mass 42 kDa. Recombinant Pcal_0630 was produced as inclusion bodies in E. coli which were tried to be refold by denaturing in guanidine hydrochloride and urea but the refolded sample was inactive when assayed using cellobiose or pnitrophenol- α-D-glucopyranoside (PNPG) substrates. Pcal_0672, the second enzyme I studied, was composed of 1458 nucleotides which encoded a protein of 485 amino acids with an approximate molecular mass of 54 kDa. This gene was annotated as a glycosyl hydrolase belonging to family GH57 and showed a high homology of 81% with uncharacterized members of thermophilic bacteria and archaea. The sequence homology was very low with the characterized members of family GH57 like 4-α-GTase from T. litoralis (19%), branching enzyme from T. kodakarensis (18%), and α-amylase from P. furiosus (19%). Recombinant Pal_0672 was also produced as insoluble aggregates in E. coli. However, these insoluble aggregates exhibited the enzyme activity when analyzed by gel staining. Nucleotide analysis of the Pcal_0768 gene, the third enzyme of my study, showed that it consists of 1407 nucleotides encoding a protein of 468 amino acids with a calculated molecular mass of 53 kDa. Pcal_0768 was annotated as 4-α-GTase belonging to family GH77. The amino acid sequence of Pcal_0768 showed the highest homology of 75% with P. aerophilum amylomaltase. Four highly conserved regions characteristics of the α-amylase superfamily were also conserved in Pcal_0768. Amino acid residues constituting the catalytic triad were also conserved in Pcal_0768 at positions Asp270, Glu317 and Asp370. The gene was cloned and expressed in E.coli BL21 CodonPlus (DE3)-RIL and the recombinant protein was produced in soluble form which was further purified upto apparent homogeneity on SDS-PAGE. Recombinant Pcal_0768 exhibited maximum activity at 80 °C. Although the enzyme was active over the broad range of pH (3 – 10), its optimum pH was 7.0 and more than 50% residual activity was observed at pH 10. Pcal_0768 was highly stable at 80 °C. The half-life at 100 °C was 6.5 h. Pcal_0768 exhibited the highest ever reported disproportionation activity towards maltotriose (1951 U mg-1) as well as quite high transglycosylation activity (23 U mg-1). The activity of Pcal_0768 was independent of any metal ions. The recombinant enzyme was equally able to transglycosylate the high mass polysaccharides (in the presence or absence of an acceptor) to small linear chain glucans as well as disproportionate maltooligosaccharides (G2 – G7) to large amylaceous products. Pullulan, pannose and cyclodextrins (α, β and γ) were not the substrates of the enzyme. Pcal_0768 displayed transglycosylation activity on various maltooligosaccharides, among them the smallest donor and acceptor molecules were maltose and glucose, respectively whereas the smallest transferred unit was glucose. The enzyme was also efficient in disproportionating maltose into a mixture of maltooligosaccharides. These facts suggested that Pcal_0768 is a type V amylomaltase. Pcal_0768 was stable in the presence of ionic (1 mM SDS) as well as nonionic (2% Triton X-100 or Tween 20) detergents. However, loss of activity was observed in the presence of 5 mM SDS. When the protein was incubated with various concentrations of urea it was found that there was no loss of activity even at 8 M urea. The docked complex of urea and the enzyme active pocket revealed that due to the presence of charged/polar residue around the enzyme active pocket urea was unable to denature the enzyme. Pcal_0768 activity was markedly affected in the presence of DTT, p-chloromesrcuric benzoic acid and NBS which suggested that cysteine and tryptophan residues are critically involved in the enzyme catalysis. When Pcal_0768 was studied for its industrial applications it was found that it can successfully be used in the production of thermoreversible gels that are anticipated as gelatin/fat replacers in the food industry. Though cycloamyloses were not synthesized by the action of Pcal_0768 but there were strong indications for the synthesis of small linear chain maltooligosaccharides which can be used as prebiotics. However a detailed study on these aspects is needed. In conclusion, three starch processing enzymes have been studied. Among them, Pcal_0768 is highly efficient and a novel addition in type V amylomaltases. The enzyme is highly stable at high temperatures and in the presence of detergents and urea. Pcal_0768 is capable of forming white compact thermoreversible starch gels. These properties of Pcal_0768 make it a potential candidate for starch processing industry.
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