Mutagenesis Of Microbial Transglutaminase For QTAG-KTAG Conjugation

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dc.contributor.authorChan, Soo Khim
dc.date.accessioned2019-04-17T01:22:45Z
dc.date.available2019-04-17T01:22:45Z
dc.date.issued2018-05
dc.description.abstractThe initial discovery of microbial transglutaminase (mTGase) was to soothe the high cost of mammalian transglutaminase in food processing industries. This enzyme has become an important tool in biotechnological applications due to its robustness, availability in high purity, bypass the need of calcium ions for activation and high reaction rate. However, mTGase has low substrate specificity due to its wide active site cleft which impeding its diverse applications in biotechnological use. Therefore, mutagenesis of mTGase to obtain substrate specific enzyme is highly desirable for site-specific conjugations. Three residues (V65, W69, and Y75) that are crucial for substrate recognition were mutated to all 20 amino acids to generate the first mutant library. Mutations at respective sites of the active mutants were then combined at all possible combinations to construct the combinatorial mutant library. MTG 120 mutant with the highest specific activity was used to pan against a 20-mer NNK peptide phage library to isolate glutamine-containing peptide. MTG 120 with mutations of leucine (L), methionine (M), and glutamic acid (E) at residue 65, 69, and 75, respectively, exhibited the highest specific activity. Also, MTG 120 showed significant reduction in random cross-linking with mTGase substrates including bovine serum albumin, and in-house alkaline phosphatase. A high affinity binder against MTG 120, peptide phage P58 has been successfully isolated through biopanning process. The motif RVGQL derived from the isolated peptide P58 was fused with in-house alkaline phosphatase to perform conjugation with penta-lysine tag anti-Ubiquitin scFv. The two proteins were successfully showed conjugation in Western blot to give a band around 85 kDa. The conjugated product also showed retained of functionality in enzyme-linked immunosorbent assay (ELISA). Mutagenesis of mTGase coupled with selection of mutant specific peptide has successfully improved the substrate specificity of mTGase for applications in biotechnological fields.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/8020
dc.language.isoenen_US
dc.publisherUniversiti Sains Malaysiaen_US
dc.subjectMutagenesis of microbial transglutaminaseen_US
dc.subjectfor QTAG-KTAG conjugationen_US
dc.titleMutagenesis Of Microbial Transglutaminase For QTAG-KTAG Conjugationen_US
dc.typeThesisen_US
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