Study of Homocysteine γ-Lyase (Hcy γ-Lyase) Production

Choong, Mel June (2016) Study of Homocysteine γ-Lyase (Hcy γ-Lyase) Production. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

Homocysteine γ-lyase is a pyridoxal 5’-phosphate-dependent enzyme that is involved in the degradation of sulfur containing amino acids. It cleaves homocysteine, L-methionine and cysteine to produce hydrogen sulfide, methanethiol, NH3 and relative acids. To be on par with international academic standards, various sources of soil from different places were used to study the possibility of finding a new Hcy γ-lyase with the desired properties for Hcy measurement in bacteria. The effects of temperature, pH, inducers and carbon sources on enzyme production were studied. L- Methionine was selected as the best inducer and glucose was selected as the best carbon source. Maximum production of Hcy γ-lyase was obtained when the isolate was cultured at 37°C at pH 7.0 for about 72 hours in the medium with L-methionine and glucose as inducer and carbon source respectively. The enzyme, Hcy γ-lyase also displayed a relatively high affinity to homocysteine, followed by cysteine and β-mercaptoethanol, confirming its potency against homocyteinuria related disease and as an anti-cardiovascular agent and a specific biosensor for homocyteinuria. One of the enzymes from the soil of Bukit Taman Melawati showed its maximum affinity for L-methionine (Km = 0.903 nM, Vmax = 0.028 s-1) and homocysteine (Km = 0.676 nM, Vmax = 0.034 s-1). Besides, Hcy γ-lyase was also strongly inhibited by hydroxylamine, confirming its pyridoxal 5’-phosphate (PLP) identity, yet no inhibited by EDTA. Among 4 2 types of soils, bacillus and coccus were most likely good candidates which produced Hcy γ-lyase. The preliminary identification was based on their morphology using gram staining technique. Throughout this study, it was also indicated that the bacteria could be a good source for production of Hcy γ-lyase. Hence, these bacterial Hcy γ-lyase provides potential application in clinical treatments.