Isolation and Identification of L-Methioninase Producing Bacteria from Cheese

Tai, Jie Yi (2021) Isolation and Identification of L-Methioninase Producing Bacteria from Cheese. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

L-methioninase is a pyridoxal phosphate (PLP)- dependent enzyme that has wide contribution to the traditionally fermented food which is cheese by imparting a special aroma to cheeses. The distinctive aroma is imparted when the L-methionine is degraded to release volatile sulfur compounds (VSCs). Furthermore, this enzyme gets much attraction as it acts as a potent anticancer agent against various types of cancers. The objectives of this study were to identify the L-methioninase producing bacteria, to isolate and screen the L-methioninase bacteria for their L-methioninase productivities, to identify the bacterial strains by molecular characterization and to purify the enzyme. L-methioninase can catalyze the direct conversion of L-methionine to methanethiol, ⍺-ketobutyrate and ammonia. Bacteria such as Brevibacterium linens and Hafnia alvei were identified to be able to produce the enzyme L-methioninase in cheese. Brevibacterium linens was the first cheese-ripening bacterium identified to produce methanethiol from methionine and it is a gram-positive bacterium with rod coccus shape. By using rapid assay plate method, the ammonia produced by L-methioninase resulted to the formation of a pink to a reddish halo around colonies that were spread on the modified M9 medium and the pink colour formation indicated the L-methioninase producers. Besides, 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) assay is an alternative method to screen for L-methioninase producers and this assay involved the DTNB as a screening dye. For L-methioninase producing bacteria, the expected outcome of DTNB assay was the formation of yellow colored aryl mertcaptan (2-nitro-5-thiobenzoate or TNB) around the bacteria colonies. In a molecular characterization of bacterium Hafnia alvei, Sanger sequencing was used to compare the amplified sequence to NCBI database using BLAST and the result identified that the isolate was Hafnia alvei with 97.7–100% identity and 100% coverage. Therefore, Hafnia alvei was chosen as the best isolate to secrete L-methioninase. In the gel electrophoresis under denaturing conditions by SDS-PAGE, the purification of L-methioninase produced by Brevibacterium linens yielded a single band with an approximate molecular mass of 43 kDa.