Isolation and Identification of L-Metioninsae Producing Bacteria From Cheeses and Partial Purification of L-Metioninsae

Loo, Lok Wenn (2018) Isolation and Identification of L-Metioninsae Producing Bacteria From Cheeses and Partial Purification of L-Metioninsae. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

L-methioninase has a major role in food industry by imparting a distinctive aroma to many traditional fermented foods. Besides, it is also a potent anticancer agent against many types of cancers. The objectives of this study were to identify those bacteria that produce L-methioninase, to determine the cultivation period best for the enzyme specific activity and to partially purify the enzyme. 58 isolates were obtained from cheddar cheese and 6 from limburger cheese. 47 out of 58 isolates and the other all 6 isolates were capable to grow and utilize L- methionine as the only source of nitrogen. Throughout the quantitative rapid plate assay test, none of these isolates shown pink or with a pink zone surrounding their colonial growth when cultured in modified TGY agar which contained 0.07% phenol red. Instead, the isolates retained their original pigment as shown in the unmodified TGY agar. There was an exception for those isolates originally showed pink colonial growth in an unmodified medium. 17 + 3 isolates from the above isolates were selected for Day 3 enzymatic assay test and out of these 20, 7 isolates (No. C7, A10, C11, A16, A2, B1 and B2) which had obtained an absorbance higher than 0.2000 (0.3655, 0.3486, 0.4019, 0.213, 0.4314, 0.4116 and 0.4737 respectively) at 412 nm, were selected for Day 3 and 5 protein concentration, enzyme formation, and specific activity determinations. All determinations decreased on Day 5. The enzyme concentrations of isolates No. C7, A10 and C11 decreased slightly after partial purification, from 2.1782 ± 0.1788, 2.9038 ± 0.0873, and 2.2227 ± 0.2336 mg/ ml respectively to 1.7572 ± 0.2621, 2.8923 ± 0.1036, and 2.0374 ± 0.0812 mg/ml respectively. The partial purified enzymes each yielded a band of approximately 80 kDa, with an approximate 25 kDa enzyme subunit when checked by SDS-PAGE. These isolates were identified by ribotyping 16s rRNA followed by BLAST analysis and confirmed as Uncultured Chryseobacterium sp. (No. C7), Bacillus cereus (No.C11), Acinetobacter sp. (No.A16) and Bacillus megaterium (No.B2). KEYWORDS: L-Methioninase, cheese, purification, isolation, 16s RNA.