An enzyme with broad substrate specificity would be an asset for industrial application. T1 lipase apparently has the same active site residues as polyhydroxyalkanoates (PHA) depolymerase. Sequences of both enzymes were studied and compared, and a conserved lipase box pentapeptide region around the nucleophilic serine was detected. The alignment of 3-D structures for both enzymes showed their active site residues were well aligned with an RMSD value of 1.981 Å despite their sequence similarity of only 53.8%. Docking of T1 lipase with P(3HB) gave forth high binding energy of 5.4 kcal/mol, with the distance of 4.05 Å between serine hydroxyl (OH) group of TI lipase to the carbonyl carbon of the substrate, similar to the native PhaZ7 Pl . This suggests the possible ability of T1 lipase to bind P(3HB) in its active site. The ability of T1 lipase in degrading amorphous P(3HB) was investigated on 0.2% (w/v) P(3HB) plate. Halo zone was observed around the colony containing the enzyme which confirms that T1 lipase is indeed able to degrade amorphous P(3HB). Results obtained in this study highlight the fact that T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation activity but amorphous P(3HB) degradation activity as well.
A broad substrate specificity enzyme that can act on a wide range of substrates would be an asset in industrial application. T1 lipase known to have broad substrate specificity in its native form apparently exhibits the same active sites as polyhydroxylalkanoate (PHA) depolymerase. PhaZ6Pl is one of the PHA depolymerases that can degrade semicrystalline P(3HB). The objective of this study is to enable T1 lipase to degrade semicrystalline P(3HB) similar to PhaZ6Pl while maintaining its native function. A structural study on PhaZ6Pl contains no lid in its structure and therefore T1 lipase was designed with removal of its lid region. BSLA lipase was chosen as the reference protein for T1 lipase modification since it contains no lid. Initially, structures of both enzymes were compared via protein-protein superimposition in 3D-space and the location of the lid region of T1 lipase was highlighted. A total of three variants of T1 lipase without lid were successfully designed by referring to BSLA lipase (a lipase without lid). The ability of T1 lipase without lid variants in degrading P(3HB) was investigated quantitatively. All the variants showed activity towards the substrate which confirmed that T1 lipase without lid is indeed able to degrade P(3HB). In addition, D2 was recorded to have the highest activity amongst other variants. Results obtained in this study highlighted the fact that native T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation but also P(3HB) by simply removing the lid region.
Aquaculture is an important component of the world food supply and a significant source of protein. However, this industry faces numerous problems. Including poor fish feed digestion and uneconomic nutrient utilization. This can result in unsatisfactory growth rates and poor stock performance. Utilizing probiotics, which are beneficial microbes that can enhance digestive systems and general fish health, is one possible way to address these issues. This study was designed to identify and evaluate a novel strain of Bacillus as a promising probiotic. The strain of Bacillus rugosus that was examined and coded NM007 showed promising probiotic characteristics that could help fish digest and utilize their feed more efficiently, reduce feed waste, and improve their digestive systems. B. rugosus NM007 exhibited the ability to produce digestive enzymes like protease, amylase, and lipase, which are the main digestive enzymes. It showed strong auto-aggregation activity and co-aggregation activity with Aeromonas sp. and Streptococcus sp. It also demonstrated tolerance to the presence of bile salt, acidic pH, and salinity up to 60 ppt. The sensitivity analysis towards antibiotics, hemolytic activity and the safety assessment on Nile tilapia fish (Oreochromis niloticus) confirmed the safety of this isolate. Based on the findings of this investigation and the isolate's characterization, Bacillus rugosus NM007 could serve as a new promising probiotic bacterium for aquaculture.