Title: Enzyme Engineering for High-Yielding Amide Formation: Lipase-Catalyzed Synthesis of N-Acyl Glycine in Aqueous Media Kua GKB, Nguyen GKT, Li Z Ref: Angew Chem Int Ed Engl, :, 2023 : PubMed
Amide syntheses remain a key challenging green chemistry reaction. For instance, green synthesis of N-acyl glycine as biosurfactants and therapeutics are highly desirable to replace chemical pathway using toxic phosgene. Here, we report a novel concept for enzymatic amidation in an aqueous system via glycerol-activation of fatty acid and its subsequent aminolysis with glycine to synthesize N-acyl glycine. We then engineer an enzyme (proRML) by reshaping its catalytic pocket to enhance its aminolysis activity and catalytic efficiency by 103-fold and 465-fold, respectively. The evolved proRML (D156S/L258K/L267N/S83D/L58K/R86K/W88V) catalyzed the amidation of fatty acid by glycine to N-lauroylglycine with high yield (80%). It accepts a broad range of medium to long-chain fatty acids (C8-C18), giving high yields of N-decanoyl, N-myristoyl, and N-oleoylglycine. The developed amidation concept may be general, and the engineered enzyme is useful for green synthesis of N-acyl glycine.
        
Title: Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags Chow JY, Nguyen GKT Ref: Int J Mol Sci, 23:, 2022 : PubMed
1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The Rhizopus oryzae lipase (ROL) has recently gained interest because this enzyme possesses high selectivity and catalytic efficiency. However, its low thermostability limits its use towards reactions that work at lower temperature. Most importantly, the enzyme cannot be used for the production of 1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1,3-stearoyl-2-oleoyl-glycerol (SOS) due to the high melting points of the substrates used for the reaction. Despite various engineering efforts used to improve the thermostability of ROL, the enzyme is unable to function at temperatures above 60 degreesC. Here, we describe the rational design of ROL to identify variants that can retain their activity at temperatures higher than 60 degreesC. After two rounds of mutagenesis and screening, we were able to identify a mutant ROL_10x that can retain most of its activity at 70 degreesC. We further demonstrated that this mutant is useful for the synthesis of SOS while minimal product formation was observed with ROL_WT. Our engineered enzyme provides a promising solution for the industrial synthesis of structured lipids at high temperature.
        
Title: A Novel Lipase from Lasiodiplodia theobromae Efficiently Hydrolyses C8-C10 Methyl Esters for the Preparation of Medium-Chain Triglycerides' Precursors Ng AMJ, Yang RL, Zhang HF, Xue B, Yew WS, Nguyen GKT Ref: Int J Mol Sci, 22:, 2021 : PubMed
Medium-chain triglycerides (MCTs) are an emerging choice to treat neurodegenerative disorders such as Alzheimer's disease. They are triesters of glycerol and three medium-chain fatty acids, such as capric (C8) and caprylic (C10) acids. The availability of C8-C10 methyl esters (C8-C10 ME) from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs. However, there are few reports on enzymes that can efficiently hydrolyse C8-C10 ME to industrial specifications. Here, we report the discovery and identification of a novel lipase from Lasiodiplodia theobromae fungus (LTL1), which hydrolyses C8-C10 ME efficiently. LTL1 can perform hydrolysis over pH ranges from 3.0 to 9.0 and maintain thermotolerance up to 70C. It has high selectivity for monoesters over triesters and displays higher activity over commercially available lipases for C8C10 ME to achieve 96.17% hydrolysis within 31 h. Structural analysis by protein X-ray crystallography revealed LTL1's well-conserved lipase core domain, together with a partially resolved N-terminal subdomain and an inserted loop, which may suggest its hydrolytic preference for monoesters. In conclusion, our results suggest that LTL1 provides a tractable route towards to production of C8-C10 fatty acids from methyl esters for the synthesis of MCTs
        
Title: A Novel Lipase from Lasiodiplodia theobromae Efficiently Hydrolyses C8-C10 Methyl Esters for the Preparation of Medium-Chain Triglycerides' Precursors Ng AMJ, Yang R, Zhang H, Xue B, Yew WS, Nguyen GKT Ref: Int J Mol Sci, 22:, 2021 : PubMed
Medium-chain triglycerides (MCTs) are an emerging choice to treat neurodegenerative disorders such as Alzheimer's disease. They are triesters of glycerol and three medium-chain fatty acids, such as capric (C8) and caprylic (C10) acids. The availability of C8-C10 methyl esters (C8-C10 ME) from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs. However, there are few reports on enzymes that can efficiently hydrolyse C8-C10 ME to industrial specifications. Here, we report the discovery and identification of a novel lipase from Lasiodiplodia theobromae fungus (LTL1), which hydrolyses C8-C10 ME efficiently. LTL1 can perform hydrolysis over pH ranges from 3.0 to 9.0 and maintain thermotolerance up to 70 degreesC. It has high selectivity for monoesters over triesters and displays higher activity over commercially available lipases for C8-C10 ME to achieve 96.17% hydrolysis within 31 h. Structural analysis by protein X-ray crystallography revealed LTL1's well-conserved lipase core domain, together with a partially resolved N-terminal subdomain and an inserted loop, which may suggest its hydrolytic preference for monoesters. In conclusion, our results suggest that LTL1 provides a tractable route towards to production of C8-C10 fatty acids from methyl esters for the synthesis of MCTs.
        
Title: Zymography for Picogram Detection of Lipase and Esterase Activities Ng AMJ, Zhang H, Nguyen GKT Ref: Molecules, 26:, 2021 : PubMed
Lipases and esterases are important catalysts with wide varieties of industrial applications. Although many methods have been established for detecting their activities, a simple and sensitive approach for picogram detection of lipolytic enzyme quantity is still highly desirable. Here we report a lipase detection assay which is 1000-fold more sensitive than previously reported methods. Our assay enables the detection of as low as 5 pg and 180 pg of lipolytic activity by direct spotting and zymography, respectively. Furthermore, we demonstrated that the detection sensitivity was adjustable by varying the buffering capacity, which allows for screening of both high and low abundance lipolytic enzymes. Coupled with liquid chromatography-mass spectrometry, our method provides a useful tool for sensitive detection and identification of lipolytic enzymes.