Site‐directed mutagenesis studies of hydrophobic residues in the lid region of T1 lipase

Abstract

T1 lipase is a potential biocatalyst for industrial application since it is highly thermostable and displays optimal activity at 60–75°C. Structural analysis of T1 lipase shows that its lid region undergoes a spatial displacement along with a distinct secondary structure reorganization upon activation. To study structure/function of this atypical lid, we performed site‐directed mutagenesis on the hydrophobic residues in the lid region. These residues were mutated to hydrophilic ones and biochemical properties of mutants were investigated. Results showed that F181 might be an important residue for enzyme‐substrate binding. Mutants A186S and A190S had 35–50% increase in catalytic efficiencies compared to wild‐type T1, without compromising their functions at high temperatures. In general, mutagenesis did not cause large changes to chain‐length preference in T1 lipase. Mutants A186S and V187N were inactive towards long‐chain pNP esters (p‐Nitrophenyl stearate) and V187N showed lower activity towards long‐chain triacylglycerols than wt T1, which makes it a potential catalyst in dairy industry. Thermostability of mutants were affected at different extent due to the influence of hydrophobic contact between the lid and the protein core. These findings not only shed light on the lipase structure/function relationship but also lay the framework for further engineering to gain more potent, stable, and selective lipases. Practical applications: A thermostable T1 lipase owns an atypical lid and hydrophobic residues in the lid influence its catalytic properties. Here, we screened mutants A186S and A190S with 35–50% increase in catalytic efficiency, which have industrial application in the modification of fats and oils. Moreover, mutant V187N showed lower activity towards long‐chain TAGs than wt T1. This mutant can be a potential biocatalyst in dairy industry which promote generation of short‐chain fatty acids from milk fats to increase flavors in dairy products. The lid region of thermostable T1 lipase undergoes a spatial displacement along with a distinct secondary structure reorganization upon activation (PDB ID: 2DSN, 2W22 [16,17]). Mutagenesis on hydrophobic residues in the lid affect catalytic properties of T1 lipase.

Structure-Guided Rational Design of a Mono- and Diacylglycerol Lipase from Aspergillus oryzae: A Single Residue Mutant Increases the Hydrolysis Ability

“…The kinetic constants were determined using the spectrophotometric method at 405 nm as described by Tang et al . pNP-C10 with varying substrate concentrations from 0.05–2.0 mM was used.…”

“…According to a method reported earlier, the melting temperature assay was conducted by monitoring the fluorescence intensity changes of SYPRO Orange mixed with each enzyme in the CFX96 Real-Time PCR system (Bio-Rad). 31 The kinetic constants were determined using the spectrophotometric method at 405 nm as described by Tang et al 32 pNP-C10 with varying substrate concentrations from 0.05−2.0 mM was used. GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA) was used to determine the best-fit value of V max , and K m .…”

Rebuilding the lid region from conformational and dynamic features to engineering applications of lipase in foods: Current status and future prospects

“…These studies directly elucidated the reason for the inactivity of lipases in water and their high activity in organic solvents. However, aqueous activation of thermostable lipase from Bacillus stearothermophilus (T1 lipase) has been unexpectedly reported (Tang et al, 2017). Although water with a high dielectric constant ( 80) is theoretically unfavorable for lid opening, water elimination from organic solvents can inactivate the lipase activity for producing esters.…”

“…Many studies have investigated the role of lids as a covering or a modulator of the lipase active site as well as their functional roles in lipase catalysis (Tang, Lan, Yang, Khan, & Wang, 2017; Willems, Lelimousin, Skjold‐Jorgensen, Svendsen, & Sansom, 2018) (Table S2). As revealed by numerous studies, the amphiphilicity and amino acid composition of the lid impacted the enzyme binding with the interface and substrates, which are particularly correlated with the interfacial orientation, activation, and catalytic activity (Yang et al., 2015; Zhu et al., 2013).…”

“…In this way, special demands from the reaction in food industry would be satisfied, for example, the production of the medium–long–medium‐structured lipids that typically contain medium‐chain fatty acids at sn‐1,3 and long‐chain fatty acids (C14–C24) at sn‐2 positions. For example, constructing the mutants A186S and V187N of T1 lipase weakened the lipase activity toward long‐chain esters and long‐chain triacylglycerols, making it a promising catalyst in the dairy industry by promoting the generation of short‐chain fatty acids from milk fats to improve the flavors of dairy products (Tang, Lan, Yang, Khan, & Wang, 2017). Notably, even the performed site saturation mutation only constructs 19 mutations of lipase at one time by replacing the targeted residues with the unique amino acids at the target location.…”