Abstract
In order to improve the thermostability of lipases derived from Rhizopus chinensis, we identified lipase (Lipr27RCL) mutagenesis sites that were associated with enhanced flexibility based upon B-factor analysis and multiple sequence alignment. We found that two mutated isoforms (Lipr27RCL-K64N and Lipr27RCL-K68T) exhibited enhanced thermostability and improved residual activity, with respective thermal activity retention values of 37.88% and 48.20% following a 2 h treatment at 50°C relative to wild type Lipr27RCL. In addition, these Lipr27RCL-K64N and Lipr27RCL-K68T isoforms exhibited 2.4- and 3.0-fold increases in enzymatic half-life following a 90 min incubation at 60°C. Together these results indicate that novel mutant lipases with enhanced thermostability useful for industrial applications can be predicted based upon B-factor analysis and constructed via site-directed mutagenesis.
Highlights
Lipases mediate the hydrolysis of triglycerides into monoglycerides, fatty acids, diglycerides, and glycerol at oil-water interfaces (Naik et al, 2010), and can further catalyze interesterification, esterification, alcoholysis, acidolysis, and aminolysis in non-aqueous environments (Pandey et al, 1999; Yu et al, 2014)
The resultant Lipr27RCLK64N and Lipr27RCL-K68T proteins, which were designed via site-directed mutagenesis, exhibited both higher thermostability and superior catalytic efficiency
Normalized B-factor analysis of Lipr27RCL was the key step in the design of thermostable recombinant isoforms of this enzyme, given that B-factors offer insight into protein fluctuations, which are indicative of atom rigidity relative to their corresponding positions (Ringe and Petsko, 1986), and previous work has shown that improved thermostability depends upon achieving a higher degree of rigidity (Podar and Reysenbach, 2006)
Summary
Lipases (triacylglycerol acyl hydrolases, EC 3. 1. 1. 3) mediate the hydrolysis of triglycerides into monoglycerides, fatty acids, diglycerides, and glycerol at oil-water interfaces (Naik et al, 2010), and can further catalyze interesterification, esterification, alcoholysis, acidolysis, and aminolysis in non-aqueous environments (Pandey et al, 1999; Yu et al, 2014). 3) mediate the hydrolysis of triglycerides into monoglycerides, fatty acids, diglycerides, and glycerol at oil-water interfaces (Naik et al, 2010), and can further catalyze interesterification, esterification, alcoholysis, acidolysis, and aminolysis in non-aqueous environments (Pandey et al, 1999; Yu et al, 2014). Owing to their unique catalytic properties, lipases are utilized in a wide range of industrial contexts, such as in the production of food, leather, pharmaceuticals, and bioenergy (Persson et al, 2002; Ferreira-Dias et al, 2013). We conducted site-directed mutagenesis to improve R. chinensis lipase thermostability
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