Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR

Abstract

Lipases catalyze the hydrolysis of fats and oils, and have been widely used in various industrial fields. However, bacterial lipases have a lower thermostability in industrial processes, which was a limiting factor in their industrial application. In this study, we obtained an improve variant of Pseudomonas fluorescens lipase (PFL) with enhanced thermostability using classical error-prone PCR. Wild-type PFL showed an optimal temperature and pH of 50°C and pH 7.5, respectively. Due to the low thermostability of PFL, a library containing over 3000 individual mutants as constructed using error-prone PCR. Screening for thermotolerance yielded the mutants L218P and P184C/M243C with T m values of 62.5 and 66.0°C, which was 2.5 and 6°C higher than that of the WT, respectively. The combination of the two mutants (P184C/M243C/L218P) resulted in an approximately additive effect with a T m value of 68.0°C. Although the increase of T m was not substantial, the mutant also had dramatically increased methanol tolerance. Structural analysis revealed that the introduction of a disulfide bond between P184C and M243C and the substitution of Pro to reduce the flexibility of a loop increased the thermostability of PFL, which provides a theoretical foundation for improving the thermostability and methanol tolerance of lipase family I.1 to resist the harsh conditions of industrial processes.

Microbial Lipases and Their Potential in the Production of Pharmaceutical Building Blocks

“…as a consequence of the adaptation of its produced microorganisms to the environmental conditions, which in some cases can be extreme [ 14 ]. Furthermore, microbial lipases are typically active as monomers, relatively small (19–65 kDa) and have at least 170 structures resolved to date (rcsb.org): this makes it easier to elucidate their mechanisms of action and design variants in a more rational way by using tools of molecular biology such as directed evolution, among others [ 15 , 16 , 17 , 18 ]. This also has favored their heterologous production in easily cultured organisms such as fungi and bacteria, allowing their large-scale production [ 10 , 11 , 16 ].…”

Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges

“…Directed evolution based on random mutagenesis and high-throughput screening approaches is a general strategy for improving enzyme thermostability [ 4 ]. This effective method has been successfully applied to, for example Pseudomonas fluorescens lipase, Agrobacterium tumefaciens uronate dehydrogenase and Bacillus clausii alkaline protease [ 5 , 6 , 7 ]. Nevertheless, directed evolution involves multiple rounds of mutations and creating a large mutant library, which is a labor- and time-consuming process [ 8 ].…”

Forty years of directed evolution and its continuously evolving technology toolbox: A review of the patent landscape

“…Since introduction of the epPCR technology, new methods for producing mutant proteins and nucleic acids keep on evolving rapidly (Guan et al, 2020). In 2000, Cline and Hogrefe (2000) developed a novel method for nucleic acid PCR mutagenesis that uses a 3′–5′ exonuclease‐deficient archaeal exo‐DNA polymerases, and a protein PCR enhancing factor.…”

“…Since introduction of the epPCR technology, new methods for producing mutant proteins and nucleic acids keep on evolving rapidly (Guan et al, 2020). In 2000, Cline and Hogrefe (2000) Sequence Saturation Mutagenesis (SeSaM) by Wong et al (2004) is another scientific approach avoiding limitations of epPCR.…”