Improvement in thermostability of an alkaline lipase I from Penicillium cyclopium by directed evolution

Liu, Yihan; Líu, Hao; Huang, Lin; Gui, Shuang; Dong, Zheng; Jia, Leibo; Fu, Yu; Lu, Fuping

doi:10.1039/c7ra06307e

Cited by 23 publications

(17 citation statements)

References 49 publications

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“…Far-UV CD spectra in the range of 190–260 nm were recorded to determine the secondary structure of PFL. Figure 2C shows a strong positive absorption peak at 196–197 nm, which is a typical feature for α-helices, whereas a negative absorption peaks centered around 192–194 and 215–220 nm are a typical feature β-sheet structures ( Liu et al, 2017 ). The secondary structure percentages of PFL obtained from CD spectra were 18.6% α-helix, 37.9% β-sheet, 9.2% β-turn structures, and 34.6% unstructured regions.…”

Section: Resultsmentioning

confidence: 99%

“…The T m values of the mutants L218P and P184C/M243C was increased from 60°C to 62.5 and 66.0°C, which was 2.5 and 6°C higher than that of the WT, respectively ( Figure 5B ). A combination of mutants with higher thermostability sometimes results in a cumulative effect ( Liu et al, 2017 ). Therefore, the combined triple mutant P184C/M243C/L218P was generated using site-directed mutagenesis using the plasmid P184C/M243C as the template.…”

Section: Resultsmentioning

confidence: 99%

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

Guan¹,

Gao²,

Li³

et al. 2020

Front. Bioeng. Biotechnol.

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Guan¹,

Gao²,

Li³

et al. 2020

Front. Bioeng. Biotechnol.

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“…A study by Mohammadi et al [ 31 ] reported substitution of proline residue at position 2 in the N -terminal of lipase A from Serratia marcescens revealed a 2.3-fold higher activity at half-life of the mutant compared to the wild-type. Liu et al [ 42 ] also reported an increase of 7-fold in half-life at 45 °C of mutant alkaline lipase I from Penicillium cyclopium with proline substitution at position 41, far from the catalytic site. In both studies, the introduction of proline residue improved the stability of the structure, thus prolonging the half-life of the protein.…”

Section: Discussionmentioning

confidence: 99%

Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase

et al. 2020

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Rational design is widely employed in protein engineering to tailor wild-type enzymes for industrial applications. The typical target region for mutation is a functional region like the catalytic site to improve stability and activity. However, few have explored the role of other regions which, in principle, have no evident functionality such as the N-terminal region. In this study, stability prediction software was used to identify the critical point in the non-functional N-terminal region of L2 lipase and the effects of the substitution towards temperature stability and activity were determined. The results showed 3 mutant lipases: A8V, A8P and A8E with 29% better thermostability, 4 h increase in half-life and 6.6 °C higher thermal denaturation point, respectively. A8V showed 1.6-fold enhancement in activity compared to wild-type. To conclude, the improvement in temperature stability upon substitution showed that the N-terminal region plays a role in temperature stability and activity of L2 lipase.

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“…For example: enantioselectivity (Engström et al 2010;K. Liebeton et al 2000a;Reetz et al 1997), stability in organic compounds (Dror et al 2014;Korman et al 2013), thermostability (Augustyniak et al 2012;Liu et al 2017;Yu et al 2012;Zhang et al 2003), substrate specificity (Fujii et al 2005).…”

Section: Sequence Analysismentioning

confidence: 99%

Evolving a lipase for hydrolysis of natural triglycerides along with enhanced tolerance towards a protease and surfactants

Alfaro-Chávez

Liu

Stevenson

et al. 2019

Protein Engineering, Design and Selection

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In the accompanying paper, we described evolving a lipase to the point where variants were soluble, stable and capable of degrading C8 TAG and C8 esters. These variants were tested for their ability to survive in an environment that might be encountered in a washing machine. Unfortunately, they were inactivated both by treatment with a protease used in laundry detergents and by very low concentrations of sodium dodecyl sulfate (SDS). In addition, all the variants had very low levels of activity with triglycerides with long aliphatic chains and with naturally occurring oils, like olive oil. Directed evolution was used to select variants with enhanced properties. In the first 10 rounds of evolution, the primary screen was selected for variants capable of hydrolyzing olive oil whereas the secondary screen was selected for enhanced tolerance towards a protease and SDS. In the final six rounds of evolution, the primary and secondary screens identified variants that retained activity after treatment with SDS. Sixteen cycles of evolution gave variants with greatly enhanced lipolytic activity on substrates that had both long (C16 and C18) as well as short (C3 and C8) chains. We found variants that were stable for more than 3 hours in protease concentrations that rapidly degrade the wild-type enzyme. Enhanced tolerance towards SDS was found in variants that could break down naturally occurring lipid and resist protease attack. The amino acid changes that gave enhanced properties were concentrated in the cap domain responsible for substrate binding.

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Improvement in thermostability of an alkaline lipase I from Penicillium cyclopium by directed evolution

Abstract: A novel alkaline-stable lipase I from Penicillium cyclopium with improved thermostability was prepared by molecular modification.

Cited by 23 publications

References 49 publications

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

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

Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase

Evolving a lipase for hydrolysis of natural triglycerides along with enhanced tolerance towards a protease and surfactants

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