Enantioselectivity and catalysis improvements of Pseudomonas cepacia lipase with Tyr and Asp modification

Li, Jing; Yue, Lei; Li, Chang; Pan, Yuanjiang; Yang, Lirong

doi:10.1039/c5cy00110b

Cited by 10 publications

(9 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The T9D mutation preferred glyceryl trioctanoate (C8) among these three triglycerides. Until now, most studies have investigated the functions of the lid and pocket regions and have not elucidated the propeptide functions, such as substrate specificity. With the improved mutants designed on the propeptide, we believe that the regulation of the propeptide to shift the substrate specificity will have great potential application in the future.…”

Section: Resultsmentioning

confidence: 99%

Propeptide in Rhizopus chinensis Lipase: New Insights into Its Mechanism of Activity and Substrate Selectivity by Computational Design

Wang

2021

J. Agric. Food Chem.

View full text Add to dashboard Cite

Most fungal lipases contain a propeptide, which is very important for their function and substrate selectivity. In the present study, Rhizopus chinensis lipase (RCL) was used as a research model to explore the mechanism of the propeptide of the lipase. Conventional molecular dynamics (MD) and metadynamics simulations were used to explore the mechanism by which the propeptide affects the activity of the lipase, which was subsequently verified by mutation experiments. MD simulations indicated that the propeptide had an inhibitory effect on the lid movement of RCL and found a key region (Val5-Thr10) on the propeptide. Subsequently, site-directed mutations were created in this region. The mutations enhanced the lipase catalytic efficiency to 700% and showed the potential for the propeptide to shift the substrate specificity of RCL. The specificity and activity of RCL mutants also had similar trends to wild-type RCL toward triglycerides with varying chain lengths. The mutual corroboration of simulation and sitedirected mutagenesis results revealed the vital role of the key propeptide region in the catalytic activity and substrate specificity of the lipase.

show abstract

Section: Resultsmentioning

confidence: 99%

Propeptide in Rhizopus chinensis Lipase: New Insights into Its Mechanism of Activity and Substrate Selectivity by Computational Design

Wang

2021

J. Agric. Food Chem.

View full text Add to dashboard Cite

show abstract

“…The homology results reveal the remarkable research significance of the novel recombinant enzyme including enzyme characterizations. The LipPS1 was stable at pH 6.0-10.0 compared with that of Pseudomonas aeruginosa SL-72 (pH 7.0-8.0) (Verma et al 2012), Pseudomonas fluorescens (pH 7.0) (Panizza et al 2013), Pseudomonas cepacia (pH 7.0) (Li et al 2015) Pseudomonas gessardii (pH 5.0) (Ramani et al 2010) and Pseudomonas stutzeri PS59 (pH 8.5) (Li et al 2014). The optimum temperature (40°C) of the recombinant lipase is similar to lipase from P. gessardii, P. fluorescens, P. fragi and P. mendoncina, which were found to be optimally active within 35-45°C (Ramani and Sekaran 2012).…”

Section: Discussionmentioning

confidence: 95%

Biodegradation of waste greases and biochemical properties of a novel lipase from Pseudomonas synxantha PS1

et al. 2016

View full text Add to dashboard Cite

A lipase-producing bacterial strain was isolated from oil-well-produced water in Shengli oilfield (Shandong province, China) and was identified as Pseudomonas synxantha by 16S rDNA sequence analysis (named Pseudomonas synxantha PS1). Strain PS1 showed a maximum lipase activity of 10.8 U/mL after culturing for 48 h at 30 °C, with lactose (4 g/L) as carbon source, tryptone (8 g/L) as nitrogen source, olive oil (0.5%, v/v) as inductor, and the initial pH 8.0. Meanwhile, the lipase gene from P. synxantha PS1 was cloned and expressed in Escherichia coli BL21 with the vector pET28a. The novel gene (lipPS1) has an open reading frame of 1425 bp and encodes a 474 aa lipase (LipPS1) sharing the most identity (87%) with the lipase in Pseudomonas fluorescens. LipPS1 preferably acted on substrates with a long chain (C10-C18) of fatty acids. The optimum pH and temperature of the recombinant enzyme were 8.0 and 40 °C, respectively, towards the optimum substrate p-nitrophenyl palmitate. The LipPS1 showed remarkable stability under alkaline conditions and was stable at pH 7.0-10.0 (retaining more than 60% activity). From the organic solvents tests, the lipase was activated by 15% (v/v) methanol (112%), 15% ethanol (127%), and 15% n-butyl alcohol (116%). LipPS1 presented strong biodegradability of waste grease; 93% of waste grease was hydrolyzed into fatty acid after 12 h at 30 °C. This is the first report of the lipase activity and lipase gene obtained from P. synxantha (including wild strain and recombinant strain) and of the recombinant LipPS1 with the detailed enzymatic properties. Also a preliminary study of the biodegradability of waste greases shows the potential value in industry applications.

show abstract

“…Modification of the Tyr residues from Pseudomonas cepacian lipase (PCL) using I 3 − and N-acetyl imidazole in the absence or presence of 10% v/v n-hexane has been demonstrated to improve enantioselectivity toward secondary alcohols. 193 I 3 − reagents mediate the replacement of the ortho-hydrogen atom while N-acetyl imidazole reacts with the hydroxyl group. These initial reactions allow for a rearrangement that allows for the hydrogen atom to be substituted by acetyl selectively.…”

Section: Tyrosinementioning

confidence: 99%

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

et al. 2021

View full text Add to dashboard Cite

The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chemical diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chemical modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and critical evaluation of the applications, recent advances, and technical breakthroughs in biocatalysis for three approaches: (i) chemical modification of cAAs, (ii) incorporation of ncAAs, and (iii) chemical modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.

show abstract

Enantioselectivity and catalysis improvements of Pseudomonas cepacia lipase with Tyr and Asp modification

Cited by 10 publications

References 47 publications

Propeptide in Rhizopus chinensis Lipase: New Insights into Its Mechanism of Activity and Substrate Selectivity by Computational Design

Propeptide in Rhizopus chinensis Lipase: New Insights into Its Mechanism of Activity and Substrate Selectivity by Computational Design

Biodegradation of waste greases and biochemical properties of a novel lipase from Pseudomonas synxantha PS1

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

Contact Info

Product

Resources

About