Sequence‐based proline incorporation improves the thermostability of <i>Candida albicans</i> lipase Lip5

Yuan, Dongjuan; Zhao, Zhongyang; Wang, Xiumei; Guo, Shaohua; Yang, Bo; Wang, Yonghua

doi:10.1002/ejlt.201500273

Cited by 13 publications

(10 citation statements)

References 24 publications

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“…The t 1/2 , activation free energy (DDG), K m and k cat were calculated using methods described previously [43]. The kinetic constants were determined by pNP-C8 with different concentrations ranging from 0.05 to 5 mM.…”

Section: General Activity Assaymentioning

confidence: 99%

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Crystal structure of a lipase from Streptomyces sp. strain W007 – implications for thermostability and regiospecificity

et al. 2017

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Section: General Activity Assaymentioning

confidence: 99%

“…The kinetic constants were determined by pNP-C8 with different concentrations ranging from 0.05 to 5 mM. The t 1/2 , activation free energy (DDG), K m and k cat were calculated using methods described previously [43]. PRISM 5.0 (GraphPad Software Inc., La Jolla, CA, USA) was used to determine the best-fit value of all parameters.…”

Section: General Activity Assaymentioning

confidence: 99%

Crystal structure of a lipase from Streptomyces sp. strain W007 – implications for thermostability and regiospecificity

et al. 2017

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“…However, no activity was found after incubating for 3 h at 60 °C. The half-life ( t 1/2 ) of rGZEL was calculated according to the formula t 1/2 = ln2/ к ( к was defined as thermal inactivation rate constant) descried previously [ 12 ]. As shown in Figure 3 A, the t 1/2 value of rGZEL was 37.6 min at 60 °C.…”

Section: Resultsmentioning

confidence: 99%

Recombinant Lipase from Gibberella zeae Exhibits Broad Substrate Specificity: A Comparative Study on Emulsified and Monomolecular Substrate

Wang

Zhang

Zhao

et al. 2017

IJMS

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Using the classical emulsified system and the monomolecular film technique, the substrate specificity of recombinant Gibberella zeae lipase (rGZEL) that originates from Gibberella zeae was characterized in detail. Under the emulsified reaction system, both phospholipase and glycolipid hydrolytic activities were observed, except for the predominant lipase activity. The optimum conditions for different activity exhibition were also determined. Compared with its lipase activity, a little higher ratio of glycolipid hydrolytic activity (0.06) than phospholipase activity (0.02) was found. rGZEL preferred medium chain-length triglycerides, while lower activity was found for the longer-chain triglyceride. Using the monomolecular film technique, we found that the preference order of rGZEL to different phospholipids was 1,2-diacyl-sn-glycero-3-phospho-l-serine (PS) > 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (PG) > 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) > l-α-phosphatidylinositol (PI) > cardiolipin (CL) > 3-sn-phosphatidic acid sodium salt (PA) > l-α-phosphatidylethanolamine (PE), while no hydrolytic activity was detected for sphingomyelin (SM). Moreover, rGZEL showed higher galactolipase activity on 1,2-distearoyimonoglactosylglyceride (MGDG). A kinetic study on the stereo- and regioselectivity of rGZEL was also performed by using three pairs of pseudodiglyceride enantiomers (DDGs). rGZEL presented higher preference for distal DDG enantiomers than adjacent ester groups, however, no hydrolytic activity to the sn-2 position of diglyceride analogs was found. Furthermore, rGZEL preferred the R configuration of DDG enantiomers. Molecular docking results were in concordance with in vitro tests.

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“…Increasing the conformational stability of proteins is an important goal for both basic research and industrial applications . The changes in protein stability upon mutations using in silico approach can be estimated .…”

Section: Resultsmentioning

confidence: 99%

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

Tang

Lan

Yang

et al. 2016

Euro J Lipid Sci & Tech

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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.

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Sequence‐based proline incorporation improves the thermostability of Candida albicans lipase Lip5

Cited by 13 publications

References 24 publications

Crystal structure of a lipase from Streptomyces sp. strain W007 – implications for thermostability and regiospecificity

Crystal structure of a lipase from Streptomyces sp. strain W007 – implications for thermostability and regiospecificity

Recombinant Lipase from Gibberella zeae Exhibits Broad Substrate Specificity: A Comparative Study on Emulsified and Monomolecular Substrate

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

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