Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability

Veno, Jiivittha; Leow, Adam Thean Chor; Ali, Mohd Shukuri Mohamad; Masomian, Malihe; Rahman, Raja Noor Zaliha Raja Abd

doi:10.3390/ijms18112202

Cited by 16 publications

(16 citation statements)

References 41 publications

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“…Therefore, a reliable and effective screening system is needed to find the rare variants with desired properties [ 20 , 21 ]. To date, there have been reported successes in improving the activity, specificity, or tolerance to extremes of pH or temperature of enzymes by directed evolution [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. For example, endo-β-1, 4-xylanase mutants with improved catalytic efficiencies were obtained through using error-prone PCR and a high-throughput screening system.…”

Section: Introductionmentioning

confidence: 99%

Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution

Chen

Liang

et al. 2017

Molecules

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The glycoside hydrolase LXYL-P1–2 from Lentinula edodes can specifically hydrolyze 7-β-xylosyltaxanes to form 7-β-hydroxyltaxanes for the semi-synthesis of paclitaxel. In order to improve the catalytic properties of the enzyme, we performed error-prone PCR to construct the random mutant library of LXYL-P1–2 and used the methanol-induced plate method to screen the mutants with improved catalytic properties. Two variants, LXYL-P1–2-EP1 (EP1, S91D mutation) and LXYL-P1–2-EP2 (EP2, T368E mutation), were obtained from the library and exhibited 17% and 47% increases in their catalytic efficiencies on 7-β-xylosyl-10-deacetyltaxol. Meanwhile, compared with LXYL-P1–2, EP1 and EP2 showed elevated stabilities in the range of pH ≥ 6 conditions. After treatment at pH 12 for 48 h, EP1 and EP2 retained 77% and 63% activities, respectively, while the wild-type only retained 33% activity under the same condition. Molecular docking results revealed that the S91D mutation led to a shorter distance between the R-chain and the substrate, while the T368E mutation increased negative charge at the surface of the enzyme, and may introduce alterations of the loop near the active pocket, both of which may result in improved stabilities and catalytic activities of enzymes. This study provides a practical directed evolution method for exploring catalytically improved glycoside hydrolase.

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Section: Introductionmentioning

confidence: 99%

Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution

Chen

Liang

et al. 2017

Molecules

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“…In earlier studies, the C-terminal truncated lipase from S. epidemidis AT2 (rT-M386) was mutated using error-prone PCR. From the resultant mutant library, one mutant with a single point mutation (G210C) showed more than 85% (401 U/mg) stability at 50 °C, shifting the temperature profile of this cold-adapted lipase [12].…”

Section: Resultsmentioning

confidence: 99%

“…Previously, mutant G210C from S. epidermidis AT2 lipase was laboratory evolved and demonstrated a significant improvement in its thermostability profile [12]. The cold-adapted enzyme, which is initially active at 25 °C shows about 85% stability at 50 °C (while retaining its activity at 25 °C) after a random introduction of a cysteine at residue 210 in replacement of glycine.…”

Section: Introductionmentioning

confidence: 99%

Insight into Improved Thermostability of Cold-Adapted Staphylococcal Lipase by Glycine to Cysteine Mutation

et al. 2019

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Thermostability remains one of the most desirable traits in many lipases. Numerous studies have revealed promising strategies to improve thermostability and random mutagenesis often leads to unexpected yet interesting findings in engineering stability. Previously, the thermostability of C-terminal truncated cold-adapted lipase from Staphylococcus epidermidis AT2 (rT-M386) was markedly enhanced by directed evolution. The newly evolved mutant, G210C, demonstrated an optimal temperature shift from 25 to 45 °C and stability up to 50 °C. Interestingly, a cysteine residue was randomly introduced on the loop connecting the two lids and accounted for the only cysteine found in the lipase. We further investigated the structural and mechanistic insights that could possibly cause the significant temperature shift. Both rT-M386 and G210C were modeled and simulated at 25 °C and 50 °C. The results clearly portrayed the effect of cysteine substitution primarily on the lid stability. Comparative molecular dynamics simulation analysis revealed that G210C exhibited greater stability than the wild-type at high temperature simulation. The compactness of the G210C lipase structure increased at 50 °C and resulted in enhanced rigidity hence stability. This observation is supported by the improved and stronger non-covalent interactions formed in the protein structure. Our findings suggest that the introduction of a single cysteine residue at the lid region of cold-adapted lipase may result in unexpected increased in thermostability, thus this approach could serve as one of the thermostabilization strategies in engineering lipase stability.

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“…The mutation of S141G in Pseudomonas fragi lipase also had its rigidity relinquished, causing the structure to destabilize [39]. Therefore, amino acid substitution could successfully increase protein thermostability and has been reported by Veno et al [40]. Through amino acid substitution of Gly210Cys in lipase derived from Staphylococcus epidermidis, the thermostability has been increased.…”

Section: Discussionmentioning

confidence: 99%

Changes of Thermostability, Organic Solvent, and pH Stability in Geobacillus zalihae HT1 and Its Mutant by Calcium Ion

Ishak

Masomian

Leow

et al. 2019

IJMS

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Thermostable T1 lipase from Geobacillus zalihae has been crystallized using counter-diffusion method under space and Earth conditions. The comparison of the three-dimensional structures from both crystallized proteins show differences in the formation of hydrogen bond and ion interactions. Hydrogen bond and ion interaction are important in the stabilization of protein structure towards extreme temperature and organic solvents. In this study, the differences of hydrogen bond interactions at position Asp43, Thr118, Glu250, and Asn304 and ion interaction at position Glu226 was chosen to imitate space-grown crystal structure, and the impact of these combined interactions in T1 lipase-mutated structure was studied. Using space-grown T1 lipase structure as a reference, subsequent simultaneous mutation D43E, T118N, E226D, E250L, and N304E was performed on recombinant wild-type T1 lipase (wt-HT1) to generate a quintuple mutant term as 5M mutant lipase. This mutant lipase shared similar characteristics to its wild-type in terms of optimal pH and temperature. The stability of mutant 5M lipase improved significantly in acidic and alkaline pH as compared to wt-HT1. 5M lipase was highly stable in organic solvents such as dimethyl sulfoxide (DMSO), methanol, and n-hexane compared to wt-HT1. Both wild-type and mutant lipases were found highly activated in calcium as compared to other metal ions due to the presence of calcium-binding site for thermostability. The presence of calcium prolonged the half-life of mutant 5M and wt-HT1, and at the same time increased their melting temperature (Tm). The melting temperature of 5M and wt-HT1 lipases increased at 8.4 and 12.1 °C, respectively, in the presence of calcium as compared to those without. Calcium enhanced the stability of mutant 5M in 25% (v/v) DMSO, n-hexane, and n-heptane. The lipase activity of wt-HT1 also increased in 25% (v/v) ethanol, methanol, acetonitrile, n-hexane, and n-heptane in the presence of calcium. The current study showed that the accumulation of amino acid substitutions D43E, T118N, E226D, E250L, and N304E produced highly stable T1 mutant when hydrolyzing oil in selected organic solvents such as DMSO, n-hexane, and n-heptane. It is also believed that calcium ion plays important role in regulating lipase thermostability.

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Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability

Cited by 16 publications

References 41 publications

Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution

Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution

Insight into Improved Thermostability of Cold-Adapted Staphylococcal Lipase by Glycine to Cysteine Mutation

Changes of Thermostability, Organic Solvent, and pH Stability in Geobacillus zalihae HT1 and Its Mutant by Calcium Ion

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