Kinetic studies of Gly28:Ser mutant form of Bacillus pumilus lipase: Changes in kcat and thermal dependence

Bustos‐Jaimes, Ismael; Mora-Lugo, Rodrigo; Calcagno, Mario L.; Farrés, Amelia

doi:10.1016/j.bbapap.2010.09.001

Cited by 24 publications

(8 citation statements)

References 32 publications

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“…BSLA belongs to the lipase subfamily I-4, contains 181 amino acids in its mature form and shares 74-80% sequence identity with other members of this subfamily (Arpigny and Jaeger 1999;Bustos-Jaimes et al 2010). An SSM library covering all natural diversity with a single amino acid exchange (named 'BSLA-SSM library') has been previously reported Fulton et al 2015).…”

Section: Introductionmentioning

confidence: 99%

Exploring the full natural diversity of single amino acid exchange reveals that 40–60% of BSLA positions improve organic solvents resistance

Frauenkron-Machedjou

Fulton

Zhao

et al. 2018

Bioresour. Bioprocess.

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Objectives: Protein engineering has been employed to successfully improve organic solvent resistance of enzymes. Exploration of nature's full potential (how many beneficial positions/beneficial substitutions of the target enzyme) to improve organic solvent resistance of enzymes by a systematic study was performed. Results:We report the results of screening the previously generated BSLA (Bacillus subtilis lipase A)-SSM (site saturation mutagenesis) library (covering the full natural diversity of BSLA with one amino acid exchange) in presence of three cosolvents. The potential of single amino acid substitution that nature offers to improve the cosolvent resistance of BSLA was determined by analyzing the number of beneficial positions/substitutions, accessibility and chemical compositions. Conclusion:Lessons learned from analysis of BSLA-SSM library are: (1) 41-59% of BSLA positions with in total 4-10% of all possible substitutions improve the cosolvent resistance against TFE, DOx, and DMSO; (2) charged substitutions are preferred to improve DOx and TFE resistance whereas polar ones are preferred for DMSO; (3) charged substitutions on the surface predominantly improved resistance while polar ones were preferred in buried "regions". (4) Interestingly, 58-93% of beneficial substitutions led to chemically different amino acids.

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Section: Introductionmentioning

confidence: 99%

Exploring the full natural diversity of single amino acid exchange reveals that 40–60% of BSLA positions improve organic solvents resistance

Frauenkron-Machedjou

Fulton

Zhao

et al. 2018

Bioresour. Bioprocess.

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“…Meanwhile, ARM lipase had k cat value of 16.7 sec -1 at 50 °C. BustosJaimes et al [36] reported that the changes in k cat value might be due to noticeable changes in K M . The highest K M value was 900 mM for ARM lipase at 50 °C.…”

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confidence: 99%

The Role of Arg157Ser in Improving the Compactness and Stability of ARM Lipase

Salleh¹

2012

JCSB

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Consensus approach is an efficient strategy to identify hot residue important for compactness and stability of protein. Structure of ARM lipase was modeled to explore the possible effect of critical point mutation towards structure and function. The significant difference of amino acid at position 157 between ARM lipase (Arg157) and other thermostable lipases (Ser157) was targeted as a critical residue. Using YASARA software, Arg157 was substituted to Ser and subsequently the energy minimized. Both ARM and R157S lipases were analyzed by MD simulations at different temperatures (50ºC,60ºC, and 70ºC). MD simulation result showed that R157S lipase had lower value of RMSD, RMSF, solvent accessible surface area (SASA) and radius of gyration than native ARM lipase. It indicated that R157S lipase had higher compactness in the structure leading to enhanced stability. To validate the computational data, the substitution of Arg157 to Ser has been conducted using site-directed mutagenesis experimentally. The catalytic efficiency (k cat /K M ) of R157S lipase was refold better than ARM lipase of 70°C. Circular dichorism study revealed that R157S lipase had increased thermostability with higher T m value (71.6°C) than its wildtype (63.9°C) indicating a better compactness as revealed by spectrofluorocence study. Thus the rational design of substituting Arg157 with Ser improved the protein folding of mutant lipase as shown in MD simulations and subsequently increased the catalytic effectiveness and thermodynamic stability. during evolution. The substitution of critical residue was analyzed using in silico to avoid unnecessary conformational distortion in engineered lipase and validate with kinetic study and thermodynamic study. Methods Preparation of 3D structures of ARM and R157S lipasesThe 3D structure of ARM lipase was predicted by comparative modeling. The first step in comparative modeling is the assignment of the unknown ARM lipase structure to a protein family with thousands of sequences already stored in protein databases (PDB). The structure of ARM lipase was modeled using available software packages such as HOMOLOGY and MODELLER, Accelrys, San Diego (http://www. accelrys.com). The unknown ARM lipase structure (target protein) was built from the known 3D structure of T1 lipase (2DSN) as a template. The final model of ARM lipase was evaluated using Ramachandran plot. Using YASARA, the three-dimensional structure of the mutant (R157S lipase) was modeled by substituting Arg157 to Ser and energy minimization was used to release bad contact. MD simulations of ARM and R157S lipasesMD simulations were performed on ARM lipase and R157S lipase structures using Amber03 force field in YASARA package. The periodic box was filled with explicit water molecules to a density of 0.98320 g/l. The whole system was neutralized by adding counter ions and all ionizable protein groups were protonated according to their tabulated pKa values at pH 7 of the medium. Water molecules were relaxed by simulated annealing procedure. Minimiza...

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“…The specific activity of B. pumilus B26 was reported to be 138 U/mg [12]. The k cat value of B. pumilus GMA1 was 1 s -1 [4]. In comparison, lipases BPL1 and BPL3 have higher activities than the most known Bacillus lipases.…”

Section: Purification Of Lipases Bpl1 and Bpl3mentioning

confidence: 99%

“…Among the known bacterial lipases, Bacillus lipases have been classified as subfamily I. 4. They are of interest from a research point of view and because of the industrial prowess.…”

Section: Introductionmentioning

confidence: 99%

Expression and Biochemical Characterization of Cold-Adapted Lipases from Antarctic Bacillus pumilus Strains

Litantra

Lobionda²,

Yim³

et al. 2013

J. Microbiol. Biotechnol.

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Two lipase genes (bpl1 and bpl3) from Antarctic Bacillus pumilus strains were expressed in Bacillus subtilis. Both recombinant lipases BPL1 and BPL2 were secreted to the culture medium and their activities reached 3.5 U/ml and 5.0 U/ml, respectively. Their molecular masses apparent using SDS-PAGE were 23 kDa for BPL1 and 19 kDa for BPL3. Both lipases were purified to homogeneity using ammonium sulfate precipitation and HiTrap SP FF column and Superose 12 column chromatographies. The final specific activities were estimated to be 328 U/mg for BPL1 and 310 U/mg for BPL3. Both lipases displayed an optimum temperature of 35°C, similar to other mesophilic enzymes. However, they maintained as much as 70% and 80% of the maximum activities at 10°C. Accordingly, their calculated activation energy at a temperature range of 10-35°C was 5.32 kcal/mol for BPL1 and 4.26 kcal/mol for BPL3, typical of cold-adapted enzymes. The optimum pH of BPL1 and BPL3 was 8.5 and 8.0, respectively, and they were quite stable at pH 7.0-11.0, showing their strong alkaline tolerance. Both lipases had a preference toward medium chain length (C6-C10) fatty acid substrates. These results indicate the potential for the two Antarctic B. pumilus lipases as catalysts in bioorganic synthesis, food, and detergent industries.

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Kinetic studies of Gly28:Ser mutant form of Bacillus pumilus lipase: Changes in kcat and thermal dependence

Cited by 24 publications

References 32 publications

Exploring the full natural diversity of single amino acid exchange reveals that 40–60% of BSLA positions improve organic solvents resistance

Exploring the full natural diversity of single amino acid exchange reveals that 40–60% of BSLA positions improve organic solvents resistance

The Role of Arg157Ser in Improving the Compactness and Stability of ARM Lipase

Expression and Biochemical Characterization of Cold-Adapted Lipases from Antarctic Bacillus pumilus Strains

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