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

Frauenkron-Machedjou, Victorine Josiane; Fulton, Alexander; Zhao, Jing; Weber, Lina; Jaeger, Karl‐Erich; Schwaneberg, Ulrich; Zhu, Leilei

doi:10.1186/s40643-017-0188-y

Cited by 28 publications

(43 citation statements)

References 45 publications

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“…Chemically competent Escherichia coli DH5a and Escherichia coli BL21‐Gold (DE3) (Agilent Technologies; Santa Clara, USA) were used as hosts for plasmids amplification and protein expression, respectively. The plasmid pET22b(+)‐bsla wild type was constructed in the previous work . A detailed description of the “BSLA‐SSM” library generation and the activity assay with pNPB in 96‐well MTP was reported in our previous studies .…”

Section: Computational and Experimental Sectionmentioning

confidence: 99%

“…Exploring the effects of all interactions possibilities offered by nature is likely a prerequisite to discover general design principles and thereby solve the enzyme‐OSs interaction puzzle. Such an SSM library has been reported for BSLA in our previous work, termed as “BSLA‐SSM” library (181 positions, in total 3440 variants) . The “BSLA‐SSM” library screened towards improved resistance in three OSs (22 % (v/v) 1,4‐dioxane (DOX), 60 % (v/v) DMSO, 12 % (v/v) 2,2,2‐trifluoroethanol (TFE)) .…”

Section: Introductionmentioning

confidence: 99%

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How to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study

et al. 2020

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Expanding synthetic capabilities to routinely employ enzymes in organic solvents (OSs) is a dream for protein engineers and synthetic chemists. Despite significant advances in the field of protein engineering, general and transferable design principles to improve the OS resistance of enzymes are poorly understood. Herein, we report a combined computational and directed evolution study of Bacillus subtlis lipase A (BSLA) in three OSs (i. e., 1,4‐dioxane, dimethyl sulfoxide, 2,2,2‐trifluoroethanol) to devise a rational strategy to guide engineering OS resistant enzymes. Molecular dynamics simulations showed that OSs reduce BSLA activity and resistance in OSs by (i) stripping off essential water molecules from the BLSA surface mainly through H‐bonds binding; and (ii) penetrating the substrate binding cleft leading to inhibition and conformational change. Interestingly, integration of computational results with “BSLA‐SSM” variant library (3439 variants; all natural diversity with amino acid exchange) revealed two complementary rational design strategies: (i) surface charge engineering, and (ii) substrate binding cleft engineering. These strategies are most likely applicable to stabilize other lipases and enzymes and assist experimentalists to design organic solvent resistant enzymes with reduced time and screening effort in lab experiments.

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Section: Computational and Experimental Sectionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

How to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study

et al. 2020

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“…Conformational changes in the structure of the enzyme are the main reason for deactivation by organic solvents, due to an impaired balance of hydrophilic-hydrophobic interactions. Moreover, polar (hydrophilic) solvents can penetrate the hydrophilic core of the enzyme, affecting secondary and tertiary conformational changes while stripping structured water molecules from the protein hydration shell (2,(8)(9)(10)(11)(12)(13)(14)(15).…”

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confidence: 99%

“…Protein engineering methods include (i) random mutagenesis, (ii) rational design, and (iii) semirational design (2,8,(18)(19)(20). It was previously shown that these approaches can be applied separately or in combination to tailor enzymes to enhance their stability in organic solvents (1,9,12,18,21,22).…”

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Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol

Gihaz

Kanteev

Pazy

et al. 2018

Appl Environ Microbiol

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Enhanced stability in organic solvents is a desirable feature for enzymes implemented under industrial conditions. Lipases potential as biocatalysts is mainly limited by denaturation in polar alcohols. In this study we focused on selected solvent tunnels in lipase from T6 to improve its stability in methanol during biodiesel synthesis. Using rational mutagenesis, bulky aromatic residues were incorporated in order to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. Each solvent tunnel was systematically analyzed with respect to its chemical and structural characteristics. Selected residues were replaced with Phe, Tyr or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed elevated stability up to 81-fold compared with wild-type. All stabilizing mutations were found in the longest tunnel detected in the "closed-lid" x-ray structure. Combining the Phe substitutions created double mutant A187F/L360F with an increase in T of +7°C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. Kinetic analysis with -nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates ( ), though mostly their stability properties determined the transesterification capability. Seven crystal structures of different variants were solved disclosing new π-π or CH/π intramolecular interactions emphasizing the significance of aromatic interactions to improved solvent stability. This rational approach could be implemented in other enzymes for stabilization in organic solvents. Enzymatic synthesis in organic solvents holds increasing industrial opportunities in many fields, however, one major obstacle is the limited stability of biocatalysts in such a denaturing environment. Aromatic interactions play a major role in protein folding and stability and we were inspired by this to re-design enzyme voids. Rational protein engineering of solvent tunnels of lipase from is presented here, offering a promising approach to introduce new aromatic interactions within the enzyme core. We discovered that longer tunnels leading from the surface to the enzyme active site were more beneficial targets for mutagenesis for improving lipase stability in methanol during biodiesel biosynthesis. Structural analysis of the variants confirmed the generation of new interactions involving aromatic residues. This work provides insights into stability-driven enzyme design by targeting solvent channels void.

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“…The substrate range clearly qualifies BSLA as a lipase, as does the stability in the presence of solvents [9,[26][27][28][29]. This stability has even been significantly improved in recent mutational studies and BSLA mutants can be very stable in the presence of water-miscible solvents, such as dimethyl sulfoxide (DMSO), dioxane and trifluoroethanol [30,31]. Studies on BSLA in dry organic solvents are, however, missing.…”

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confidence: 99%

Bacillus subtilis Lipase A—Lipase or Esterase?

et al. 2020

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The question of how to distinguish between lipases and esterases is about as old as the definition of the subclassification is. Many different criteria have been proposed to this end, all indicative but not decisive. Here, the activity of lipases in dry organic solvents as a criterion is probed on a minimal α/β hydrolase fold enzyme, the Bacillus subtilis lipase A (BSLA), and compared to Candida antarctica lipase B (CALB), a proven lipase. Both hydrolases show activity in dry solvents and this proves BSLA to be a lipase. Overall, this demonstrates the value of this additional parameter to distinguish between lipases and esterases. Lipases tend to be active in dry organic solvents, while esterases are not active under these circumstances.Catalysts 2020, 10, 308 2 of 17 lipase A (BSLA) [9]. BSLA is a small (181 amino acids, 19 kDa) serine hydrolase ( Figure 1). It is neither interfacially activated nor does it have a lid (criteria one and two) [9,[22][23][24] and sequence data are not conclusive, but it is a minimal α/β hydrolase fold enzyme [9,23,25]. The substrate range clearly qualifies BSLA as a lipase, as does the stability in the presence of solvents [9,[26][27][28][29]. This stability has even been significantly improved in recent mutational studies and BSLA mutants can be very stable in the presence of water-miscible solvents, such as dimethyl sulfoxide (DMSO), dioxane and trifluoroethanol [30,31]. Studies on BSLA in dry organic solvents are, however, missing. As an experimental parameter, we demonstrate the activity of BSLA in dry toluene. Toluene is not water-miscible and has a logP of 2.5 [32]. It is commonly used in organic synthesis and is highly suitable for lipases and also other enzymes with an α/β hydrolase fold. To date, only lipases were shown to be active in toluene with a very low a w [1,3].Catalysts 2019, 9, x FOR PEER REVIEW 2 of 17 interfacially activated nor does it have a lid (criteria one and two) [9,[22][23][24] and sequence data are not conclusive, but it is a minimal α/β hydrolase fold enzyme [9,23,25]. The substrate range clearly qualifies BSLA as a lipase, as does the stability in the presence of solvents [9,[26][27][28][29]. This stability has even been significantly improved in recent mutational studies and BSLA mutants can be very stable in the presence of water-miscible solvents, such as dimethyl sulfoxide (DMSO), dioxane and trifluoroethanol [30,31]. Studies on BSLA in dry organic solvents are, however, missing. As an experimental parameter, we demonstrate the activity of BSLA in dry toluene. Toluene is not watermiscible and has a logP of 2.5 [32]. It is commonly used in organic synthesis and is highly suitable for lipases and also other enzymes with an α/β hydrolase fold. To date, only lipases were shown to be active in toluene with a very low aw [1,3].

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

Cited by 28 publications

References 45 publications

How to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study

How to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol

Bacillus subtilis Lipase A—Lipase or Esterase?

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