Organic Solvent Tolerance Research Articles

Characterization and Application of a Novel Glucose Dehydrogenase with Excellent Organic Solvent Tolerance for Cofactor Regeneration in Carbonyl Reduction.

An efficient cofactor regeneration system has been developed to provide a hydride source for the preparation of optically pure alcohols by carbonyl reductase-catalyzed asymmetric reduction. This system employed a novel glucose dehydrogenase (BcGDH90) from Bacillus cereus HBL-AI. The gene encoding BcGDH90 was found through the genome-wide functional annotation. Homology-built model study revealed that BcGDH90 was a homo-tetramer, and each subunit was composed of βD-αE-αF-αG-βG motif, which was responsible for substrate binding and tetramer formation. The gene of BcGDH90 was cloned and expressed in Escherichia coli. The recombinant BcGDH90 exhibited maximum activity of 45.3 U/mg at pH 9.0 and 40°C. BcGDH90 showed high stability in a wide pH range of 4.0-10.0 and was stable after the incubation at 55°C for 5h. BcGDH90 was not a metal ion-dependent enzyme, but Zn2+ could seriously inhibit its activity. BcGDH90 displayed excellent tolerance to 90% of acetone, methanol, ethanol, n-propanol, and isopropanol. Furthermore, BcGDH90 was applied to regenerate NADPH for the asymmetric biosynthesis of (S)-(+)-1-phenyl-1,2-ethanediol ((S)-PED) from hydroxyacetophenone (2-HAP) with high concentration, which increased the final efficiency by 59.4%. These results suggest that BcGDH90 is potentially useful for coenzyme regeneration in the biological reduction.

Purification and characterization of lipase enzyme from endophytic Bacillus pumilus WSS5 for application in detergent industry

Lipolytic enzymes from endophytic microbes have been the focus of intense and growing research in view of the applicability of such enzymes in various industrial applications and endophytic microorganisms may emerge as a potential source of lipases with distinct characteristics. The current work involved purification and characterization of the lipase from an endophytic-bacteria isolated from the stem of Withania somnifera L. Dunal. Based on the analysis of morphology and 16S rDNA sequence, the endophytic bacteria was identified as Bacillus pumilus and designated as B. pumilus WSS5. Purified lipase from the B. pumilus WSS5 was found to be of 27KDa as revealed by SDS-PAGE with optimum activity at 37 °C and pH 8. The stability of the enzyme in presence of various metal ions, surfactants, inhibitors, and organic solvents suggested that this enzyme could be exploited in leather and detergent industries. In addition, the stain removal potential of the crude and purified lipase was determined on cotton fabric pieces stained with vegetable oil. Lipase added to water along with detergent significantly enhanced the stain removal efficiency of a commercially available detergent. The current findings suggest the potential of B. pumilusWSS5 lipase owing to promising properties such as the alkali-thermostability, organic solvent-tolerance, and broad substrate specificity that makes it a strong candidate for application in detergent formulations, biotransformation, industries, and medicine. To our knowledge, this is the first report on the purification and characterization of lipase from bacterial endophyte isolated from the stem of Withania somnifera L. Dunal.

Adenylate Kinase Fused to Spidroin as a Catalyst for Decreasing Leakage out of 3D-Bioprinted Hydrogels and for ATP Regeneration.

Numerous metabolic reactions and pathways use adenosine 5'-triphosphate (ATP) as an energy source and as a phosphorous or pyrophosphorous donor. Based on three-dimensional (3D)-printing, enzyme immobilization can be used to improve ATP regeneration and operability and reduce cost. However, due to the relatively large mesh size of 3D-bioprinted hydrogels soaked in a reaction solution, the lower-molecular-weight enzymes cannot avoid leaking out of the hydrogels readily. Here, a chimeric adenylate-kinase-spidroin (ADK-RC) is created, with ADK serving as the N-terminal domain. The chimera is capable of self-assembling to form micellar nanoparticles at a higher molecular scale. Although fused to spidroin (RC), ADK-RC remains relatively consistent and exhibits high activity, thermostability, pH stability, and organic solvent tolerance. Considering different surface-to-volume ratios, three shapes of enzyme hydrogels are designed, 3D bioprinted, and measured. In addition, a continuous enzymatic reaction demonstrates that ADK-RC hydrogels have higher specific activity and substrate affinity but a lower reaction rate and catalytic power compared to free enzymes in solution. With ATP regeneration, the ADK and ADK-RC hydrogels significantly increase the production of d-glucose-6-phosphate and obtain an efficient usage frequency. In conclusion, enzymes fused to spidroin might be an efficient strategy for maintaining activity and reducing leakage in 3D-bioprinted hydrogels under mild conditions.

Role of N-Terminal Extensional Long α-Helix in the Arylesterase from Lacticaseibacillus rhamnosus GG on Catalysis and Stability

In the α/β hydrolases superfamily, the extra module modulated enzymatic activity, substrate specificity, and stability. The functional role of N-terminal extensional long α-helix (Ala2-Glu29, designated as NEL-helix) acting as the extra module in the arylesterase LggEst from Lacticaseibacillus rhamnosus GG had been systemically investigated by deletion mutagenesis, biochemical characterization, and biophysical methods. The deletion of the NEL-helix did not change the overall structure of this arylesterase. The deletion of the NEL-helix led to the shifting of optimal pH into the acidity and the loss of thermophilic activity. The deletion of the NEL-helix produced a 10.6-fold drop in catalytic activity towards the best substrate pNPC10. NEL-Helix was crucial for the thermostability, chemical resistance, and organic solvents tolerance. The deletion of the NEL-helix did not change the overall rigidity of enzyme structure and only reduced the local rigidity of the active site. Sodium deoxycholate might partially replenish the loss of activity caused by the deletion of the NEL-helix. Our research further enriched the functional role of the extra module on catalysis and stability in the α/β hydrolase fold superfamily.

A novel aerobic denitrifying phosphate-accumulating bacterium efficiently removes phthalic acid ester, total nitrogen and phosphate from municipal wastewater

Simultaneous removal of nitrogen, phosphate and emerging pollutants are critical for safe reuse of wastewater, but research in this field is limited. In the present study, a novel aerobic denitrifying phosphate-accumulating bacterial strain RL-GZ01 was found to be able to utilize phthalic acid esters (PAEs) as carbon resource for cell growth. Based on 16S rRNA gene analysis, physiological and biochemical characterization, and genome-based average nucleotide identity calculation, RL-GZ01 was identified as Rhodococcus pyridinivorans. Strain RL-GZ01 showed high DEHP degradation in alkaline conditions and good tolerance of salinity and organic solvents. The degradation of DEHP by RL-GZ01 fitted well with a modified Gompertz model (R2 = 0.9985). Metabolic intermediates of DEHP were identified via UHPLC-MS/MS analysis and the catabolic pathway was proposed thereafter. Genes and gene clusters contributed to the utilization of DEHP were analyzed through genomic analysis. Analysis of KEGG nitrogen metabolism pathway indicated that nitrate and nitrite were further transformed into ammonium which was further used for the biosynthesis of l-glutamine and L-glutamate. Strain RL-GZ01 was further identified as a denitrifying phosphate accumulating organism which can accumulate phosphate by generating polyphosphate. Finally, strain RL-GZ01 was applied to municipal wastewater treatment for simultaneous removal of nitrogen, phosphate and DEHP. The removal percentages of DEHP (5 mg/L), TN (71.2 mg/L), NH4+-N (70.9 mg/L), PO43−-P (10.89 mg/L) and COD (622.4 mg/L) by strain RL-GZ01 were 89.94 %, 64.45 %, 64.94 %, 76.30 % and 63.23 % within 84 h, respectively. These demonstrated the capability of strain RL-GZ01 for the biological treatment of wastewater containing PAEs.

Engineered living hydrogels enable efficient biocatalysis beyond just buffers

In the October issue of <i>Cell Reports Physical Science</i>, Gao and coworkers demonstrate smart engineered living hydrogels as a versatile platform for robust and tunable biocatalysis with improved tolerance of pure organic solvents.

An Overview of the Role of Membrane Proteins in Microbial Solvents Tolerance.

Solvent tolerance is a desired feature of microorganisms for their application in biotechnology. Organic solvent-tolerant microorganisms are able to thrive in the presence of organic solvents. Several mechanisms have been proposed to elucidate their intrinsic tolerance to organic solvents. The present review aims to summarize the state of the art of the roles of membrane proteins in microbial organic solvent tolerance. Strategies and challenges for improving the protective function of membrane proteins in organic solvent stress are also proposed. Membrane proteins related to transporter, signal transduction, and material and energy metabolism are involved in solvent tolerance. Optimization of the expression level of membrane proteins and engineering of membrane proteins are utilized to tackle the toxicity caused by organic solvents. Membrane proteins occupy a strikingly important position in microbial solvent tolerance. Further research on novel methods in membrane proteins, trade-offs among overexpression and toxicity of membrane proteins and solvent yield, and a direct relationship between signaling pathways and solvent tolerance will advance the utilization of organic solvent-tolerant microorganisms in biotechnology.

Isolation of atrazine nanobodies enhanced by depletion of anti-carrier protein phages and performance comparison between the nanobody and monoclonal antibody derived from the same immunogen

Nanobody, a single domain antibody, has been shown a great promise for immunoassay (IA) applications. To improve the panning efficiency so as to obtain a valuable nanobody, anti-carrier protein phages in a phage display library were depleted to enhance the selection of nanobodies against the herbicide atrazine by using immunomagnetic beads conjugated with bovine serum albumin (IMB-BSA). The depletion of anti-carrier protein phages from the atrazine phage display library tripled the number of atrazine positive phage clones after four rounds of panning. One of the most sensitive phage clones Nb3 selected from the IMB-BSA depleted library was used to compare the performance with the monoclonal antibody (mAb 5D9) developed from the same immunogen. The Nb3-based IA exhibited similar specificity with the mAb 5D9-based IA, but greater thermostability and organic solvent tolerance. The half-maximum inhibition concentration (IC50) of the former was 3.5-fold greater than that of the latter (36.7 ng/mL versus 10.2 ng/mL). Because the Nb3-based IA was more robust than the mAb 5D9-based IA, the method detection limit of the two assays was 7.8 ng/mL of atrazine in river samples. The depletion strategy can increase the chance to acquire high quality nanobody and can be applicable for effective development of nanobodies against other small molecules.

Specific and efficient hydrolysis of all outer glucosyls in protopanaxadiol type and protopanaxatriol type ginsenosides by a β-glucosidase from Thermoclostridium stercorarium

To develop a new method for enzymatic preparation of minor ginsenosides, T. stercorarium β-glucosidase (Tsbgl) was characterized and its activities of deglycosylation towards natural ginsenosides were examined. The substrates of 1 mmol l−1 were incubated with the enzyme of 38.3 U ml−1 at 65 ℃ and pH 5.0. The Km values of Tsbgl for ginsenosides Rb1, Rg1 and pNPG were 0.37 ± 0.03, 3.26 ± 0.19, and 1.24 ± 0.03 mmol l−1, and the Vmax values were 183.63 ± 7.15, 85.03 ± 4.90, and 117.66 ± 1.96 μmol mg−1 min−1, respectively. The molar conversion of ginsenosides Rb1, Rb2, Rb3, Rc, Re, Rg1, and Rf by Tsbgl within 6 h was 100%, 50.1%, 42.7%, 92.0%, 57.3%, 67.9%, and 76.8%, respectively. The yield of aglycone protopanaxadiol was 35.5 μmol l−1 h−1 for Rb1, while the yields of aglycone protopanaxatriol were 64.2 and 70.4 μmol l−1 h−1 for Rg1 and Rf. Tsbgl with good organic solvent tolerance, mild reaction conditions and broad substrate specificity, could completely remove all outer glucosyls at the C-3 and C-20 hydroxyls of protopanaxadiol-type ginsenosides, and the C-6 and C-20 hydroxyls of protopanaxatriol-type ginsenosides through various pathways, providing a specific and efficient way to produce minor ginsenosides.

An MnO2-ZIF-67 immobilized acetylcholinesterase method for acetylcholinesterase activity assay and inhibitor screening from Inula macrophylla based on capillary electrophoresis

The inhibition of acetylcholinesterase (AChE) from decomposing acetylcholine (ACh) is regarded as a common therapeutic strategy for Alzheimer's disease (AD). Screening AChE inhibitors is of great significance for AD treatment. In this work, AChE was physically absorbed on the synthesized MnO2-ZIF-67 through electrostatic interaction for the first time, and the fabricated MnO2-ZIF-67 immobilized AChE coupled with capillary electrophoresis (CE) was applied to screening AChE inhibitor. The established MnO2-ZIF-67 immobilized AChE with excellent pH, thermal and organic solvents tolerance stability was evaluated by Ellman's method. Huperzine A was used as model inhibitor to investigate the inhibition kinetics of the immobilized AChE, which possess the feasibility and applicability for its inhibitor screening. The screening mechanism of AChE inhibitor was based on the Ellman's method and measured by the changes of 5-thio-2-nitrobenzoic acid (TNB), which was produced by the reaction of thiocholine (TCh) with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB). Benefiting from the advantages of convenience and high sensitivity, the proposed platform was employed to screen AChE inhibitors from 11 compounds isolated from Inula macrophylla, and macrophyllic acid A (compound 1), dibutyl phthalate (compound 2) exhibited strong AChE inhibitory activity. Thus, this work not only provides a promising platform for monitoring AChE activity, but also holds great potential for the further exploration of active components with AChE inhibitory activity from Inula macrophylla.

Molecular characterization of transesterification activity of novel lipase family I.1.

Lipase’s thermostability and organic solvent tolerance are two crucial properties that enable it to function as a biocatalyst. The present study examined the characteristics of two recombinant thermostable lipases (Lk2, Lk3) based on transesterification activity. Conversion of C12-C18 methyl ester with paranitrophenol was investigated in various organic solvent. Both lipases exhibited activity on difference carbon chain length (C12 - C18, C18:1, C18:2) of substrates. The activity of Lk2 was higher in each of substrate compared with that of Lk3. Experimental findings showed that the best substrates for Lk2 and Lk3 are C18:1 and C18:2 respectively, in agreement with the computational analysis. The activity of both enzymes prefers on nonpolar solvent. On nonpolar solvent the enzymes are able to keep its native folding shown by the value of radius gyration, solvent–enzyme interaction and orientation of triad catalytic residues. Lk3 appeared to be more thermostable, with maximum activity at 55°C. The presence of Fe3+ increased the activity of Lk2 and Lk3. However, the activity of both enzymes were dramatically decreased by the present of Ca2+ despite of the enzymes belong to family I.1 lipase known as calcium dependent enzyme. Molecular analysis on His loop of Lk2 and Lk3 on the present of Ca2+ showed that there were shifting on the orientation of catalytic triad residues. All the data suggest that Lk2 and Lk3 are novel lipase on the family I.1 and both lipase available as a biocatalyst candidate.

Synthesis of 1H-Pyrazolo[1,2-b]Phthalazine-5,10-Dione and 1H-Pyrazolo[1,2-a]Pyridazine-5,8-Dione Derivatives by Bovine Serum Albumin in Water

Bovine Serum Albumin (BSA) was used as a green, inexpensive and efficient biocatalyst, for synthesis of 1H-pyrazolo[1,2-b]phthalazine-5,10-dione and 1H-pyrazolo[1,2-a]pyridazine-5,8-dione derivatives through a one-pot three component reaction between phthalhydrazide or maleic hydrazide, Aryl aldehydes, and malononitrile in good to excellent yield (73–93%). The remarkable features of this approach include green catalyst, no organic solvent, metal-free, mild reaction, easy work-up and good functional group tolerance.

Engineered living hydrogels for robust biocatalysis in pure organic solvents

Engineered living hydrogels that can protect cells from harsh environments have achieved preliminary successes in biomedicine and environmental remediation. However, their biocatalytic applications in pure organic solvents have not been explored. Here, living hydrogels were engineered by integrating genetically modified Escherichia coli cells into alginate hydrogels for robust biocatalysis in pure organic solvents. The biocompatible hydrogels could not only support cell growth and diminish cell escape but could also act as protective matrices to improve organic solvent tolerance, thereby prolonging catalytic activity of whole-cell biocatalysts. Moreover, the influence of hydrogel microenvironments on biocatalytic efficiency was thoroughly investigated. Importantly, the versatility of engineered living hydrogels paves the way to achieve robust biocatalytic efficiency in a variety of pure organic co-solvents. Overall, we are able to engineer living hydrogels for regio-selective synthesis in pure organic solvents, which may be particularly useful for the innovation of living hydrogels in biocatalysis. • Living hydrogels are engineered by integrating microbial cells and alginate hydrogels • Hydrogel microenvironments modulate catalytic efficiency of engineered living hydrogels • Engineered living hydrogels enable improved solvent tolerance in pure organic solvents • The versatility of engineered living hydrogels for robust biocatalysis is verified A versatile strategy to improve the organic solvent tolerance of whole-cell biocatalysts is highly demanded. Gao et al. design engineered living hydrogels using genetically modified microbial cells for improved organic solvent tolerance and robust biocatalysis in pure organic solvents.

Chitosan-regulated biomimetic hybrid nanoflower for efficiently immobilizing enzymes to enhance stability and by-product tolerance

Organic-inorganic hybrid nano-materials have been considered to be promising immobilization matrixes for enzymes due to their significantly enhanced reusability and stability of enzymes. Herein, we constructed a novel organic-inorganic hybrid nanoflower via biomacromolecule-regulated biomimetic mineralization to immobilize sucrose phosphorylase (SPase). It was found that chitosan (CS) effectively regulated the biomimetic mineralization of calcium phosphate (CaP), leading to the formation of flower-like hybrid materials for the entrapment of SPase via self-assembly to establish a nano-biocatalyst (CS-CaP@SPase). Upon immobilization, the obtained CS-CaP@SPase exhibited excellent pH, by-product and organic solvents tolerance, and storage stability. Specifically, at acidic condition (pH 4), CS-CaP@SPase performed over 80 % of initial activity, which was 2.42-folds higher than that of free SPase. The catalytic activity of free SPase was severely inhibited about 30 % in the presence of fructose (1.2 M), but CS-CaP@SPase only lost 5 % relative activity. The CS-CaP@SPase retained over 80 % of its relative activity, while the free SPase maintained <20 % of its relative activity in acetonitrile. The relative activity of CS-CaP@SPase was still retained about 80 % after 10 cycles and maintained 75 % after 15 days. Based on Raman spectra analysis, it was also found that the increased β-folding component of SPase in the secondary structure after immobilization was the main factor for its enhanced stability. It is reasonable to believe that biomacromolecule-regulated biomimetic mineralization could be potentially used as a promising method to immobilize enzymes with excellent stability and recyclability, thereby facilitating the preparation of highly efficient catalysts for industrial biocatalysts, biosensing, and biomedicine.

Production and characterization of novel thermo- and organic solvent-stable keratinase and aminopeptidase from Pseudomonas aeruginosa 4-3 for effective poultry feather degradation.

Feather biodegradation is an important premise for efficient resource development and utilization, in which keratinase plays an important role. However, there are few keratinases that combine the high activity, thermal stability, and organic solvent tolerance required for industrialization. This paper reported an efficient feather-degrading Pseudomonas aeruginosa 4-3 isolated from slaughterhouses. After 48h of fermentation by P. aeruginosa 4-3 in a feather medium at 40°C, pH 8.0, keratinase was efficiently produced (295.28 ± 5.42 U/mL) with complete feather degradation (95.3 ± 1.5%). Moreover, the keratinase from P. aeruginosa 4-3 showed high optimal temperature (55°C), good thermal stability, wide pH tolerance, and excellent organic solvent resistance. In addition, P. aeruginosa 4-3-derived aminopeptidases also exhibit excellent thermal stability and organic solvent tolerance. Encouragingly, the reaction of crude keratinase and aminopeptidase with feathers for 8h resulted in a 78% degradation rate of feathers. These properties make P. aeruginosa 4-3 keratinase and aminopeptidase ideal proteases for potential applications in keratin degradation, as well as provide ideas for the synergistic degradation of keratin by multiple enzymes.

Halohydrin dehalogenase immobilization in magnetic biochar for sustainable halocarbon biodegradation and biotransformation

Regarding the fact that halohydrin dehalogenase (HHDH) displays great potential for both biotransformation and biodegradation, developing immobilized HHDH that is suitable for those applications will be of great importance. Herein, the functionalized magnetic biochar was prepared for efficient enzyme immobilization, easy separation, and reuse. The immobilized halohydrin dehalogenase (HheC) was characterized using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM). The results showed that the relative enzyme activity retention was 85% upon immobilization in biochar (HheC-N-MBC600). After 70 days of storage at 4 °C, the HheC-N-MBC600 retained 50% of its initial activity, while only 8% of the activity was retained for the free enzyme. HheC-N-MBC600 displayed a strong organic solvent tolerance as it retained activity above 92% after incubation in 50% organic solvent for 12 h. Moreover, the immobilized catalyst retained more than 70% activity after a consecutive 30 cycles of reuse, indicating excellent recyclability of the immobilized biocatalyst. Moreover, the immobilized enzyme displayed the same enantioselective behavior as the free enzyme. Together with the immobilized epoxide hydrolyase (EchA-MBC600), HheC-N-MBC600 completely converted 1,3-dichloro-2-propanol (1,3-DCP) to a nontoxic compound. Our results showed that the immobilized HheC-N-MBC600 could be a green and industrially viable tool to degrade halogenated compounds and make useful chiral compounds.

N-acylhomoserine lactonase-based hybrid nanoflowers: a novel and practical strategy to control plant bacterial diseases

BackgroundThe disease caused by plant pathogenic bacteria in the production, transportation, and storage of many crops has brought huge losses to agricultural production. N-acylhomoserine lactonases (AHLases) can quench quorum-sensing (QS) by hydrolyzing acylhomoserine lactones (AHLs), which makes them the promising candidates for controlling infections of QS-dependent pathogenic bacteria. Although many AHLases have been isolated and considered as a potentially effective preventive and therapeutic agents for bacterial diseases, the intrinsically poor ambient stability has seriously restricted its application.ResultsHerein, we showed that a spheroid enzyme-based hybrid nanoflower (EHNF), AhlX@Ni3(PO4)2, can be easily synthesized, and it exhibited 10 times AHL (3OC8-HSL) degradation activity than that with free AhlX (a thermostable AHL lactonase). In addition, it showed intriguing stability even at the working concentration, and retained ~ 100% activity after incubation at room temperature (25 °C) for 40 days and approximately 80% activity after incubation at 60 °C for 48 h. Furthermore, it exhibited better organic solvent tolerance and long-term stability in a complicated ecological environment than that of AhlX. To reduce the cost and streamline production processes, CSA@Ni3(PO4)2, which was assembled from the crude supernatants of AhlX and Ni3(PO4)2, was synthesized. Both AhlX@Ni3(PO4)2 and CSA@Ni3(PO4)2 efficiently attenuated pathogenic bacterial infection.ConclusionsIn this study, we have developed N-acylhomoserine lactonase-based hybrid nanoflowers as a novel and efficient biocontrol reagent with significant control effect, outstanding environmental adaptability and tolerance. It was expected to overcome the bottlenecks of poor stability and limited environmental tolerance that have existed for over two decades and pioneered the practical application of EHNFs in the field of biological control.Graphical

Screening and characterizing flavone synthases and its application in biosynthesizing vitexin from naringenin by a one-pot enzymatic cascade

C-glycosylated flavonoids are important structural derivatives of flavonoids and have a variety of physiological activities. Flavone synthase is a key enzyme for producing C-glycosylated flavonoids. In this study, three flavone synthase genes were cloned, overexpressed and characterized in E. coli. By analyzing the enzymatic properties of the enzymes, Aethusa cynapium flavone synthase (AcFNS) was better than Apium graveolens flavone synthase (AgFNS) and Petroselinum crispum flavone synthase (PcFNS) in terms of catalytic ability, organic solvent tolerance and stability. Then, a one-pot enzymatic cascade was developed to synthesize vitexin from naringenin by using AcFNS, C-glycosyltransferase (TcCGT) from Trollius chinensis, and sucrose synthase (GmSUS) from Glycine max. The effects of enzyme ratios, substrate concentrations, cofactors, and reaction conditions on vitexin production were determined. The highest vitexin production reached 935.6 mg/L with a corresponding molar conversion of 78.7 % for (2 S)-naringenin. Thus, this is the first report of a one-pot enzymatic cascade for vitexin production from naringenin in vitro.

Different Effects of Salt Bridges near the Active Site of Cold-Adapted Proteus mirabilis Lipase on Thermal and Organic Solvent Stabilities

Organic solvent-tolerant (OST) enzymes have been discovered in psychrophiles. Cold-adapted OST enzymes exhibit increased conformational flexibility in polar organic solvents resulting from their intrinsically flexible structures. Proteus mirabilis lipase (PML), a cold-adapted OST lipase, was used to assess the contribution of salt bridges near the active site involving two arginine residues (R237 and R241) on the helix η1 and an aspartate residue (D248) on the connecting loop to the thermal and organic solvent stabilities of PML. Alanine substitutions for the ion pairs (R237A, R241A, D248A, and R237A/D248A) increased the conformational flexibility of PML mutants compared to that of the wild-type PML in an aqueous buffer. The PML mutants became more susceptible to denaturation after increasing the dimethyl sulfoxide or methanol concentration than after a temperature increase. Methanol was more detrimental to the structural stability of PML compared to dimethyl sulfoxide. These results suggest that direct interactions of dimethyl sulfoxide and methanol with the residues near the active site can have a destructive effect on the structure of PML compared with the global effect of heat on the protein structure. This study provides insight into the conformational changes within an OST enzyme with different effects on its thermal and organic solvent stabilities.

Improving E. coli organic solvent tolerance by 1,4-dihydroxy-2-naphthoic acid.

Improving the organic solvent tolerance of bacteria is beneficial for the bioproduction of various valuable chemicals. In this study, we found that 1,4-dihydroxy-2-naphthoic acid (DHNA), known as a prebiotic, increased organic solvent tolerance in Escherichia coli. The AcrAB-TolC multidrug efflux pump contributes to the intrinsic organic solvent tolerance of E. coli. The addition of DHNA increased this pump's expression level. Transcriptional activators MarA, SoxS, and Rob proteins are known to control the expression of marA/soxS/rob regulon genes, including acrAB and tolC. Evaluation of the organic solvent tolerances of ΔmarA mutant, ΔsoxS mutant, and Δrob mutant showed that ΔmarA mutant and ΔsoxS mutant did not improve organic solvent tolerance by the addition of DHNA. In addition, DHNA increased the promoter activities of both marA and soxS. These results indicated that DHNA induces the AcrAB-TolC pump through both the marRAB system and the soxRS system.