Expression and characterization of a thermostable monoacylglycerol lipase from thermophilic Geobacillus kaustophilus.

Thermophilic Geobacillus kaustophilus HTA426 genome possesses a monoacylglycerol lipase (MAGL) gene. MAGLs can synthesize emulsifiers for use in the food and pharmaceutical industries from fatty acids and glycerol. They can also be used to analyze monoacylglycerol (MAG) levels in serum and food. The MAGL gene from strain HTA426 was artificially synthesized and heterologously expressed in Escherichia coli BL21(DE3). The recombinant His-tag fused MAGL (GkMAGL) was purified using a Ni2+-affinity column. The purified enzyme showed a temperature optimum at 65 °C and was stable up to 75 °C after 30 min incubation. In addition, the enzyme exhibited a pH optimum of 7.5 and was stable from pH 5.0 to 11.0. The enzyme hydrolyzed monoacylglycerols and showed the highest activity toward 1-monolauroylglycerol. The enzyme was stable in the presence of various organic solvents and detergents. The addition of Triton X-100 significantly increased GkMAGL activity. The thermal stability of the enzyme was higher than that of thermostable MAGL from Geobacillus sp. 12AMOR1 (12AMOR1_MAGL). Circular dichroism spectral analysis showed that the conformational stability of the GkMAGL was higher than that of 12AMOR1_MAGL at higher temperatures. These results indicate that the GkMAGL has useful features that can be used for various biotechnological applications.

Expression and characterization of cholesterol oxidase with high thermal and pH stability from Janthinobacterium agaricidamnosum.

Cholesterol oxidases (COXases) have a diverse array of applications including analysis of blood cholesterol levels, synthesis of steroids, and utilization as an insecticidal protein. The COXase gene from Janthinobacterium agaricidamnosum was cloned and expressed in Escherichia coli. The purified COXase showed an optimal temperature of 60 °C and maintained about 96 and 72% of its initial activity after 30 min at 60 and 70 °C, respectively. In addition, the purified COXase exhibited a pH optimum at 7.0 and high pH stability over the broad pH range of 3.0-12.0. The pH stability of the COXase at pH 12.0 was higher than that of highly stable COXase from Chromobacterium sp. DS-1. The COXase oxidized cholesterol and ß-cholestanol at higher rates than other 3ß-hydroxysteroids. The Km, Vmax, and kcat values for cholesterol were 156 µM, 13.7 µmol/min/mg protein, and 14.4 s-1, respectively. These results showed that this enzyme could be very useful in the clinical determination of cholesterol in serum and the production of steroidal compounds. This is the first report to characterize a COXase from the genus Janthinobacterium.

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.

Improving the organic solvent tolerance of Escherichia coli with vanillin, and the involvement of an AcrAB-TolC efflux pump in vanillin tolerance.

Vanillin is used as a flavor ingredient in a diverse range of food categories and is known to inhibit microbial fermentation. In this study, we found that vanillin improved the hydrophobic organic solvent tolerance of Escherichia coli and showed that the AcrAB-TolC efflux pump is implicated in that tolerance. The expression level of the pump was enhanced by the addition of vanillin. AcrAB-TolC efflux pump expression is known to be regulated by transcription activators such as MarA, SoxS, and Rob. Among these three transcription factors, marA transcription was significantly elevated by the addition of vanillin. We found that the AcrAB-TolC efflux pump is involved also in vanillin tolerance. The ΔacrB mutant was more sensitive to vanillin than the parent strain. A complementation test revealed that the introduction of the acrB gene recovered the vanillin tolerance of the ΔacrB mutant.

Involvement of catalase and superoxide dismutase in hydrophobic organic solvent tolerance of Escherichia coli.

Escherichia coli strains are generally sensitive to hydrophobic organic solvents such as n-hexane and cyclohexane. Oxidative stress in E. coli by exposure to these hydrophobic organic solvents has been poorly understood. In the present study, we examined organic solvent tolerance and oxygen radical generation in E. coli mutants deficient in reactive oxygen species (ROS)-scavenging enzymes. The organic solvent tolerances in single gene mutants lacking genes encoding superoxide dismutase (sodA, sodB, and sodC), catalase (katE and katG), and alkyl hydroperoxide reductase (ahpCF) were similar to that of parent strain BW25113. We constructed a BW25113-based katE katG double mutant (BW25113∆katE∆katG) and sodA sodB double mutant (BW25113sodA∆sodB). These double-gene mutants were more sensitive to hydrophobic organic solvents than BW25113. In addition, the intracellular ROS levels in E. coli strains increased by the addition of n-hexane or cyclohexane. The ROS levels in BW25113∆katE∆katG and BW25113∆sodA∆sodB induced by exposure to the solvents were higher than that in BW25113. These results suggested that ROS-scavenging enzymes contribute to the maintenance of organic solvent tolerance in E. coli. In addition, the promoter activities of sodA and sodB were significantly increased by exposure to n-hexane.

Cholesterol oxidase from Rhodococcus erythropolis with high specificity toward ß-cholestanol and pytosterols.

Two genes (choRI and choRII) encoding cholesterol oxidases belonging to the vanillyl-alcohol oxidase (VAO) family were cloned on the basis of putative cholesterol oxidase gene sequences in the genome sequence data of Rhodococcus erythropolis PR4. The genes corresponding to the mature enzymes were cloned in a pET vector and expressed in Escherichia coli. The two cholesterol oxidases produced from the recombinant E. coli were purified to examine their properties. The amino acid sequence of ChoRI showed significant similarity (57%) to that of ChoRII. ChoRII was more stable than ChoRI in terms of pH and thermal stability. The substrate specificities of these enzymes differed distinctively from one another. Interestingly, the activities of ChoRII toward ß-cholestanol, ß-sitosterol, and stigmasterol were 2.4-, 2.1-, and 1.7-fold higher, respectively, than those of cholesterol. No cholesterol oxidases with high activity toward these sterols have been reported so far. The cholesterol oxidation products from these two enzymes also differed. ChoRI and ChoRII oxidized cholesterol to form cholest-4-en-3-one and 6ß-hydroperoxycholest-4-en-3-one, respectively.

Production of styrene oxide from styrene by a recombinant Escherichia coli with enhanced AcrAB-TolC efflux pump level in an aqueous-organic solvent two-phase system.

The AcrAB-TolC efflux pump is involved in the organic solvent tolerance of Escherichia coli. Most E. coli strains are highly sensitive to organic solvents such as n-hexane and cyclohexane. Here, a recombinant E. coli transformed with an expression plasmid containing acrAB and tolC became tolerant to n-hexane and cyclohexane. The levels of AcrA, AcrB, and TolC in the recombinant increased by 3- to 5-fold compared to those in the control strain without the plasmid for acrAB or tolC. To investigate the usability of the recombinant as a biocatalyst in an aqueous-organic solvent two-phase system, we further introduced xylMA xylene monooxygenase genes from Pseudomonas putida mt-2 into the recombinant and examined the production of styrene oxide from styrene. The resulting recombinant produced 1.8 mg and 1.0 mg styrene oxide mL-1 of medium in a medium overlaid with a 25% volume of n-hexane and cyclohexane containing 10% (wt vol-1) styrene, respectively.

Cholesterol oxidase with high catalytic activity from Pseudomonas aeruginosa: Screening, molecular genetic analysis, expression and characterization.

An extracellular cholesterol oxidase producer, Pseudomonas aeruginosa strain PA157, was isolated by a screening method to detect 6ß-hydroperoxycholest-4-en-3-one-forming cholesterol oxidase. On the basis of a putative cholesterol oxidase gene sequence in the genome sequence data of P. aeruginosa strain PAO1, the cholesterol oxidase gene from strain PA157 was cloned. The mature form of the enzyme was overexpressed in Escherichia coli cells. The overexpressed enzyme formed inclusion bodies in recombinant E. coli cells grown at 20 °C and 30 °C. A soluble and active PA157 enzyme was obtained when the recombinant cells were grown at 10 °C. The purified enzyme was stable at pH 5.5 to 10 and was most active at pH 7.5-8.0, showing optimal activity at pH 7.0 and 70 °C. The enzyme retained about 90% of its activity after incubation for 30 min at 70 °C. The enzyme oxidized 3ß-hydroxysteroids such as cholesterol, ß-cholestanol, and ß-sitosterol at high rates. The Km value and Vmax value for the cholesterol were 92.6 µM and 15.9 µmol/min/mg of protein, respectively. The Vmax value of the enzyme was higher than those of commercially available cholesterol oxidases. This is the first report to characterize a cholesterol oxidase from P. aeruginosa.

Improvement of organic solvent tolerance by disruption of the lon gene in Escherichia coli.

The Lon ATP-dependent protease plays an important role in regulating many biological processes in bacteria. In this study, we examined the organic solvent tolerance of a Δlon mutant of Escherichia coli K-12 and found that the mutant showed remarkably higher organic solvent tolerance than the parent strain. Δlon mutants are known to overproduce capsular polysaccharide, resulting in the formation of mucoid colonies. We considered that this increase in capsular polysaccharide production might be involved in the organic solvent tolerance in E. coli. However, a ΔlonΔwcaJ double-gene mutant displaying a nonmucoid phenotype was as tolerant to organic solvents as the Δlon mutant, suggesting that capsular polysaccharide is not involved in organic solvent tolerance. On the other hand, the Lon protease is known to exhibit proteolytic activity against the transcriptional activators MarA and SoxS, which can enhance the expression level of the AcrAB-TolC efflux pump. We found that the Δlon mutant showed a higher expression level of AcrB than the parent strain. In addition, the ΔlonΔacrB double-gene mutant showed a significant decrease in organic solvent tolerance. Thus, it was shown that organic solvent tolerance in the Δlon mutant depends on the AcrAB-TolC pump but not capsular polysaccharide. E. coli strain JA300 acrRIS marR overexpresses the AcrAB-TolC pump and exhibits high-level solvent tolerance. In an attempt to further improve the solvent tolerance of JA300 acrRIS marR, a lon gene disruptant of this strain was constructed. However, the resulting mutant JA300 acrRIS marR Δlon showed lower solvent tolerance than JA300 acrRIS marR.

Hyper-activation of foldase-dependent lipase with lipase-specific foldase.

The LST-03 lipase from Pseudomonas aeruginosa LST-03 requires lipase-specific foldase for activation. Abundant expression of the active lipase was successfully accomplished with individual expression of the lipase and foldase in a heterologous host and subsequent in vitro activation. Although the activity of the native lipase from culture supernatant of P. aeruginosa LST-03 was 110 kI.U./g, that after in vitro activation using individually expressed lipase and foldase was 228 kI.U./g. Furthermore, the activity after in vitro activation with afterwards adding calcium ions was 359 kI.U./g. However, the incubation of the lipase with the foldase in the presence of calcium ions resulted in a small conformational transition and low activation levels of the lipase by the foldase. The lipase showed high affinity for the foldase in the presence of calcium ions. The results indicate that in a cellular environment that contains calcium ions, the lipase would not become a hyperactive form by the foldase.

Halarchaeum rubridurum sp. nov., a moderately acidophilic haloarchaeon isolated from commercial sea salt samples.

Six halo-acidophilic archaeal strains were isolated from four commercial salt samples obtained from seawater in the Philippines, Indonesia (Bali) and Japan (Okinawa) on agar plates at pH 4.5. Cells of the six strains were pleomorphic, and stained Gram-negative. Two strains were pink-red pigmented, while four other strains were orange-pink pigmented. Strain MH1-16-3(T) was able to grow at 9-30% (w/v) NaCl [with optimum at 18% (w/v) NaCl], at pH 4.5-6.8 (optimum, pH 5.5) and at 20-50 °C (optimum, 42 °C). The five other strains grew at slightly different ranges. The six strains required at least 1 mM Mg(2+) for growth. The 16S rRNA gene sequences of the six strains were almost identical, sharing 99.9 (1-2 nt differences) to 100% similarity. The closest relatives were Halarchaeum acidiphilum MH1-52-1(T) and Halarchaeum salinum MH1-34-1(T) with 97.7% similarity. The DNA G+C contents of the six strains were 63.2-63.7 mol%. Levels of DNA-DNA relatedness amongst the six strains were 79-86%, while those between MH1-16-3(T) and H. acidiphilum MH1-52-1(T) and H. salinum MH1-34-1(T) were both 43 and 45% (reciprocally), respectively. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the six isolates represent a novel species of the genus Halarchaeum, for which the name Halarchaeum rubridurum sp. nov. is proposed. The type strain is MH1-16-3(T) ( =JCM 16108(T) =CECT 7535(T)).

Halarchaeum salinum sp. nov., a moderately acidophilic haloarchaeon isolated from commercial sea salt.

Three halophilic archaeal strains, MH1-34-1(T), MH1-16-1 and MH1-224-5 were isolated from commercial salt samples produced from seawater in Indonesia, the Philippines and Japan, respectively. Cells of the three strains were pleomorphic and stained Gram-negative. Strain MH1-34-1(T) was orange-red pigmented, while MH1-16-1 and MH1-224-5 were pink-pigmented. Strain MH1-34-1(T) was able to grow at 12-30 % (w/v) NaCl (with optimum at 18 % NaCl, w/v) at pH 4.5-7.2 (optimum, pH 5.2-5.5) and at 15-45 °C (optimum, 42 °C). Strains MH1-16-1 and MH1-224-5 grew in slightly different ranges. These strains required at least 1 mM Mg(2+) for growth. The 16S rRNA gene sequences of strains MH1-34-1(T), MH1-16-1 and MH1-224-5 were almost identical (99.8-99.9 % similarities), and the closest relative was Halarchaeum acidiphilum MH-1-52-1(T) with 98.4 % similarities. The DNA G+C contents of MH1-34-1(T), MH1-16-1 and MH1-224-5 were 59.3, 60.8 and 61.0 mol%, respectively. The level of DNA-DNA relatedness amongst the three strains was 90-91 %, while that between each of the three strains and Halarchaeum acidiphilum MH1-52-1(T) was 51-55 %. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the isolates should represent a novel species of the genus Halarchaeum, for which the name Halarchaeum salinum sp. nov. is proposed. The type strain is MH1-34-1(T) ( = JCM 16330(T) = CECT 7574(T)).

Contributions of mutations in acrR and marR genes to organic solvent tolerance in Escherichia coli.

The AcrAB-TolC efflux pump is involved in maintaining intrinsic organic solvent tolerance in Escherichia coli. Mutations in regulatory genes such as marR, soxR, and acrR are known to increase the expression level of the AcrAB-TolC pump. To identify these mutations in organic solvent tolerant E. coli, eight cyclohexane-tolerant E. coli JA300 mutants were isolated and examined by DNA sequencing for mutations in marR, soxR, and acrR. Every mutant carried a mutation in either marR or acrR. Among all mutants, strain CH7 carrying a nonsense mutation in marR (named marR109) and an insertion of IS5 in acrR, exhibited the highest organic solvent-tolerance levels. To clarify the involvement of these mutations in improving organic solvent tolerance, they were introduced into the E. coli JA300 chromosome by site-directed mutagenesis using λ red-mediated homologous recombination. Consequently, JA300 mutants carrying acrR::IS5, marR109, or both were constructed and named JA300 acrRIS, JA300 marR, or JA300 acrRIS marR, respectively. The organic solvent tolerance levels of these mutants were increased in the following order: JA300 < JA300 acrRIS < JA300 marR < JA300 acrRIS marR. JA300 acrRIS marR formed colonies on an agar plate overlaid with cyclohexane and p-xylene (6:4 vol/vol mixture). The organic solvent-tolerance level and AcrAB-TolC efflux pump-expression level in JA300 acrRIS marR were similar to those in CH7. Thus, it was shown that the synergistic effects of mutations in only two regulatory genes, acrR and marR, can significantly increase organic solvent tolerance in E. coli.

Structural characterization of the organic solvent-stable cholesterol oxidase from Chromobacterium sp. DS-1.

Cholesterol oxidase is of significant commercial interest as it is widely used as a biosensor for the detection of cholesterol in clinical samples, blood serum and food. Increased stability of this enzyme with regards to temperature and different solvent conditions are of great importance to the reliability and versatility of its applications. We here report the crystal structure of the cholesterol oxidase of Chromobacterium sp. DS-1 (CHOLOX). In contrast to other previously characterized cholesterol oxidases, this enzyme retains high activity in organic solvents and detergents at temperatures above 85 degrees C despite its mesophilic origin. With the availability of one other homologous oxidase of known three-dimensional structure, a detailed comparison of its sequence and structure was performed to elucidate the mechanisms of stabilization. In contrast to factors that typically contribute to the stability of thermophilic proteins, the structure of CHOLOX exhibits a larger overall cavity volume, less charged residues and less salt bridge interactions. Moreover, the vast majority of residue substitutions were found on or near the protein's solvent exposed surface. We propose that the engineering of enhanced stability may also be accomplished through selective engineering of the protein periphery rather than by redesigning its entire core.

Enhancement of the aspartame precursor synthetic activity of an organic solvent-stable protease.

The PST-01 protease is highly stable and catalyzes the synthesis of the aspartame precursor with high reaction yields in the presence of organic solvents. However, the synthesis rate using the PST-01 protease was slower than that observed when thermolysin was used. Structural comparison of both enzymes showed particular amino acid differences near the active center. These few residue differences in the PST-01 protease were mutated to match those amino acid types found in thermolysin. The mutated PST-01 proteases at the 114th residue from tyrosine to phenylalanine showed enhancement of synthetic activity. This activity was found to be similar to thermolysin. In addition, mutating the residue in the PST-01 protease with arginine and serine showed more improvement of the activity. The mutant PST-01 protease should be more useful than thermolysin for the synthesis of the aspartame precursor, because this enzyme has higher stability and activity in the presence of organic solvents. The results show the potential of organic solvent-stable enzymes as industrial catalysts.

Characteristics and biotechnological applications of microbial cholesterol oxidases.

Microbial cholesterol oxidase is an enzyme of great commercial value, widely employed by laboratories routinely devoted to the determination of cholesterol concentrations in serum, other clinical samples, and food. In addition, the enzyme has potential applications as a biocatalyst which can be used as an insecticide and for the bioconversion of a number of sterols and non-steroidal alcohols. The enzyme has several biological roles, which are implicated in the cholesterol metabolism, the bacterial pathogenesis, and the biosynthesis of macrolide antifungal antibiotics. Cholesterol oxidase has been reported from a variety of microorganisms, mostly from actinomycetes. We recently reported cholesterol oxidases from gram-negative bacteria such as Burkholderia and Chromobacterium. These enzymes possess thermal, detergent, and organic solvent tolerance. There are two forms of cholesterol oxidase, one containing a flavin adenine dinucleotide cofactor non-covalently bound to the enzyme (class I) and the other containing the cofactor covalently linked to the enzyme (class II). These two enzymes have no significant sequence homology. The phylogenetic tree analyses show that both class I and class II enzymes can be further divided into at least two groups.

Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase.

Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53-62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. K(m) and V(max) values of the cholesterol oxidase were estimated from Lineweaver-Burk plots. The V(max)/K(m) ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures.

A maltooligosaccharide-forming amylase gene from Brachybacterium sp. strain LB25: cloning and expression in Escherichia coli.

Brachybacterium sp. strain LB25 produces a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents. The enzyme hydrolyzed starch to produce maltotriose primarily. The structural gene encoding the amylase from strain LB25 was cloned and sequenced. The amino acid sequence of the product showed significant similarity (45 to 49%) to amylases from the genus Streptomyces. The amylase gene was expressed in Escherichia coli, but the specific activity of the recombinant amylase was lower than that of the amylase purified from strain LB25.

Purification and characterization of Chromobacterium sp. DS-1 cholesterol oxidase with thermal, organic solvent, and detergent tolerance.

A new screening method for 6beta-hydroperoxycholest-4-en-3-one (HCEO)-forming cholesterol oxidase was devised in this study. As the result of the screening, a novel cholesterol oxidase producer (strain DS-1) was isolated and identified as Chromobacterium sp. Extracellular cholesterol oxidase of strain DS-1 was purified from the culture supernatant. The molecular mass of the purified enzyme was 58 kDa. This enzyme showed a visible adsorption spectrum having peaks at 355 and 450 nm, like a typical flavoprotein. The enzyme oxidized cholesterol to HCEO, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for every 1 mol of cholesterol oxidized. The enzyme oxidized 3beta-hydroxysteroids such as cholesterol, beta-cholestanol, and pregnenolone at high rates. The Km value for cholesterol was 26 microM. The enzyme was stable at pH 3 to 11 and most active at pH 7.0-7.5, showing optimal activity at pH 7.0 and 65 degrees C. The enzyme retained about 80% of its activity after incubation for 30 min at 85 degrees C. The thermal stability of the enzyme was the highest among the cholesterol oxidases tested. Moreover, the enzyme was more stable in the presence of various organic solvents and detergents than commercially available cholesterol oxidases.

Characterization of recombinant glyoxylate reductase from thermophile Thermus thermophilus HB27.

A glyoxylate reductase gene from the thermophilic bacterium Thermus thermophilus HB27 (TthGR) was cloned and expressed in Escherichia coli cells. The recombinant enzyme was highly purified to homogeneity and characterized. The purified TthGR showed thermostability up to 70 degrees C. In contrast, the maximum reaction condition was relatively mild (45 degrees C and pH 6.7). Although the kcat values against co-enzyme NADH and NADPH were similar, the Km value against co-enzyme NADH was approximately 18 times higher than that against NADPH. TthGR prefers NADPH rather than NADH as an electron donor. These results indicate that a phosphate group of a co-enzyme affects the binding affinity rather than the reaction efficiency, and TthGR demands appropriate amount of phosphate for a high activity. Furthermore, it was found that the half-lives of TthGR in the presence of 25% dimethyl sulfoxide and diethylene glycol were significantly longer than that in the absence of an organic solvent.