Jinyeong Kim

Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases

Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen. Therefore, we have generated mutants of Est-AF that retained high thermostability whilst improving enantioselectivity. A library of Est-AF mutants was created by error-prone polymerase chain reaction, and mutants with improved enantioselectivity were isolated by site-saturation mutagenesis. The regions of Est-AF containing amino acid mutations were analyzed by homology modeling of its three-dimensional structure, and structure-based explanations for the changes in enantioselectivity are proposed. Finally, we isolated two mutants showing improved enantioselectivity over Est-AF (ee% = −16.2 ± 0.2 and E = 0.7 ± 0.0): V138G (ee% = 35.9 ± 1.0 and E = 3.0 ± 0.1) and V138G/L200R (ee% = 89.2 ± 0.2 and E = 19.5 ± 0.5). We also investigated various characteristics of these mutants and found that the mutants showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.

Optimization of dilute sulfuric acid pretreatment of corn stover for enhanced xylose recovery and xylitol production

Pretreatment steps are necessary for the bioconversion of corn stover (CS) to xylitol. In order to optimize the pretreatment parameters, the sulfuric acid concentration, sulfuric acid residence time, and solid slurry concentration were evaluated, based on the glucose and xylose recovered from CS at the relatively low temperature of 120°C. The optimum conditions were found to be pretreatment with 2.5% (w/v) sulfuric acid for 1.5 h, with a solid slurry concentration of 90 g/L. Under these conditions, the hydrolysis rates of glucan and xylan were approximately 26.0 and 82.8%, respectively. High xylitol production (10.9 g/L) and conversion yield (0.97 g/g) were attained from corn stover hydrolysate (CSH) without detoxification and any nutrient addition. Our results were similar for xylitol production in synthetic medium under the same conditions. The non-necessity of both the hydrolysate detoxification step and nutrient addition to the CSH is undoubtedly promising for scale-up application on an industrial scale, because this medium-based manufacturing process is expected to reduce the production cost of xylitol. The present study demonstrates that value-added xylitol could be effectively produced from CS under optimized pretreatment conditions, especially with CSH as the substrate material.

Cloning and characterization of a novel thermostable esterase from Bacillus gelatini KACC 12197

A novel gene encoding a thermostable esterase (designated as Est-gela) was isolated from the moderate thermophile Bacillus gelatini KACC 12197. The open reading frame of this gene (1170 bp) encodes 389 amino acid residues, and the molecular weight of Est-gela is approximately 42 kDa. The protein sequence of Est-gela shows similarity with β-lactamases and esterases (⩽ 43%). Est-gela contains the Ser-X-X-Lys conserved sequence (Ser58-Met59-Thr60-Lys61) and belongs to family VIII of esterases. We overexpressed Est-gela in Escherichia coli XL1-blue and purified this protein using a His tag. Est-gela showed a strong enzymatic activity toward p-nitrophenyl esters with short acyl chains (⩽ C4) and the strongest activity toward p-nitrophenyl butyrate. Est-gela showed an enhanced enzymatic activity at 65-75 °C and retained more than 90% of the activity after incubation at 65 °C for 180 min. These results indicated that Est-gela was thermostable. In addition, Est-gela showed the maximal activity at pH 10. We also evaluated the effects of surfactants and organic solvents. Surfactants were more effective at improving the enzymatic activity than were organic solvents. Finally, Est-gela hydrolyzed (R,S)-ketoprofen ethyl ester (Kcat/Km = 5.0 ± 0.2 s(-1) mM(-1), mean ± standard error) with enantioselectivity toward (S)-ketoprofen ethyl ester rather than (R)-ketoprofen ethyl ester.

Improved 1,3-propanediol production by Escherichia coli from glycerol due to Co-expression of glycerol dehydratase reactivation factors and succinate addition

Escherichia coli was engineered to produce 1,3-propanediol (1,3-PDO) from glycerol, an inexpensive carbon source. This was done by introducing a synthetic pathway consisting of glycerol dehydratase, glycerol dehydratase reactivation factor, and 1,3-propanediol oxidoreductase isoenzyme. The JM-30BY15AB harboring pQE30/dhaB123, yqhD and pQE15A/gdrA, gdrB produced 1,3-PDO (7.2 g/L) from glycerol, at a level higher than that produced by JM-30BY harboring pQE30/dhaB123, yqhD (4.1 g/L). When 10mM succinate was added to the medium, the titer of 1,3-PDO and the glycerol consumption increased to 9.9 and 23.84 g/L, respectively. In addition, the ratio of NADH to NAD+ increased by 43%. The titer of 1,3-PDO and glycerol consumption were 145.6 and 86.6% higher, respectively, than those from the control which harbors one vector system without gdrAB and did not include succinate addition. Under fed-batch fermentation conditions, the titer of 1,3-PDO and its conversion yield from glycerol were 13.11 g/L and 0.49 g/g, respectively. This dual-vector system may be a useful approach for the co-expression of recombinant proteins. Further, succinate addition is a promising route for the biotechnological production of NADH-dependent microbial metabolites.