Chun-Xiu Li

Highly efficient synthesis of chiral alcohols with a novel NADH-dependent reductase from Streptomyces coelicolor.

An NADH-dependent reductase (ScCR) from Streptomyces coelicolor was discovered by genome mining for carbonyl reductases. ScCR was overexpressed in Escherichia coli BL21, purified to homogeneity and its catalytic properties were studied. This enzyme catalyzed the asymmetric reduction of a broad range of prochiral ketones including aryl ketones, α- and β-ketoesters, with high activity and excellent enantioselectivity (>99% ee) towards β-ketoesters. Among them, ethyl 4-chloro-3-oxobutanoate (COBE) was efficiently converted to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important pharmaceutical intermediate, in water/toluene biphasic system. As much as 600 g/L (3.6M) of COBE was asymmetrically reduced within 22 h using 2-propanol as a co-substrate for NADH regeneration, resulting in a yield of 93%, an enantioselectivity of >99% ee, and a total turnover number (TTN) of 12,100. These results indicate the potential of ScCR for the industrial production of valuable chiral alcohols.

Efficient Synthesis of a Chiral Precursor for Angiotensin-Converting Enzyme (ACE) Inhibitors in High Space-Time Yield by a New Reductase without External Cofactors

A new reductase, CgKR2, with the ability to reduce ethyl 2-oxo-4-phenylbutyrate (OPBE) to ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE), an important chiral precursor for angiotensin-converting enzyme (ACE) inhibitors, was discovered. For the first time, (R)-HPBE with >99% ee was produced via bioreduction of OPBE at 1 M without external addition of cofactors. The space-time yield (700 g·L(-1)·d(-1)) was 27 times higher than the highest record.

Biocatalytic properties of a recombinant aldo-keto reductase with broad substrate spectrum and excellent stereoselectivity

In the screening of 11 E. coli strains overexpressing recombinant oxidoreductases from Bacillus sp. ECU0013, an NADPH-dependent aldo-keto reductase (YtbE) was identified with capability of producing chiral alcohols. The protein (YtbE) was overexpressed, purified to homogeneity, and characterized of biocatalytic properties. The purified enzyme exhibited the highest activity at 50°C and optimal pH at 6.5. YtbE served as a versatile reductase showing a broad substrate spectrum towards different aromatic ketones and keto esters. Furthermore, a variety of carbonyl substrates were asymmetrically reduced by the purified enzyme with an additionally coupled NADPH regeneration system. The reduction system exhibited excellent enantioselectivity (>99% ee) in the reduction of all the aromatic ketones and high to moderate enantioselectivity in the reduction of α- and β-keto esters. Among the ketones tested, ethyl 4,4,4-trifluoroacetoacetate was found to be reduced to ethyl (R)-4,4,4-trifluoro-3-hydroxy butanoate, an important pharmaceutical intermediate, in excellent optical purity. To the best of our knowledge, this is the first report of ytbE gene-encoding recombinant aldo-keto reductase from Bacillus sp. used as biocatalyst for stereoselective reduction of carbonyl compounds. This study provides a useful guidance for further application of this enzyme in the asymmetric synthesis of chiral alcohol enantiomers.

An efficient bioprocess for enzymatic production of l-menthol with high ratio of substrate to catalyst using whole cells of recombinant E. coli

A gene encoding an esterase of Bacillus subtilis ECU0554 previously isolated from soil was cloned and overexpressed in Escherichia coli BL21. The recombinant esterase (recBsE) showed the best enantioselectivity (E>100) towards DL-menthyl acetate, in contrast to DL-menthyl esters propionate and butyrate. A high ratio of substrate to catalyst (S/C-ratio, ≥50) was achieved in the kinetic resolution of DL-menthyl acetate by using whole cells of recombinant E. coli BL21. Some key parameters of the biocatalytic process, including amount of cosolvent, catalyst loading and substrate loading, were optimized. Compared with the process catalyzed by wild-type whole cells of B. subtilis ECU0554, the second-generation bioprocess using whole cells of recombinant E. coli BL21 afforded a 40-fold improvement in S/C-ratio and a 75-fold improvement in the volumetric productivity per biocatalyst loading. Moreover, the substrate loading was increased up to 200 g L(-1) (∼1 M), the biocatalyst loading was reduced to 2.5 g L(-1) and the space-time yield was improved from 54 g L(-1) d(-1) to 202 g L(-1) d(-1).

A new thermostable β-glucosidase mined from Dictyoglomus thermophilum: properties and performance in octyl glucoside synthesis at high temperatures.

A new β-glucosidase (DtGH) representing 40% identity with an apple seed glycosidase (ASG) was cloned from Dictyoglomus thermophilum. DtGH showed extremely high thermostability in aqueous solution, with half-lives of 533, 44, and 5 h measured at 70, 80 and 90 °C, respectively. Therefore it was used for direct glycosylation of n-octanol at 70 °C instead of 50 °C as usually. As a result, the glucose based conversion was increased by 27%, but the time spent to reach equilibrium was decreased from 7 d to 3 d. This enzyme also exhibited excellent stability under the reaction environment, retaining 70-80% of its initial activity after 7 d of incubation at 70 °C in either 1.7 M glucose solution or octanol-aqueous (85:15, v/v) system. It could retain part of synthetic activity even in boiling water. Owing to the strong glucose-tolerance and extremely high thermostability, DtGH should be promising for various glucosides synthesis.

Cloning of Thermostable Cellulase Genes of Clostridium thermocellum and Their Secretive Expression in Bacillus subtilis

Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.

Crumb Rubber–Modified Asphalt: Microwave Treatment Effects

Abstract Crumb rubber pretreatment by microwave irradiation before blending with an asphalt matrix improves the properties of rubber–asphalt blends. This treatment cleft the surface vulcanization network, resulting in higher surface activity of the crumb rubber and improved compatibility with asphalt. This new rubber blend exhibited minor temperature susceptibility behaviors and high ductility at 5°C and an improved viscoelastic nature and storage stability. Characterization has also been performed to elucidate the mechanism for this improvement using a microwave treatment technique.

A Novel (R)-Imine Reductase fromPaenibacillus lactisfor Asymmetric Reduction of 3 H-Indoles

A novel (R)‐imine reductase (PlRIR) from Paenibacillus lactis was heterologously overexpressed in Escherichia coli, purified and characterized. The purified PlRIR exhibited relatively high catalytic efficiency (kcat/Km=1.58 s−1 mm−1) towards 2,3,3‐trimethylindolenine. A panel of 3H‐indoles and 3H‐indole iodides were reduced by PlRIR to yield the corresponding products with good‐to‐excellent enantioselectivity (66–98 % ee). In addition, PlRIR also possesses good activities toward other types of imines such as pyrroline, tetrahydropyridine, and dihydroisoquinoline, indicating a reasonably broad substrate acceptance. In a 100 mg scale preparative reaction, 100 mm 2,3,3‐trimethylindolenine was converted efficiently to afford (R)‐2,3,3‐trimethylindoline with 96 % ee and 81 % yield.

Combinatorial evolution of phosphotriesterase toward a robust malathion degrader by hierarchical iteration mutagenesis

Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR‐based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling. Finally, guided by in silico energy calculations (FoldX), thermostability of the variant was improved. The mutations extend from the core region to the enzyme surface during the evolutionary pathway. After screening <9,000 mutants, the best variant PoOPHM9 showed 25‐fold higher activity than wild‐type PoOPHM2, with a thermostability (T5015) of 67.6°C. Thus, PoOPHM9 appears to be an efficient and robust candidate for malathion detoxification. Biotechnol. Bioeng. 2016;113: 2350–2357.

Engineering 7β-Hydroxysteroid Dehydrogenase for Enhanced Ursodeoxycholic Acid Production by Multiobjective Directed Evolution

Ursodeoxycholic acid (UDCA) is the main active ingredient of natural bear bile powder with multiple pharmacological functions. 7β-Hydroxysteroid dehydrogenase (HSDH) is a key biocatalyst for the synthesis of UDCA. However, all the 7β-HSDHs reported commonly suffer from poor activity and thermostability, resulting in limited productivity of UDCA. In this study, a multiobjective directed evolution (MODE) strategy was proposed and applied to improve the activity, thermostability, and pH optimum of a 7β-HSDH. The best variant (V3-1) showed a specific activity 5.5-fold higher than and a half-life 3-fold longer than those of the wild type. In addition, the pH optimum of the variant was shifted to a weakly alkaline value. In the cascade reaction, the productivity of UDCA with V3-1 increased to 942 g L-1 day-1, in contrast to 141 g L-1 day-1 with the wild type. Therefore, this study provides a useful strategy for improving the catalytic efficiency of a key enzyme that significantly facilitated the bioproduction of UDCA.