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Liu J.,Nanjing University of Technology | Liu J.,National Engineering Technique Research Center for Biotechnology | Guo T.,Central South University of forestry and Technology | Yang T.,Central South University of forestry and Technology | And 8 more authors.
International Journal of Biochemistry and Cell Biology | Year: 2017

Clostridium beijerinckii 4693:int with high ferulic acid (FA) tolerance was engineered and characterized in our lab. In this study, the minimum inhibition concentrations of FA against C. beijerinckii NCIMB 8052 (wild-type) and 4693:int were 1.0 and 1.5 g/l, respectively; cell viability was 18.5% and 106.7%, respectively, in the presence of 0.5 g/l FA. A comparative transcriptome analysis was carried out at two different growth stages to evaluate sensitivity to FA. Genes that were differentially expressed included those related to redox and associated cofactors, riboflavin metabolism, two-component system, glycolysis and butanoate metabolism, and DNA replication as well as those encoding ATP-binding cassette transporters. Cbei_2134 and Cbei_2135 encoding alkyl hydroperoxide reductases are thought to be involved in antibacterial and adaptation mechanisms in C. beijerinckii in the presence of FA. © 2017 Elsevier Ltd


Wu J.,Nanjing University of Technology | Wu J.,National Engineering Technique Research Center for Biotechnology | Chen C.,Research Institute of Benzene Chemical
Acta Crystallographica Section E: Structure Reports Online | Year: 2014

In the title compound, C21H26O2SSe, the S atom adopts a pyramidal geometry (bond-angle sum = 304°) and the n-butyl chain shows an extended conformation. An intramolecular C-H⋯O hydrogen bond closes an S(8) ring. In the crystal, inversion dimers are formed with molecules linked by pairs of O-H⋯O=S hydrogen bonds, generating R 2 2(14) loops. Weak C-H⋯O interactions also occur.


Ye Q.,State Key Laboratory of Materials Oriented Chemical Engineering | Ye Q.,Nanjing University of Technology | Ye Q.,National Engineering Technique Research Center for Biotechnology | Ouyang P.,State Key Laboratory of Materials Oriented Chemical Engineering | And 5 more authors.
Applied Microbiology and Biotechnology | Year: 2011

Ethyl (S)-4-chloro-3-hydroxybutanoate ester ((S)-CHBE) is a precursor of enantiopure intermediates used for the production of chiral drugs, including the cholesterol-lowering 3-hydroxy-3-methyl-glutaryl CoA reductase inhibitors (statins). The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate ester (COBE) to (S)-CHBE by biocatalysis has several positive attributes, including low cost, mild reaction conditions, high yield, and a high level of enantioselectivity. During genome database mining of the yeast Pichia stipitis, our group found two novel carbonyl reductases (PsCRI and PsCRII) that have a promising future for the industrial production of (S)-CHBE with >99% enantiomeric excess. This review covers the main process of biosynthesis of (S)-CHBE: screening of microorganisms that catalyze the reduction of COBE to (S)-CHBE (I); gene cloning, expression, and characterization of carbonyl reductases for the production of (S)-CHBE in Escherichia coli (II); development of cofactor generation systems for regenerating cofactors (III); and biocatalysis of COBE to (S)-CHBE by recombinant E. coli (IV). © 2010 Springer-Verlag.


Zhu C.,Nanjing University of Technology | Zhu C.,National Engineering Technique Research Center for Biotechnology | Zhang Z.,Nanjing University of Technology | Zhang Z.,National Engineering Technique Research Center for Biotechnology | And 12 more authors.
Green Chemistry | Year: 2014

With the aid of the highly active nitroxyl radical AZADO (2-azaadamantane N-oxyl), a simple method for the aerobic catalytic oxidation of alcohols is presented. The oxidations could typically proceed under practical ambient conditions (room temperature, air atmosphere, no moisture effect, metal-free, etc.) with a broad generality of the alcohol substrates, and especially for the oxidation of complex and highly functionalized alcohols. An ionic mechanism is proposed for the present system. © 2014 The Royal Society of Chemistry.


Ye Q.,State Key Laboratory of Materials Oriented Chemical Engineering | Ye Q.,Nanjing University of Technology | Ye Q.,National Engineering Technique Research Center for Biotechnology | Cao H.,State Key Laboratory of Materials Oriented Chemical Engineering | And 15 more authors.
Bioresource Technology | Year: 2010

Biocatalysis of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] was carried out using Escherichia coli co-expressing a carbonyl reductase gene from Pichia stipitis and a glucose dehydrogenase gene from Bacillus megaterium. An efficient polycistronic plasmid with a high-level of enzyme co-expression was constructed by changing the order of the genes, altering the Shine-Dalgarno (SD) regions, and aligned spacing (AS) between the SD sequence and the translation initiation codon. The optimal SD sequence was 5-TAAGGAGG-3, and the optimal AS distance was eight nucleotides. Asymmetric reduction of COBE to (S)-CHBE with more than 99% enantiomeric excess was demonstrated by transformants, using a water/ethyl caprylate system. The recombinant cells produced 1260 mM product in the organic phase, and the total turnover number, defined as moles (S)-CHBE formed per mole NADP+, was 12,600, which was more than 10-fold higher than in aqueous systems. © 2010 Elsevier Ltd. All rights reserved.


Li H.,Nanjing University of Technology | Xu H.,Nanjing University of Technology | Li S.,Nanjing University of Technology | Guo C.,Nanjing University of Technology | And 3 more authors.
Biotechnology and Bioprocess Engineering | Year: 2010

Low-energy nitrogen ion beam implantation technique was used for the strain improvement of Alcaligenes sp. NX-3 for the production of exopolysaccharide welan gum. A high welan gum producing mutant, Alcaligenes sp. NX-3-1, was obtained through 20 keV N + ion beam irradiation. Starting at a concentration of 50 g/L of glucose, mutant NX-3-1 produced 25.0 g/L of welan gum after 66 h of cultivation in a 7.5 L bioreactor, which was 34.4% higher than that produced by the wild-type strain. The results of metabolic flux analysis showed that the glucose-6-phosphate and acetyl coenzyme A nodes were the principle and flexible nodes, respectively. At the glucose-6-phosphate node, the fraction of carbon measured from glucose-6-phosphate to glucose-1-phosphate was enhanced after mutagenesis, which indicated that more flux was used to synthesize welan gum in the mutant. By analyzing the activities of related enzymes in the biosynthetic pathway of sugar nucleotides essential for welan gum production, we found that the specific activities of phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-glucose dehydrogenase, and dTDP-glucose pyrophosphorylase in the mutant strain were higher than those in the wild-type strain. These improvements in enzyme activities could be due to the affected of ion beam implantation. © 2010 The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg.


Cao H.,State Key Laboratory of Materials Oriented Chemical Engineering | Cao H.,Nanjing University of Technology | Mi L.,Nanjing University of Technology | Ye Q.,State Key Laboratory of Materials Oriented Chemical Engineering | And 16 more authors.
Bioresource Technology | Year: 2011

A novel NADH-dependent dehydrogenases/reductases (SDRs) superfamily reductase (PsCRII) was isolated from Pichia stipitis. It produced ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] in greater than 99% enantiomeric excess. This enzyme was purified to homogeneity by ammonium sulfate precipitation followed by Q-Sepharose chromatography. Compared to similar known reductases producing (S)-CHBE, PsCR II was more suitable for production since the purified PsCRII preferred the inexpensive cofactor NADH to NADPH as the electron donor. Furthermore, the Km of PsCRII for ethyl 4-chloro-3-oxobutanoate (COBE) was 3.3mM, and the corresponding Vmax was 224μmol/mg protein/min. The catalytic efficiency is the highest value ever reported for NADH-dependent reductases from yeasts that produce CHBE with high enantioselectivity. In addition, this enzyme exhibited broad substrate specificity for several β-keto esters using NADH as the coenzyme. The properties of PsCRII with those of other carbonyl reductases from yeasts were also compared in this study. © 2010 Elsevier Ltd.


Zhu C.,Nanjing University of Technology | Zhu C.,National Engineering Technique Research Center for Biotechnology | Li Q.,Nanjing University of Technology | Pu L.,Nanjing University of Technology | And 6 more authors.
ACS Catalysis | Year: 2016

The application of synthetic flavinium organocatalysts for the in situ regeneration of oxidized cofactors NAD(P)+ using O2 as the terminal oxidant without any special illumination or equipment is reported. With the aid of the highly active bridged flavinium catalyst, the rate of NAD(P)H oxidation is accelerated by 3 orders of magnitude. The results show that the catalytic activity of the bridged flavinium catalyst is not dependent on light but on only oxygen. Furthermore, this catalyst is compatible with various preparative enzymatic oxidation reactions. A hydride transfer mechanism is proposed for the presented system. © 2016 American Chemical Society.


Ren B.,Nanjing University of Technology | Li S.,Nanjing University of Technology | Xu H.,Nanjing University of Technology | Feng X.-H.,Nanjing University of Technology | And 2 more authors.
Bioprocess and Biosystems Engineering | Year: 2011

A highly selective sucrose isomerase (SIase) was purified to homogeneity from the cell-free extract of Erwinia rhapontici NX-5 with a recovery of 27.7% and a fold purification of 213.6. The purified SIase showed a high specific activity of 427.1 U mg-1 with molecular weight of 65.6 kDa. The K m for sucrose was 222 mM while V max was 546 U mg -1. The optimum pH and temperature for SIase activity were 6.0 and 30 °C, respectively. The purified SIase was stable in the temperature range of 10-40 °C and retained 65% of the enzyme activity after 2 weeks' storage at 30 °C. The SIase activity was enhanced by Mg2+ and Mn 2+, inhibited by Ca2+, Cu2+, Zn2+, and Co2+, completely inhibited by Hg2+ and Ag 2+. The purified SIase was strongly inhibited by SDS, while partially inhibited by dimethylformamide, tetrahydrofuran, and PMSF. Additionally, glucose and fructose acted as competitive inhibitors for purified SIase. © 2011 Springer-Verlag.


Lin X.,State Key Laboratory of Materials Oriented Chemical Engineering | Lin X.,Nanjing University of Technology | Lin X.,National Engineering Technique Research Center for Biotechnology | Wu J.,State Key Laboratory of Materials Oriented Chemical Engineering | And 16 more authors.
Journal of Chemical Technology and Biotechnology | Year: 2012

Background: Owing to the rapid depletion of petroleum fuel, the production of bio-butanol has attracted much attention. However, low butanol productivity severely limits its potential industrial application. It is important to establish an approach for recovering low-concentration butanol from fermentation broth. Experiments were conducted using batch adsorption mode under different conditions of initial butanol concentration and temperature. Batch adsorption data were fitted to Langmuir and Freundlich isotherms and the macropore diffusion, pseudo-first- and second-order models for kinetic study. Results: The maximum adsorption capacity of butanol onto KA-I resin increase with increasing temperature, ranged from 139.836 to 304.397 mg g -1. The equilibrium adsorption data were well fitted by the Langmuir isotherm. The adsorption kinetics was more accurately represented by the macropore diffusion model, which also clearly predicted the intraparticle distribution of the concentration. The effective pore diffusivity (D p) was dependent upon temperature, but independent of initial butanol concentration, and was 0.251 × 10 -10, 0.73 × 10 -10, 1.32 × 10 -10 and 4.31 × 10 -10 m 2 s -1 at 283.13, 293.13, 303.13 and 310.13 K, respectively. Conclusion: This work demonstrates that KA-I resin is an efficient adsorbent for the removal of butanol from aqueous solutions and available for practical applications for future in situ product recovery of butanol from ABE fermentation broth. © 2012 Society of Chemical Industry.

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