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1.
Shi-Ming Yang Wei Chen Wei-Wei Guo Shuping Jia Jian-He Sun Hui-Zhan Liu Wie-Yen Young David Z. Z. He 《PloS one》2012,7(9)
The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60–70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die. 相似文献
2.
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. 相似文献
3.
4.
Yan Zhang Jiang Pan Zheng-Jiao Luan Guo-Chao Xu Sunghoon Park Jian-He Xu 《Applied and environmental microbiology》2014,80(23):7348-7355
A novel nonheme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (S)-2,2-dimethylcyclopropanecarboxylate, was identified from a shotgun library of Rhodococcus sp. strain ECU1013. RhEst1 was overexpressed in Escherichia coli BL21(DE3), purified to homogeneity, and functionally characterized. Fingerprinting analysis revealed that RhEst1 prefers para-nitrophenyl (pNP) esters of short-chain acyl groups. pNP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for RhEst1. The Km values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively. RhEst1 could serve as an efficient hydrolase for the bioproduction of optically pure (S)-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa), which is an important chiral building block for cilastatin. As much as 0.5 M DmCpCe was enantioselectively hydrolyzed into (S)-DmCpCa, with a molar yield of 47.8% and an enantiomeric excess (ee) of 97.5%, indicating an extremely high enantioselectivity (E = 240) of this novel and unique biocatalyst for green manufacturing of highly valuable chiral chemicals. 相似文献
5.
Chiral secondary alcohols play an important role in pharmaceutical, agrochemical, and chemical industries. In recent years, impressive steps forward have been achieved towards biocatalytic ketone reduction as a green and useful access to enantiopure alcohols. An increasing number of novel and robust enzymes are now accessible as a result of the ongoing progress in genomics, screening and evolution technologies, while process engineering provides further success in areas of biocatalytic reduction in meeting synthetic challenges. The versatile platform of these techniques and strategies offers the possibility to apply high substrate loading and thus to overcome the limitation of low volumetric productivity of usual enzymatic processes which is the bottleneck for their practical application. In addition, the integration of bioreduction with other enzymatic or chemical steps allows the efficient synthesis of more complex chiral products. 相似文献
6.
Jian-Wei Zhao Hua-Lei Wu Jian-Dong Zhang Wen-Chao Gao Xiao-Jun Fan Hong-Hong Chang Wen-Long Wei Jian-He Xu 《Biotechnology letters》2018,40(2):349-358
Objectives
To investigate the efficiency of a new cascade biocatalysis system for the conversion of R, S-β-amino alcohols to enantiopure vicinal diol and β-amino alcohol.Results
An efficient cascade biocatalysis was achieved by combination of a transaminase, a carbonyl reductase and a cofactor regeneration system. An ee value of > 99% for 2-amino-2-phenylethanol and 1-phenyl-1, 2-ethanediol were simultaneously obtained with 50% conversion from R, S-2-amino-2-phenylethanol. The generality of the cascade biocatalysis was further demonstrated with the whole-cell approaches to convert 10–60 mM R, S-β-amino alcohol to (R)- and (S)-diol and (R)- and (S)-β-amino alcohol in 90–99% ee with 50–52% conversion. Preparative biotransformation was demonstrated at a 50 ml scale with mixed recombinant cells to give both (R)- and (S)-2-amino-2-phenylethanol and (R)- and (S)-1-phenyl-1, 2-ethanediol in > 99% ee and 40–42% isolated yield from racemic 2-amino-2-phenylethanol.Conclusions
This cascade biocatalysis system provides a new practical method for the simultaneous synthesis of optically pure vicinal diol and an β-amino alcohol.7.
Xin Ju Hui-Lei Yu Jiang Pan Dong-Zhi Wei Jian-He Xu 《Applied microbiology and biotechnology》2010,86(1):83-91
Substrate-directed screening was carried out to find bacteria that could deacylate O-acetylated mandelic acid from environmental samples. From more than 200 soil isolates, we identified for the first time that
Pseudomonas sp. ECU1011 biocatalytically deacylated (S)-α-acetoxyphenylacetic acid with high enantioselectivity (E > 200), yielding (S)-mandelic acid with 98.1% enantiomeric excess (ee) at a 45.5% conversion rate. The catalytic deacylation of (S)-α-acetoxyphenylacetic acid by the resting cell was optimized using a single-factor method to yield temperature and pH optima
of 30°C and 6.5, respectively. These optima help to reduce the nonselective spontaneous hydrolysis of the racemic substrate.
It was found that substrate concentrations up to 60 mM could be used. 2-Propanol was used as a moderate cosolvent to help
the substrate disperse in the aqueous phase. Under optimized reaction conditions, the ee of the residual (R)-α-acetoxyphenylacetic acid could be improved further, to greater than 99%, at a 60% conversion rate. Furthermore, using
this newly isolated strain of Pseudomonas sp. ECU1011, three kinds of optically pure analogs of (S)-mandelic acid and (R)-α-acetoxyphenylacetic acid were successfully prepared at high enantiomeric purity. 相似文献
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9.
A fungus strain ECU2002, capable of enantioselectively hydrolyzing chiral lactones to optically pure hydroxy acids, was newly
isolated from soil samples through two steps of screening and identified as Fusarium proliferatum (Matsushima) Nirenberg. From the crude extract of F. proliferatum ECU2002, a novel levo-lactonase was purified to homogeneity, with a purification factor of 460-folds and an overall yield of 9.7%, by ultrafiltration,
acetone precipitation, and chromatographic separation through DEAE-Toyopearl, Butyl-Toyopearl, Hydroxyapatite, Toyoscreen-Super
Q, and TSK-gel columns. The purified enzyme is a monomer; with a molecular mass of ca 68 kDa and a pI of 5.7 as determined by two-dimensional electrophoresis. The catalytic performance of the partially purified levo-lactonase was investigated, giving temperature and pH optima at 50°C and 7.5, respectively, for γ-butyrolactone hydrolysis.
The substrate specificity of the partially purified lactonase was also examined using several useful lactones, among which
α-hydroxy-γ-butyrolactone was the best substrate, with 448-fold higher lactonase activity as compared to γ-butyrolactone.
The F. proliferatum lactonase preferentially hydrolyzed the levo enantiomer of butyrolactones, including β-butyrolactone, α-hydroxy-γ-butyrolactone, α-hydroxy-β,β-dimethyl-γ-butyrolactone
(pantolactone), and β-hydroxy-γ-butyrolactone, affording (+)-hydroxy acids in high (94.8∼98.2%) enantiomeric excesses (ee) and good conversions (38.2∼44.2%). A simple immobilization of the crude lactonase with glutaraldehyde cross-linking led
to a stable and easy-to-handle biocatalyst for catalytic resolution of chiral lactones. The immobilized lactonase also performed
quite well in repeated batch resolution of dl-pantolactone at a concentration of 35% (w/v), retaining 67% of initial activity after ten cycles of reaction (corresponding to a half life of 20 cycles) and affording
the product in 94∼97% ee, which can be easily enhanced to >99% ee after the d-hydroxy acid was chemically converted into l-lactone and crystallized. 相似文献
10.
We optimized culture medium and batch-fed fermentation conditions to enhance production of an acetyl esterase from Pseudomonas sp. ECU1011 (PSAE). This enzyme enantioselectively deacetylates α-acetoxyphenylacetic acid. The medium was redesigned by
single-factor and statistical optimization. The addition of ZnSO4 enhanced enzyme production by 37%. Yeast extract concentration was directly associated with the enzyme production. The fermentation
was scaled up in a 5-l fermenter with the optimized medium, and the correlations between enzyme production and dissolved oxygen,
pH, and feeding strategy were investigated. The fermentation process was highly oxygen-demanding, pH sensitive and mandelic
acid-inducible. The fermentation pH was controlled at 7.5 by a pH and dissolved oxygen feedback strategy. Feeding mandelic
acid as both a pH regulator and an enzyme inducer increased the enzyme production by 23%. The results of the medium redesign
experiments were confirmed and explained in fed-batch culture experiments. Mathematical models describing the fermentation
processes indicated that the enzyme production was strongly associated with cell growth. The optimized pH and dissolved oxygen
stat fed-batch process resulted high volumetric production of PSAE (4166 U/l, 7.2-fold higher than the initial) without enantioselectivity
decline. This process has potential applications for industrial production of chiral mandelic acid or its derivatives. 相似文献