Yin-Chu Shen

Isolation, identification and characterization of Bacillus subtilis ZJB-063, a versatile nitrile-converting bacterium.

Strain ZJB-063, a versatile nitrile-amide-degrading strain, was newly isolated from soil in this study. Based on morphology, physiological tests, Biolog and the 16S rDNA sequence, strain ZJB-063 was identified as Bacillus subtilis. ZJB-063 exhibited nitrilase activity without addition of inducers, indicating that the nitrilase in B. subtilis ZJB-063 is constitutive. Interestingly, the strain exhibited nitrile hydratase and amidase activity with the addition of ɛ-caprolactam. Moreover, the substrate spectrum altered with the alteration of enzyme systems due to the addition of ɛ-caprolactam. The constitutive nitrilase was highly specific for arylacetonitriles, while the nitrile hydratase/amidase in B. subtilis ZJB-063 could not only hydrolyze arylacetonitriles but also other nitriles including some aliphatic nitriles and heterocyclic nitriles. Despite comparatively low activity, the amidase of hydratase/amidase system was effective in converting amides to acids. The versatility of this strain in the hydrolysis of various nitriles and amides makes it a potential biocatalyst in organic synthesis.

Microbial Transformation of Nitriles to High-Value Acids or Amides

Biotransformation of nitriles mediated by nitrile-amide converting enzymes has attracted considerable attention and developed tremendously in the recent years in China since it offers a valuable alternative to traditional chemical reaction which requires harsh conditions. As a result, an upsurge of these promising enzymes (including nitrile hydratase, nitrilase and amidase) has been taking place. This review aims at describing these enzymes in detail. A variety of microorganisms harboring nitrile-amide converting activities have been isolated and identified in China, some of which have already applied with moderate success. Currently, a wide range of high-value compounds such as aliphatic, alicyclic, aromatic and heterocyclic amides and their corresponding acids were provided by these nitrile-amide degrading organisms. Simultaneously, with the increasing demand of chiral substances, the enantioselectivity of the nitrilase superfamily is widely investigated and exploited in China, especially the bioconversion of optically active alpha-substituted phenylacetamides, acids and 2,2-dimethylcyclopropanecarboxamide and 2,2-dimethylcyclopropanecarboxylic acid by means of the catalysts exhibiting excellent stereoselectivity. Besides their synthetic value, the nitrile-amide converting enzymes also play an important role in environmental protection. In this context, cloning of the genes and expression of these enzymes are presented. In the near future in China, an increasing number of novel nitrile-amide converting organisms will be screened and their potential in the synthesis of useful acids and amides will be further exploited.

Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10.

Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids. In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The optimum pH and temperature for the purified nitrilase was 7-8 and 40 °C, respectively. Mg(2+) stimulated hydrolytic activity, whereas Cu(2+), Co(2+), Ni(2+), Ag(+), and Hg(2+) showed a strong inhibitory effect. The K(m) and v(max) for mandelonitrile were 4.74 mM and 15.85 μmol min(-1) mg(-1) protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(-)-mandelic acid was >99%. Characteristics investigation indicates that this nitrilase is promising in catalysis applications.

Use of glycerol for producing 1,3-dihydroxyacetone by Gluconobacter oxydans in an airlift bioreactor.

1,3-Dihydroxyacetone can be produced by biotransformation of glycerol with glycerol dehydrogenase from Gluconobacter oxydans cells. Firstly, improvement the activity of glycerol dehydrogenase was carried out by medium optimization. The optimal medium for cell cultivation was composed of 5.6g/l yeast extract, 4.7 g/l glycerol, 42.1g/l mannitol, 0.5 g/l K(2)HPO(4), 0.5 g/l KH(2)PO(4), 0.1g/l MgSO(4)·7H(2)O, and 2.0 g/l CaCO(3) with the initial pH of 4.9. Secondly, an internal loop airlift bioreactor was applied for DHA production from glycerol by resting cells of G. oxydans ZJB09113. Furthermore, the effects of pH, aeration rate and cell content on DHA production and glycerol feeding strategy were investigated. 156.3 ± 7.8 g/l of maximal DHA concentration with 89.8±2.4% of conversion rate of glycerol to DHA was achieved after 72h of biotransformation using 10g/l resting cells at 30°C, pH 5.0 and 1.5vvm of aeration rate.

Isolation of brefeldin A from Eupenicillium brefeldianum broth using macroporous resin adsorption chromatography

Brefeldin A (BFA) is a macrolide lactone antibiotic, possessing antitumor, antiviral, antifungal activities. In this work, a separation strategy involving one-step macroporous resin adsorption chromatography combined with crystallization was established for BFA purification from Eupenicillium brefeldianum CCTCC M 208113 fermentation broth. Among six macroporous resin adsorbents tested, the non-polar resin HZ830 had the best adsorption and desorption performance. The static equilibrium adsorption data fitted well with the Freundlich equation, and the adsorption kinetic followed the pseudo-second order model. Through experimental optimization of column adsorption and desorption, BFA in purity of 90.4% (w/w), 92.1% (w/w) yield was obtained by a one-step macroporous resin adsorption chromatography, using a stepwise elution protocol. Furthermore, high purity (>99%, w/w) of BFA crystals were prepared from E. brefeldianum CCTCC M 208113 fermentation broth in an overall recovery of 67.0% (w/w), using a combination of adsorption chromatography packed with non-polar macroporous adsorbent HZ830 and crystallization in acetone.

A screening system for active and enantioselective amidase based on its acyl transfer activity

A novel enantioselective amidase screening system was developed and proved to be efficient and accurate. This screening system employed acyl transfer activity of amidase in the presence of hydroxylamine, leading to the formation of hydroxamic acids, followed by spectrophotometric quantification of hydroxamic acid/iron(III) complexes. The enantioselectivities of amidase were evaluated by employing (R, S)-2, 2-dimethyl cyclopropanecarboxamide (1), (S)-2, 2-dimethyl cyclopropanecarboxamide and their mixture as substrates concurrently under the same conditions. To prove the accuracy of the screening system, enantioselectivity of acyl transfer reaction (ET) and that of hydrolytic reaction (EH) was compared. With this method, we obtained eight microorganism strains with enantioselective amidase from 523 isolates, two of which showed R-stereospecific avtivity for (R, S)-1.

A novel synthesis of iminodiacetic acid: Biocatalysis by whole Alcaligenes faecalis ZJB-09133 cells from iminodiacetonitrile

Iminodiacetic acid (IDA) has been widely used as an important intermediate in the fine chemical industry. In this study, a novel synthesis route of IDA from iminodiacetonitrile by whole microorganisms was investigated. A strain with the capability of producing nitrilase, ZJB‐09133, was isolated and identified, and later named Alcaligenes faecalis ZJB‐09133. In addition, the detailed biocatalysis of iminodiacetonitrile to produce IDA using ZJB‐09133 was investigated. The results showed that the conversion reached 65.3% in Na2HPO4‐NaH2PO4 buffer of pH 8.0 under the following conditions: cells in the amount of 0.075‐g DCW/L, 1.5% substrate, conversion time of 8 h, and a reaction temperature of 35°C. To the best of our knowledge, this is the first time that the production of IDA using a biocatalysis method has been reported.

Screening and Improving the Recombinant Nitrilases and Application in Biotransformation of Iminodiacetonitrile to Iminodiacetic Acid

In this study, several nitrilase genes from phylogenetically distinct organisms were expressed and purified in E. coli in order to study their ability to mediate the biotransformation of nitriles. We identified three nitrilases: Acidovorax facilis nitrilase (AcN); Alcaligenes fecalis nitrilase (AkN); and Rhodococcus rhodochrous nitrilase (RkN), which catalyzed iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA). AcN demonstrated 8.8-fold higher activity for IDAN degradation as compared to AkN and RkN. Based on homology modeling and previously described ‘hot spot’ mutations, several AcN mutants were screened for improved activity. One mutant M3 (F168V/L201N/S192F) was identified, which demonstrates a 41% enhancement in the conversion as well as a 2.4-fold higher catalytic efficiency towards IDAN as compared to wild-type AcN.

Actinoplanes utahensis ZJB-08196 fed-batch fermentation at elevated osmolality for enhancing acarbose production

Acarbose, a potent α-glucosidase inhibitor, is as an oral anti-diabetic drug for treatment of the type two, noninsulin-dependent diabetes. Actinoplanes utahensis ZJB-08196, an osmosis-resistant actinomycete, had a broad osmolality optimum between 309 mOsm kg(-1) and 719 mOsm kg(-1). Utilizing this unique feature, an fed-batch culture process under preferential osmolality was constructed through intermittently feeding broths with feed medium consisting of 14.0 g l(-1) maltose, 6.0 g l(-1) glucose and 9.0 g l(-1) soybean meal, at 48 h, 72 h, 96 h and 120 h. This intermittent fed-batch culture produced a peak acarbose titer of 4878 mg l(-1), increased by 15.9% over the batch culture.

One-pot, single-step deracemization of 2-hydroxyacids by tandem biocatalytic oxidation and reduction

A facile and efficient one-pot, single-step method for deracemizing a broad range of 2-hydroxyacids to (R)-2-hydroxyacids was established by combination of resting cells of an (S)-hydroxyacid dehydrogenase-producing microorganism and an (R)-ketoacid reductase-producing microorganism.