Ya-Ping Xue

Production of Octenyl Succinic Anhydride-Modified Waxy Corn Starch and Its Characterization

The objective of this work is to investigate the effects of reaction conditions on the synthesis of octenyl succinic anhydride (OSA)-modified starch from waxy corn starch and to study the characteristics of the OSA-modified starch as well as its applications. A mathematical model was developed to investigate the influences of various processing condition factors on the production of the OSA-modified waxy corn starch production and predict the optimum reaction conditions. The maximal degree of substitution (DS) of OSA-modified waxy corn starch (0.0204) was predicted to occur when the starch concentration was 31.2%, the pH was 8.6, the reaction temperature was 33.6 degrees C, and the reaction time was 18.7 h. Repeated reactions for producing OSA-modified waxy corn starch were carried out in a 5 m(3) reactor under the optimized conditions for verification of the model. The characteristics of modified waxy corn starch including infrared spectrum, scanning electron microscopy, and pasting property were tested and emulsification capacity of the OSA-modified starch were evaluated as well.

Improvement of Alcaligenes faecalis nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of (R)-(-)-mandelic acid.

Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(-)-mandelic acid after 7.5 h of conversion reached 693 mM with an enantiomeric excess of 99%, and the space-time productivity of Mut3 was 21.50-fold higher than that of wild-type nitrilase. The Km, Vmax, and k(cat) of wild-type and Mut3 for mandelonitrile were 20.64 mM, 33.74 μmol mg(-1) min(-1), 24.45 s(-1), and 9.24 mM, 47.68 μmol mg(-1) min(-1), and 34.55 s(-1), respectively. A homology modeling and molecular docking study showed that the diameter of the catalytic tunnel of Mut3 became longer and that the tunnel volume was smaller. These structural changes are proposed to improve the hydrolytic activity and pH stability of Mut3. Mut3 has the potential for industrial applications in the upscale production of (R)-(-)-mandelic acid.

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.

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.

Recent advances in biotechnological applications of alcohol dehydrogenases

Alcohol dehydrogenases (ADHs), which belong to the oxidoreductase superfamily, catalyze the interconversion between alcohols and aldehydes or ketones with high stereoselectivity under mild conditions. ADHs are widely employed as biocatalysts for the dynamic kinetic resolution of racemic substrates and for the preparation of enantiomerically pure chemicals. This review provides an overview of biotechnological applications for ADHs in the production of chiral pharmaceuticals and fine chemicals.

Biosynthesis of Iminodiacetic Acid from Iminodiacetonitrile by Immobilized Recombinant Escherichia coli Harboring Nitrilase

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacture of chelating agents, glyphosate herbicides and surfactants. In the current work, the fragment with the length of 1,110 bp encoding the Acidovorax facilis nitrilase was obtained. The recombinant nitrilase expressed in Escherichia coli BL21 (DE3) was successfully used in the production of IDA from iminodiacetonitrile. To improve the stability of operation, the recombinant cells were entrapped in polyvinyl alcohol (PVA) and sodium alginate (SA) copolymer. The maximum relative nitrilase activity with 98.1% was further observed at 1.0% SA, 8.0% PVA, 1.0% CaCl2, and 5.0% wet cells, under conditions of 1.0% iminodiacetonitrile in distilled water and a temperature of 40°C, respectively. The entrapped cells facilitated easy separation and good recycling compared with free cells. Moreover, the immobilized cells showed good operation and storage stability. This report is the first to describe IDA preparation using immobilized recombinant E. coli harboring nitrilase.

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.

Enzymatic synthesis of an ezetimibe intermediate using carbonyl reductase coupled with glucose dehydrogenase in an aqueous-organic solvent system.

(4S)-3-[(5S)-5-(4-Fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one ((S)-ET-5) is an important chiral intermediate in the synthesis of chiral side chain of ezetimibe. Recombinant Escherichia coli expressing carbonyl reductase (CBR) was successfully constructed in this study. The total E. coli biomass and the specific activity of recombinant CBR in 5L fermenter culture were 10.9gDCWL-1 and 14900.3Ug-1DCW, respectively. The dual-enzyme coupled biocatalytic process in an aqueous-organic biphasic solvent system was first constructed using p-xylene as the optimal organic phase under optimized reaction conditions, and 150gL-1 (4S)-3-[5-(4-fluorophenyl)-1,5-dioxophentyl]-4-phenyl-1,3-oxazolidin-2-one (ET-4) was successfully converted to (S)-ET-5 with a conversion of 99.1% and diastereomeric excess of 99% after 24-h, which are the highest values reported to date for the production of (S)-ET-5.

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.

Directed Evolution of Carbonyl Reductase from Rhodosporidium toruloides and Its Application in Stereoselective Synthesis of tert-Butyl (3R,5S)-6-Chloro-3,5-dihydroxyhexanoate

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl reductase RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH). For the activity of RtSCR9 to be improved, random mutagenesis and site-saturation mutagenesis were performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys, and mut-Phe156Gln). These mutants exhibited 1.94-, 3.03-, and 1.61-fold and 1.93-, 3.15-, and 1.97-fold improvement in the specific activity and kcat/Km, respectively. Asymmetric reduction of (S)-CHOH by mut-Ile144Lys coupled with glucose dehydrogenase was conducted. The yield and enantiomeric excess of (3R,5S)-CDHH reached 98 and 99%, respectively, after 8 h bioconversion in a single batch reaction with 1 M (S)-CHOH, and the space-time yield reached 542.83 mmol L-1 h-1 g-1 wet cell weight. This study presents a new carbonyl reductase for efficient synthesis of (3R,5S)-CDHH.