Jian-Miao Xu

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.

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.

Significant improvement of the nitrilase activity by semi-rational protein engineering and its application in the production of iminodiacetic acid

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacturing of chelating agents, glyphosate herbicides and surfactants. To improve activity and tolerance to the substrate for IDA production, Acidovorax facilis nitrilase was selected for further modification by the gene site saturation mutagenesis method. After screened by a two-step screening method, the best mutant (Mut-F168V/T201N/S192F/M191T/F192S) was selected. Compared to the wild-type nitrilase, Mut-F168V/T201N/S192F/M191T/F192S showed 136% improvement in specific activity. Co2+ stimulated nitrilase activity, whereas Cu2+, Zn2+ and Tween 80 showed a strong inhibitory effect. The Vmax and kcat of Mut-F168V/T201N/S192F/M191T/F192S were enhanced 1.23 and 1.23-fold, while the Km was decreased 1.53-fold. The yield of Mut-F168V/T201N/S192F/M191T/F192S with 453.2 mM of IDA reached 71.9% in 5 h when 630 mM iminodiacetonitrile was used as substrate. This study indicated that mutant nitrilase obtained in this study is promising in applications for the upscale production of IDAN.

IMPROVEMENT OF 1,3-DIHYDROXYACETONE PRODUCTION FROM Gluconobacter oxydans BY ION BEAM IMPLANTATION

Improvement of dihydroxyacetone (DHA) production by mutagenesis of ion beam implantation and medium optimization using response-surface methodology (RSM) were investigated in this work. More than 1000 mutant strains were selected through a mutagenesis method using N+ ions implantation with a dose of 60 × (2.6 × 1013) ions/cm2 and energy of 10 keV. Several high-yield mutant strains were showed the potent application for DHA production and the genetically stable mutant strain G. oxydans ZJB09113 was selected for optimization of cultivation condition by RSM. The optimal medium for DHA fermentation is composed (in g/L) of yeast extract 4.88, CaCO3 2.00, and glycerol 52.86 mL/L (initial pH 4.89). The maximal DHA concentration of 40.0 g/L was achieved after 24 hr of shaken flask fermentation at 30°C with 150 rpm, and 196.3% increase in DHA production in comparison with unoptimized conditions.

Production of l-tryptophan by enantioselective hydrolysis of d,l-tryptophanamide using a newly isolated bacterium

Bacterial strain ZJB-09211 capable of amidase production has recently been isolated from soil samples. The strain is able to asymmetrically hydrolyze l-tryptophanamide from d,l-tryptophanamide to produce l-tryptophan in high yield and with excellent stereoselectivity (enantiomeric excess > 99.9 %, and enantiomeric ratio > 200). Strain ZJB-09211 has been identified as Flavobacterium aquatile based on the cell morphology analysis, physiological tests, and the 16S rDNA sequence analysis. Optimization of the fermentation medium led to an about six-fold increase in the amidase activity of strain ZJB-09211, which reached 501.5 U L−1. Substrate specifity and stereoselectivity investigations revealed that amidase of F. aquatile possessed a broad substrate spectrum and high enantioselectivity.