Yuan-Shan Wang

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.

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.

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.

Stability study on the nitrile hydratase of Nocardia sp. 108: from resting cell to crude enzyme preparation.

In recent years nitrile hydratases (NHases) have drawn increasing attention due to their critical roles in organic synthesis. In the present paper an extensive investigation on the stability and activity of NHase from Nocardia sp. 108, which has succeeded in industrial application in China, was conducted by bioconversion of acrylonitrile to acrylamide in a batch manner. A study of cultivation demonstrated that biosynthesis of NHase changed significantly with the time of the culture, and the optimal NHase biosynthesis phase was 45 h after inoculation with NHase activity of a biomass of 1209.8 U/g. A stability study indicated that both crude enzyme preparations exhibited a good stability when exposed to a pH 7.2 tris-HCl buffer at 4°C for 4 h.

Enhanced Production of Acarbose and Concurrently Reduced Formation of Impurity C by Addition of Validamine in Fermentation of Actinoplanes utahensis ZJB-08196

Commercial production of acarbose is exclusively via done microbial fermentation with strains from the genera of Actinoplanes. The addition of C7N-aminocyclitols for enhanced production of acarbose and concurrently reduced formation of impurity C by cultivation of A. utahensis ZJB-08196 in 500-mL shake flasks was investigated, and validamine was found to be the most effective strategy. Under the optimal conditions of validamine addition, acarbose titer was increased from 3560 ± 128 mg/L to 4950 ± 156 mg/L, and impurity C concentration was concurrently decreased from 289 ± 24 mg/L to 107 ± 29 mg/L in batch fermentation after 168 h of cultivation. A further fed-batch experiment coupled with the addition of validamine (20 mg/L) in the fermentation medium prior to inoculation was designed to enhance the production of acarbose. When twice feedings of a mixture of 6 g/L glucose, 14 g/L maltose, and 9 g/L soybean flour were performed at 72 h and 96 h, acarbose titer reached 6606 ± 103 mg/L and impurity C concentration was only 212 ± 12 mg/L at 168 h of cultivation. Acarbose titer and proportion of acarbose/impurity C increased by 85.6% and 152.9% when compared with control experiments. This work demonstrates for the first time that validamine addition is a simple and effective strategy for increasing acarbose production and reducing impurity C formation.

Acarbose Isolation with Gel Type Strong Acid Cation Exchange Resin: Equilibrium, Kinetic and Thermodynamic Studies

Abstract Acarbose, a potent α-glucosidase inhibitor, is widely used as an oral anti-diabetic drug for the treatment of the type 2, non-insulin-dependent diabetes. In this work, a gel type strong acid cation exchange resin 001×4 was applied to isolate acarbose from fermentation broth. It was demonstrated that cation exchanger 001×4 displayed a large adsorption capacity and quick exchange rate for acarbose. The static adsorption equilibrium data were well fitted to the Langmuir equation. Column adsorption experiments demonstrated that high dynamic adsorption capacity was reached at bed height of 104.4 mm, feed flow rate of 1.0 ml·min −1 and acarbose concentration of 4.0 mg·ml −1 . Under the optimized conditions, the column chromatography packed with cation exchanger 001×4 recovered 74.3% (by mass) of acarbose from Actinoplanes utahensis ZJB-08196 fermentation broth with purity of 80.1% (by mass), demonstrating great potential in the practical applications in acarbose separation.

Corrigendum to “A spectrophotometric screening method for avermectin oxidizing microorganisms” [J. Microbiol. Methods 135 (2017) 93–95]

a Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang Province 310014, PR China b Engineering Research Center of Bioconversion and Biopurification, Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang Province 310014, PR China c Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, Zhejiang Province 310014, PR China d College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou, Zhejiang Province 310015, PR China

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.