| Nitrile hydratase (NHase) is one of the important enzymes of nitrile metabolism in numerous microbes that catalyses the hydration of nitriles to higher-value amides, and has been successfully adopted in the chemical industry for production of pharmaceuticals, agrochemicals and fine chemicals. 2-Amino-2,3-dimethylbutyramide (ADBA) is a key intermediate of highly potent and broad spectrum imidazolinone herbicides. Enzymatic hydration of 2-amino-2,3-dimethylbutyronitrile (ADBN) by NHase offers alternative to conventional ADBA production due to the high yield, mild conditions and environment-friendly nature. This paper focused on the bioconversion of ADBN to ADBA and investigated in respect of screening, identification, cultivation and application of the biocatalysts, as well as separation and purification of product.A combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase withα-amino nitriles andα-amino amides as substrates, respectively. Mechanisms of color changes were further proposed. It was due to the reactions involved ferrous ions, ferric ions, cyanide ions dissociated spontaneously fromα-amino nitriles solution and hydroxyl ions in theα-amino amides solution. Using this method, 3 strains with nitrile hydratase activity towards ADBN were screened from 418 isolates. 2 of them named ZA0707 and P4 were isolated from soil samples and the other one, R. boritolerans CCTCC M 208108 was selected from culture collections in our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both NHase and amidase.Strain ZA0707 screened from soil samples was identified as Rhodococcus qingshengii on the basis of its morphological and physiological features along with API identification system and 16S rDNA sequencing method. This is the first report on strains in this species with NHase activity. In addition, the cyanide-resistance, product tolerance, thermostability and NHase activity towards ADBN of strain ZA0707 and R. boritolerans CCTCC M 208108 were investigated. Consequently, R. boritolerans CCTCC M 208108 with the better results in all these aspects were selected as the optimum catalyst for the following investigation.The medium composition for NHase formation in R. boritolerans CCTCC M 208108 was optimized by single factors and response surface methodology. The optimum medium composition was as follows (g/l): sucrose 7.00, sodium citrate 3.04, beef extract 5.13, yeast extract 5.00, caprolactam 1.50, KH2PO4 1.0, K2HPO4 1.0, NaCl 1.0, FeCl3 0.005, CoCl2 0.005, MnSO4 0.005. Furthermore, the satisfactory culture conditions for cell growth and NHase production were obtain as bellow: initial pH6.5, temperature 30°C, medium volumetric ratio 16% (v/v), inoculum size 3.75% (v/v). Under these conditions, 6.21 g/l biomass and 5393 U/g CDW specific NHase activity were achieved after 60 h cultivation.The influences of reaction conditions and media on NHase activity were also investigated. It was indicated that the NHase exhibited maximal activity in Tris-HCl buffer (pH 8.9) at 30°C. The addition of 30 % (v/v) n-hexane and 1 mM Ni2+ in the reaction mixture resulted in approximately 30 and 23 %, respectively, enhancement in NHase activity. Kinetic studies on the NHase catalyzed reactions were performed and the kinetic constants were as follows: Vmax = 15.73μmol·min?1·mg?1, Km = 44.30 mM. Moreover, ADBN was found to be more stable in water at lower temperature. Especially, cyanide accumulated very slowly in the initial 10 h at 10 oC, giving concentrations of 2.01 mM, which was less than a quarter of that at 30 oC. Based on these findings, a preparative scale (800 ml) process for continuous production of ADBA in both aqueous and biphasic systems was developed and some key parameters of the biocatalytic process were optimized. Inhibition of NHase by cyanide dissociated from ADBN was successfully overcome by temperature control (at 10 oC). The product concentration, yield and catalyst productivity were further improved to 50 g·l-1, 91% and 6.3 g product/g catalyst using a 30/100 (v/v) n-hexane/water biphasic system. Furthermore, cells of R. boritolerans CCTCC M 208108 could be reused for at lease twice by stopping the continuous reaction before cyanide concentration rose to 2 mM, with the catalyst productivity increasing to 12.3 g product/g catalyst.Next, in order to improve the cyanide-resistance and reusability of the biocatalyst, expanded perlite was employed to immobilize R. boritolerans CCTCC M 208108. After optimization in terms of NHase activity, the optimum preparation conditions were set as follows: initial pH7.0, temperature 30 oC, perlite loading 40 ml (medium volume 40 ml in 250 ml flask), 54 h of immobilization time. As a result, the cyanide-resistance and reusability were greatly improved, which were 16% and 3 batches higher than those of free cells. It was the first report on immobilization of NHase-producing strain using perlite. The kinetic constants of immobilized cells including maximum reaction velocity (Vmax-固) and Michaelis constant (Km-固) were calculated to be 13.59μmol·min-1·mg-1 and 47.98 mM, respectively. Free cells showed lower Km value of 44.30 mM compared to 47.98 mM for immobilized cells. It indicated that the substrate ADBN was more accessible to free cells than immobilized cells. This also suggested that external mass transfer (ADBN from bulk to cell in immobilized matrix) resistance affected hydration of ADBN by immobilized cells and could be the limiting step in comparison to the reaction rate. It was further confirmed by calculating the values of Damk-hler number at different ADBN concentrations, which were very much higher than 1.Finally, a new method for separation and purification of ADBA was developed. Purified ADBA (98.5%) was obtained by centrifugation, impurity removal using activated carbon, reduced pressure distillation and recrystallization from ethyl acetate/n-hexane. The structure of ADBA was further confirmed by FT-IR, 1H NMR and 13C NMR spectra.
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