NHases

It is known that NHase used in industry has a lower optimum temperature," therefore many reports have been concerned with screening for thermostable NHase. Miyanaga et al. has succeeded to analyze the X-ray structure of such a thermostable NHase from Pseudomonas thermophila. Bacillus sp. BR449 producing NHase with optimum temperature of 55°C has been isolated. Similarly, Bacillus sp. RAPc8 has a growth optimum at 65°C. Takashima et al. has isolated Bacillus smithii strain SC-J05-1, with optimum temperature at 40°C, and whose NHase has an optimum temperature and pH of 50°C and 10, respectively. Its crystal structure has also been elucidated. Bacillus pallidus strain Dac521 has... 

The NHase responsible for aldoxime metabolism from the i -pyridine-3-aldoxime-degrading bacterium, Rhodococcus sp. strain YH3-3, was purified and characterized. Addition of cobalt ion was necessary for the formation of enzyme. The native enzyme had a Mr of 130000 and consisted of two subunits (a-subunit, 27 100 (3-subunit, 34500). The enzyme contained approximately 2 mol cobalt per mol enzyme. The enzyme had a wide substrate specificity it acted on aliphatic saturated and unsaturated as well as aromatic nitriles. The N-terminus of the (3-subunit showed good sequence similarities with those of other NHases. Thus, this NHase is part of the metabolic pathway for aldoximes in microorganisms. 

As described above, the procedure and some of our examples of successful isolation of microorganisms having new enzymes were briefly described. Furthermore, historic changes of the way and methods of the research on the degradation of nitrile compounds and the exploitation of some industrially important enzymes including NHase, etc., were introduced. 

Nitrile hydratase (NHase) catalyzes the hydration of nitriles to amides (Figure 1.11) and has been used for production of acrylamide and nicotinamide at large scale. NHases are roughly... 

Existing synthetic methods and commercial processes that employ nitrile hydratases (NHases) and nitrilases continue to be improved by directed evolution of existing enzymes, or by the discovery of new enzymes with improved properties, and new applications of these catalysts have recently been described. Numerous reviews have previously been published that describe applications of NHase [ 1-6] and nitrilase [ 1,4—11 ], and in this review we present examples of new applications of these nitrile-utilizing catalysts from journal articles, patent applications, and issued patents that have been published in the past 2-3 years. 

Table 8.1 Temperature stability of Rhodococcus rhodochrous J1E/393G mutant NHase compared with J1 NHase, each expressed in Escherichia coli JM109...

Acrylonitrile produced industrially via propylene ammoxidation contains trace amounts of benzene. When using Pseudonocardia thermophila JCM3095 or Rhodococcus rhodochrous J-1 as microbial NHase catalyst for conversion of acrylonitrile to acrylamide, concentrations of benzene of <4 ppm produced a significant increase in the reaction rate [16]. Maintaining the concentration of HCN and oxazole at <5 ppm and <10 ppm respectively produced high-quality acrylamide suitable for polymerization. 

While the production of acrylamide by NHase is a well-established industrial process, only a first report exists for the production of butyramide from butyronitrile. Using Rhodococcus rhodochrous PA-34 (at a loading of 1 g dew), 595 g butyramide was prepared in quantitative yield from 60% (v/v) butyronitrile in a pH 7.0, 1 L batch reaction, at 10 °C [18]. 

Table 8.2 Comparison of the thermal stability of NHase activities of Rhodococcus sp. FZ4 and GF270 to Amycolatopsis sp. NA40 and Rhodococcus rhodochrous J1 ...

A thermally stable NHase from Comamonas testosteroni 5-MGAM-4D (ATCC 55 744) [22] was recombinantly expressed in Escherichia coli, and the resulting transformant cells immobilized in alginate beads that were subsequently chemically cross-linked with glutaraldehyde and polyethylenimine. This immobilized cell catalyst (at 0.5 % dew per reaction volume) was added to an aqueous reaction mixture containing 32wt% 3-cyanopyridine at 25 °C, and a quantitative conversion to nicotinamide was obtained. The versatility of this catalyst system was further illustrated by a systematic study of substrates, which included... 

Unimmobilized Corynebacterium propinquum (CGMCC No. 0886) cells containing a cobalt-dependent NHase were employed in either batch or continuous reactions for the production of nicotinamide from 3-cyanopyridine [24]. In the continuous process, membrane filtration separated precipitated product (>5 wt%) and the microbial cell catalyst from the reaction mixture, where the catalyst was then recovered and returned to the reactor using a continuous addition of aqueous 3-cyanpyridine to maintain substrate concentration at <20% (w/v), a final conversion of >99% was obtained. 

The activities of NHases from Rhodococcus sp. Adpl2 and Gordonia sp. BR-1 strains have been partially characterized [25]. In reactions that catalyze the hydration of a-hydroxynitriles such as lactonitrile or glycolonitrile, the substrate can dissociate to produce HCN and the corresponding aldehydes. HCN can inhibit and/or inactivate NHase, and it was determined that these two enzymes remain active in the presence of cyanide ion at concentrations up to 20 him. The dependence of the NHase activity of cell-free extracts of Rhodococcus rhodochrous J1 and Gordonia sp. BR-1 on cyanide ion concentration is illustrated in Figure 8.1, demonstrating the improved cyanide stability of BR-1 NHase relative to that of Jl. 

Figure 8.1 Dependence of the NHase activity of cell-free extracts of Rhodococcus rhodochrous J1 (a) and Gordonia sp. BR-1 ( ) on cyanide ion concentration...

Figure 8.2 Residual activity of Rhodococcus Adpl2 NHase after 10 min in reaction mixtures containing 10-40% (v/v) methanol ( ), ethanol (a), ethyl acetate ( ) and n-hexane (a) at 20 °C...

Table 8.4 Residual activity of Rhodococcus NHases after incubation in 1-20 mM cyanide for 30 min Strain KCN (mM)...

Rhodococcus erythropolis NCIMB 11540 has been employed as biocatalyst for the conversion of (R)- or (.S )-cyanohydrins to the corresponding (R)- or (S)-a-hydroxycarboxylic acids with an optical purity of up to >99% enatiomeric excess (ee) [27-29] the chiral cyanohydrins can separately be produced using hydroxynitrile lyase from Hevea braziliensis or from Prunus anygdalis [30]. Using the combined NHase-amidase enzyme system of the Rhodococcus erythropolis NCIMB 11 540, the chiral cyanohydrins were first hydrolyzed to the... 

Isolated polynucleotide clusters from Rhodococcus opacus which encode four polypeptides possessing the activities of a NHase (a and /3 subunits), an auxiliary protein P15K that activates the NHase, and a cobalt transporter protein were expressed in Escherichia coli DSM 14459 cells [34]. Methionine nitrile was added continuously to a suspension of the transformant cells (5.6% w/v of wet cells) in phosphate buffer (50 mM, pH 7.5) at 20 °C, at a rate where the nitrile concentration did not exceed 15 g L 1 while maintaining the pH constant at 7.5. After 320 min, the nitrile was completely converted into amide, corresponding to a final product concentration of 176 gL1.4-Methylthio-a-hydroxybutyramide is readily hydrolyzed with calcium hydroxide, where the calcium salt of 4-methylthio-a-hydroxybutyric acid (MHA) can be directly used as a nutritional supplement in animal feed as an alternative to methionine or MHA. 

The NHase and amidase from Rhodococcus rhodochrous IFO 15 564 was studied using a series of a,a-disubstituted malononitriles. This amidase preferentially hydrolyzes the pro (R) amide of the prochiral di-amide, which is an intermediate resulting from the nonenantiotopic NHase activity on the dinitrile substrate. This transformation was combined with a Hofmann rearrangement to generate a key precursor of (A)-methyldopa in 98.2% ee and 95% yield (Figure 8.5) [41],... 

Figure 8.5 Conversion of 2-(l,3-benzodioxol-5-ylmethyl)-2-methyl-propanedinitrile to (R)-ot-(aminocarbonyl)-o -methyl-1,3-benzodioxole-5-propanoic acid using the non-enantiotopic NHase activity...

By screening 53 Rhodococcus and Pseudomonas strains, an NHase-amidase biocatalyst system was identified for the production of the 2,2-dimethylcyclopropane carboxylic acid precursor of the dehydropeptidase inhibitor Cilastatin, which is used to prolong the antibacterial effect of Imipenem. A systematic study of the most selective of these strains, Rhodococcus erythropolis ATCC25 544, revealed that maximal product formation occurs at pH 8.0 but that ee decreased above pH 7.0. In addition, significant enantioselectivity decreases were observed above 20 °C. A survey of organic solvent effects identified methanol (10% v/v) as the... 

NHase from Rhodococcus. sp. AJ270 was isolated, purified, and applied to the enantiose-lective transformation of a series of cyclopropane carbonitriles. Amides with moderate ee were isolated from conversion of many of the cyclopropane substrates, to yield the amides trans-( IR, 2/ )-3-phenylcyclopropane carbonitrile (49% conv. 22.7% ee), trans-( IS, 35)-2,2-dimethyl-3-phenylcyclopropanecarbonitrile (40% conv. 84.7% ee), trans-( IR, 3/f)-2,2-dibromo-3-phenylcy-clopropanecarbonitrile (11.6% conv. 83.8% ee), cis-( IR, 25)-3-phenylcyclopropanecarbonitrile (25.8% conv. 95.4% ee), and cis-(lR, 2S )-2,2-dimethyl-3-phenylcyclopropanecarbonitrile (7.9% conv. 3.2% ee) [43],... 

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