Amidase and peptidase activities of polyclonal immunoglobulin G present in the sera of patients with rheumatoid arthritis

Polyclonal Immunoglobulin (Ig) G from patients with rheumatoid arthritis (RA) and healthy subjects hydrolyzed carbobenzoxy−Val−Gly−Arg p-nitroanilide and D−Pro−Phe−Arg p-nitroanilide. RA IgG exhibited higher activity against the former substrate, but not the latter. On the other hand, RA IgG showed reduced activity against D−Pro−Phe−Arg methylcoumarinamide, when compared with those of the healthy controls. These results suggest that RA IgGs differ from normal IgGs in the substrate specificity of amidase activity. Preliminary studies have shown that two out of three RA IgG samples cleaved a pentapeptide—Gln−Arg−Arg−Arg−Ala−Ala— which is assumed to be associated with the risk of developing RA (Gregersen, P. K. et al. (1987), Arthritis Rheum. 30, 1205–1213). By contrast, virtually no cleavage of the same peptide was observed with IgG from healthy controls. A peptide analog, Gln−Arg−Arg−Trp−Ala, was not cleaved at all by any IgGs examined either from RA patients or healthy controls.     

Cleavage of Menger’s aliphatic amide: A model for peptidase enzyme solely explained by proximity orientation in intramolecular proton transfer

Ab initio HF/6-31G and DFT B3LYP/6-31G (d,p) calculations for the cleavage of Menger’s aliphatic amide 3 (a peptidase model) under physiological conditions, indicate that the rate limiting step in the cleavage process is a proton transfer from one of the carboxyl groups onto the amidic carbonyl oxygen. The acceleration in rates is mainly due to proximity orientation, and the effect of pseudoallylic strain relief on the rates is negligible. Moreover, the calculations reveal that the mode and the mechanism of the amide cleavage are largely dependent on the pH of the reaction. These results explain the findings that peptidase enzymes are reactive around neutral pH while their activities vanish under basic medium.     

The substrate specificity of the heat-stable stereospecific amidase from Klebsiella oxytoca

The substrate specificity of the heat-stable stereospecific amidase from Klebsiella oxytoca was investigated. In addition to the original substrate, 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide, the amidase accepted 2-hydroxy-2-(trifluoromethyl)-butanamide and 3,3,3-trifluoro-2-amino-2-methylpropanamide as substrates. Compounds with larger side chains and compounds where the hydroxyl group was substituted with a methoxy group, or in which the CF₃ group was substituted by CCl₃, were not accepted. The biotransformation is a new synthetic route to (R)-(+)-3,3,3-trifluoro-2-amino-2-methylpropanoic acid, and its related (S)-(−)-amide, and to (R)-(+)-2-hydroxy-2-(trifluoromethyl)-butanoic acid and its related (S)-(−)-amide.     

Comparative study of amidase production by free and immobilized Escherichia coli cells

Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30°C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.     

Immobilization of Rhodococcus AJ270 and Useof Entrapped Biocatalyst for the Productionof Acrylic Acid

Summary.  Rhodococcus AJ270 is adsorbed by Dowex 1 at 15.4  mg dry weight per g resin with maximum amidase specific activity observed at lower loadings. Bacteria form a monolayer on the resin surface, and adsorption is complete within 2 min. AJ270 can be entrapped in agar and agarose gels (optimum loading: 20 mg dry weight bacteria per cm³ gel). Adsorption and entrapment improve amidase thermal stability 3–4 fold, and entrapment shifts the pH optimum from 8 to 7. Adsorbed and free bacteria show similar values for K _m and V _max, but entrapped bacteria have higher K _m values. Compared with bacteria adsorbed to Dowex, the activity per cm³ of matrix of agar-entrapped AJ270 is eight-fold higher. In stirred-tank reactors, exposure to acrylic acid reduces the amidase activity of the biocatalyst in the hydrolysis of acrylamide. In column reactors, entrapped AJ270 suffers little reduction in amidase activity against 0.25 M acrylamide over 22 h continuous operation. Received November 18, 1999. Accepted December 14, 1999     

Regioselective biocatalytic hydrolysis of (E,Z)-2-methyl-2-butenenitrile for production of (E)-2-methyl-2-butenoic acid

Acidovorax facilis 72W nitrilase catalyzed the regioselective hydrolysis of (E,Z)-2-methyl-2-butenenitrile, producing only (E)-2-methyl-2-butenoic acid with no detectable conversion of (Z)-2-methyl-2-butenenitrile. (E)-2-Methyl-2-butenoic acid, produced in aqueous solution as the ammonium salt, was readily separated from (Z)-2-methyl-2-butenenitrile, and isolated in high yield and purity. The combination of nitrile hydratase and amidase activities of several Comamonas testosteroni strains were also highly regioselective for the production of (E)-2-methyl-2-butenoic acid from (E,Z)-2-methyl-2-butenenitrile.     

Efficient synthesis of highly enantiopure β-lactam derivatives from biocatalytic transformations of amides and nitriles

Catalyzed by Rhodococcus erythropolis AJ270, a nitrile hydratase and amidase containing microbial strain, under mild conditions, kinetic resolution of racemic amides provides an efficient and scalable route to highly enantioenriched R-4-carboxymethyl-β-lactams and S-4-carbamoylmethyl-β-lactams despite the substrates contain a stereogenic center that is β-positioned to amide functionality. Synthetic potential of the method was demonstrated by the constructions of novel β-lactam-fused heterocyclic compounds through convenient and practical chemical transformations.