Nocardia globerula NHB-2: a versatile nitrile-degrading organism

A versatile nitrile-degrading bacterium was isolated by enrichment culture from the soil of a forest near Manali, Himachal Pradesh, India, and was identified as Nocardia globerula. This organism contains 3 enzymes with nitrile-degrading activity: nitrilase, nitrile hydratase, and amidase. Nocardia globerula NHB-2 cells grown on nutrient broth supplemented with 1% glucose and 0.1% yeast extract exhibited nitrile hydratase–amidase activity specific for saturated aliphatic nitriles or amide, while addition of acetonitrile in nutrient broth yielded cells with nitrile hydratase–amidase that in addition to saturated aliphatic nitriles–amide also hydrolyzed aromatic amide. Nocardia globerula NHB-2 cultivated on nutrient broth containing propionitrile exhibited nitrilase activity that hydrolyzed aromatic nitrile and unsaturated aliphatic nitrile. The versatility of this organism in the hydrolysis of various nitriles and amides makes it a potential bioresource for use in organic synthesis.Key words: Nocardia globerula NHB-2, nitrilase, nitrile hydratase, amidase, nitrile–amide degradation.

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Microbial Isobutyronitrile Utilization under Haloalkaline Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.00342-07 ◽

2007 ◽

Vol 73 (17) ◽

pp. 5574-5579 ◽

Cited By ~ 21

Author(s):

Dimitry Y. Sorokin ◽

Sander van Pelt ◽

Tatjana P. Tourova ◽

Gerard Muyzer

Keyword(s):

Carboxylic Acids ◽

Nitrile Hydratase ◽

Bacterial Species ◽

Intracellular Localization ◽

Soda Lake ◽

Bacterial Strains ◽

Ph Optimum ◽

Hydrolysis Products ◽

Aliphatic Nitriles ◽

Hydrolysis Of

ABSTRACT The utilization of isobutyronitrile (iBN) as a C and N source under haloalkaline conditions by microbial communities from soda lake sediments and soda soils was studied. In both cases, a consortium consisting of two different bacterial species capable of the complete degradation and utilization of iBN at pH 10 was selected. The soda lake sediment consortium consisted of a new actinobacterium and a gammaproteobacterium from the genus Marinospirillum. The former was capable of fast hydrolysis of aliphatic nitriles to the corresponding amides and much-slower further hydrolysis of the amides to carboxylic acids. Its partner cannot hydrolyze nitriles but grew rapidly on amides and carboxylic acids, thus acting as a scavenger of products released by the actinobacterium. The soda soil consortium consisted of two Bacillus species (RNA group 1). One of them initiated nitrile hydrolysis, and the other utilized the hydrolysis products isobutyroamide (iBA) and isobutyrate (iB). In contrast to the actinobacterium, the nitrile-hydrolyzing soil Bacillus grew rapidly with hydrolysis products, but it was dependent on vitamins most probably supplied by its product-utilizing partner. All four bacterial strains isolated were moderately salt-tolerant alkaliphiles with a pH range for growth from pH 7.0 to 8.5 up to 10.3 to 10.5. However, both their nitrile hydratase and amidase activities had a near-neutral pH optimum, indicating an intracellular localization of these enzymes. Despite this fact, the study demonstrated a possibility of whole-cell biocatalytic hydrolysis of various nitriles at haloalkaline conditions.

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Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber

Molecules ◽

10.3390/molecules25041002 ◽

2020 ◽

Vol 25 (4) ◽

pp. 1002 ◽

Cited By ~ 1

Author(s):

Chunmeng Xu ◽

Lingjun Tang ◽

Youxiang Liang ◽

Song Jiao ◽

Huimin Yu ◽

...

Keyword(s):

Thermal Stability ◽

Large Scale ◽

Nitrile Hydratase ◽

Fusion Expression ◽

Solvent Tolerance ◽

Rhodococcus Ruber ◽

Bacterial Cells ◽

E Coli ◽

Nitrilase Activity ◽

Stability Enhancement

For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones RrGroEL and RrGroES from Rhodococcus ruber, a nontypical actinomycete with high organic solvent tolerance, were evaluated and applied for thermal stability and activity enhancement of a model enzyme, nitrilase. Two expression strategies, namely, fusion expression and co-expression, were compared in two different hosts, E. coli and R. ruber. In the E. coli host, fusion expression of nitrilase with either RrGroES or RrGroEL significantly enhanced nitrilase thermal stability (4.8-fold and 10.6-fold, respectively) but at the expense of enzyme activity (32–47% reduction). The co-expression strategy was applied in R. ruber via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the R. ruber genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of RrGroES or RrGroEL with a plasmid, the engineered strains R. ruber TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroES) and TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the RrGroEL and nitrilase co-expressing mutant showed the best performance, with nitrilase activity and thermal stability 1.3- and 8.4-fold greater than that of the control TH3 (pNV18.1-Pami-RrNit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.

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Enzymatic hydrolysis of cyanohydrins with recombinant nitrile hydratase and amidase from hodococcus erythropolis

Biotechnology Letters ◽

10.1007/s10529-004-3521-4 ◽

2004 ◽

Vol 26 (21) ◽

pp. 1675-1680 ◽

Cited By ~ 20

Author(s):

Ch. Reisinger ◽

I. Osprian ◽

A. Glieder ◽

H. E. Schoemaker ◽

H. Griengl ◽

...

Keyword(s):

Enzymatic Hydrolysis ◽

Nitrile Hydratase ◽

Hydrolysis Of

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The linkage between binding of the C-terminal domain of hirudin and amidase activity in human α-thrombin

Biochemical Journal ◽

10.1042/bj2890475 ◽

1993 ◽

Vol 289 (2) ◽

pp. 475-480 ◽

Cited By ~ 17

Author(s):

R de Cristofaro ◽

B Rocca ◽

B Bizzi ◽

R Landolfi

Keyword(s):

Rate Constants ◽

Binding Constant ◽

Amidase Activity ◽

Equilibrium Binding ◽

Viscosity Effects ◽

The Individual ◽

Individual Rate ◽

Hydrolysis Of ◽

Equilibrium Binding Constant

A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolyl-arginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human alpha-thrombin was developed to dissect the Michaelis-Menten parameters Km and kcat into the individual rate constants of the binding, acylation and deacylation reactions. This method was used to analyse the effect of the C-terminal hirudin (residues 54-65) [hir-(54-65)] domain on the binding and hydrolysis of the three substrates. The results showed that the C-terminal hir-(54-65) fragment affects only the acylation rate, which is increased approx. 1.2-fold for all the substrates. Analysis of the dependence of acylation rate constants on hirudin-fragment concentration, allowed the determination of the equilibrium binding constant of C-terminal hir-(54-65) (Kd approximately 0.7 microM). In addition this peptide was found to competitively inhibit thrombin-fibrinogen interaction with a Ki which is in excellent agreement with the equilibrium constant derived from viscosity experiments. These results demonstrate that binding of hir-(54-65) to the fibrinogen recognition site of thrombin does not affect the equilibrium binding of amide substrates, but induces only a small increase in the acylation rate of the hydrolysis reaction.

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Aliphatic Nitrile Hydratase fromArthrobactersp. J-1 Purification and Characterization

Agricultural and Biological Chemistry ◽

10.1080/00021369.1982.10865238 ◽

1982 ◽

Vol 46 (5) ◽

pp. 1165-1174 ◽

Cited By ~ 2

Author(s):

Yasuhisa Asano ◽

Kinya Fujishiro ◽

Yoshiki Tani ◽

Hideaki Yamada

Keyword(s):

Nitrile Hydratase ◽

Purification And Characterization ◽

Aliphatic Nitrile

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Substrate specificity and other properties of the inducible S3 secondary alkylsulphohydrolase purified from the detergent-degrading bacterium Pseudomonas C12B

Biochemical Journal ◽

10.1042/bj1870181 ◽

1980 ◽

Vol 187 (1) ◽

pp. 181-190 ◽

Cited By ~ 25

Author(s):

D J Shaw ◽

K S Dodgson ◽

G F White

Keyword(s):

Carbon Atom ◽

Deae Cellulose ◽

Maximum Activity ◽

Sulphate Group ◽

Alkyl Chains ◽

Degrading Bacterium ◽

Ester Linkage ◽

Hydrolysis Of ◽

Blue Sepharose ◽

Asymmetric Carbon

The inducible S3 secondary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (683-fold from cell-free extracts by a combination of column chromatography on DEAE-cellulose. Sephadex G-100 and Blue Sepharose CL-6B. The enzyme has a molecular weight in the region of 40000–46000, and is active over a broad range of pH from 5 to 9, with maximum activity at pH 8.2. The preferred substrates of the enzyme are the symmetrical secondary alkylsulphate esters such as heptan-4-yl sulphate and nonan-5-yl sulphate and the asymmetric secondary octyl and nonyl sulphate esters with the sulphate group attached to C-3 or C-4. However, for each asymmetric ester, the L-isomer is much more readily hydrolysed than the D-isomer. This specificity is interpreted in terms of a three-point attachment of the substrate to the enzyme's active site. The alkyl chains on either side of the esterified carbon atom are bound in two separate sites, one of which can only accommodate alkyl chains of limited size. The third site binds the sulphate group. Enzymic hydrolysis of this group is accompanied by complete inversion of configuration at the asymmetric carbon atom. The implied cleavage of the C–O bond of the C–O–S ester linkage was confirmed by 18O-incorporation studies.

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