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1.
Lauren B. Jones Xiaoqiang Wang Jaya S. Gullapalli Daniel A. Kunz 《Biochemistry and Biophysics Reports》2021
We report the purification and characterization of a nitrilase (E.C. 3.5.5.1) (Nit11764) essential for the assimilation of cyanide as the sole nitrogen source by the cyanotroph, Pseudomonas fluorescens NCIMB 11764. Nit11764, is a member of a family of homologous proteins (nitrile_sll0784) for which the genes typically reside in a conserved seven-gene cluster known as Nit1C. The physical properties and substrate specificity of Nit11764 resemble those of Nit6803, the current reference protein for the family, and the only true nitrilase that has been crystallized. The substrate binding pocket of the two enzymes places the substrate in direct proximity to the active site nucleophile (C160) and conserved catalytic triad (Glu44, Lys126). The two enzymes exhibit a similar substrate profile, however, for Nit11764, cinnamonitrile, was found to be an even better substrate than fumaronitrile the best substrate previously identified for Nit6803. A higher affinity for cinnamonitrile (Km 1.27 mM) compared to fumaronitrile (Km 8.57 mM) is consistent with docking studies predicting a more favorable interaction with hydrophobic residues lining the binding pocket. By comparison, 3,4-dimethoxycinnamonitrile was a poorer substrate the substituted methoxyl groups apparently hindering entry into the binding pocket. in situ 1H NMR studies revealed that only one of the two nitrile substituents in the dinitrile, fumaronitrile, was attacked yielding trans-3-cyanoacrylate (plus ammonia) as a product. The essentiality of Nit11764 for cyanotrophy remains uncertain given that cyanide itself is a poor substrate and the catalytic efficiencies for even the best of nitrile substrates (~5 × 103 M?1 s?1) is less than stellar. 相似文献
2.
Ludmila Martínková 《Fungal Biology Reviews》2019,33(2):149-157
Nitriles are abundant in the plant kingdom. The ability to detoxify them is beneficial for microbes living in the plant environment. Nitrilases (NLases; EC 3.5.5.-), which hydrolyze nitriles to carboxylic acids, have been well characterized in bacteria, and classified into various substrate-specificity subtypes (aromatic NLases, aliphatic NLases, arylacetoNLases). NLases also occur in filamentous fungi, mainly in Ascomycota (subdivision Pezizomycotina), as documented by genome mining. However, the investigation of NLases in fungi has been delayed compared to bacteria. Only a few NLases (aromatic NLases) were purified from native fungal strains (mainly Fusarium), which were grown under suitable induction conditions. Over a few past years, the spectrum of known fungal NLases was broadened by expressing fungal NLase genes in Escherichia coli. Thus functional NLases were reported for the first time in fungi of genera Auricularia, Macrophomina, Nectria, Neurospora, Pichia, Talaromyces, Trichoderma and Trichophyton. Two major substrate-specificity subtypes were identified in them, i.e. aromatic NLases and arylacetoNLases, apart from a few NLases with broad substrate specificities. The biotechnological impact of fungal arylacetoNLases was explored with a focus on the enantioselective hydrolysis of (R,S)-mandelonitrile, the selective hydrolysis of one cyano group in dinitriles and the hydrolysis of nitrile precursors of the taxol sidechain. Despite recent advances, the wealth of fungal NLases whose sequences have been deposited in databases has not yet been fully exploited. Overproduction in E. coli has the potential to bring these NLases to life. This will enable to estimate the natural roles of NLases in fungi and will also provide new catalysts for biotechnological uses. 相似文献
3.
Vorwerk S Biernacki S Hillebrand H Janzik I Müller A Weiler EW Piotrowski M 《Planta》2001,212(4):508-516
Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5′→3′ direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three
nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active
proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences.
All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective
substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low
affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate
breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion
to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum
around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 °C independent of the substrate analyzed
(IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 °C) and PPN (35–40 °C). A potential role for
NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform
in developing embryos and in germinating seeds, as judged from an analysis of β-glucuronidase reporter gene expression under
the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development
rather than during stratification.
Received: 13 May 2000 / Accepted: 14 August 2000 相似文献
4.
The filamentous fungus Fusarium lateritium produces cyanide hydratase when grown in the presence of cyanide. The cyanide hydratase protein produced at a high level in Escherichia coli shows a low but significant nitrilase activity with acetonitrile, propionitrile and benzonitrile. The nitrilase activity is sufficient for growth of the recombinant strain on acetonitrile, propionitrile or benzonitrile as the sole source of nitrogen. The recombinant enzyme shows highest nitrilase activity with benzonitrile. Site-directed mutagenesis of the F. lateritium cyanide hydratase gene indicates that mutations leading to a loss of cyanide hydratase activity also lead to a loss of nitrilase activity. This suggests that the active site for cyanide hydratase and nitrilase activity in the protein is the same. This is the first evidence of cyanide hydratase having nitrilase activity. 相似文献
5.
Kabaivanova L Dobreva E Dimitrov P Emanuilova E 《Journal of industrial microbiology & biotechnology》2005,32(1):7-11
Cells of the moderately thermophilic Bacillus sp. UG-5B strain, producing nitrilase (EC3.5.5.1), which converts nitriles directly to the corresponding acid and ammonia, were immobilized using different types of matrices and techniques. A variety of sol-gel silica hybrids were tested for entrapment and adsorption of bacterial cells as well as chemical binding on polysulphone membranes. Activation of the matrix surface with formaldehyde led to an increase in immobilization efficiency and operational stability of the biocatalysts. Among the supports screened, membranes gave the best results for enzyme activity and especially operational stability, with retention of 100% activity after eight reaction cycles. 相似文献
6.
Shubhangi Kaushik Utpal Mohan UC Banerjee 《International Journal of Biochemistry and Molecular Biology》2012,3(4):384-391
Nitrilases represent a very important class of enzymes having an array of applications. In the present scenario, where the indepth information about nitrilases is limited, the present work is an attempt to shed light on the residues crucial for the nitrilase activity. The nitrilase sequences demonstrating varying degree of identity with P. putida nitrilase were explored. A stretch of residues, fairly conserved throughout the range of higher (96%) to lower (27%) sequence identity among different nitrilases was selected and investigated for the possible functional role in nitrilase enzyme system. Subsequently, the alanine substitution mutants (T48A, W49A, L50A, P51A, G52A, Y53A and P54A) were generated. Substitution of the rationally selected conserved residues altered the substrate recognition ability, catalysis and affected the substrate specificity but had very little impact on enantioselectivity and pattern of nitrile hydrolysis. 相似文献
7.
随着基因工程技术的快速发展,通过对不同菌株腈水解酶基因的分析,将其克隆到表达菌株内,可以构建高效并且稳定的基因工程菌。对腈水解酶进行分子改造可以明显提高酶的活性、稳定性、底物耐受性和底物特异性等性能,为腈水解酶的工业化应用提供了可能。综述了腈水解酶的来源、结构、催化机制、克隆表达、固定化及分子改造等方面的研究进展。同时对腈水解酶的研究进行了展望,具有重要的指导意义。 相似文献
8.
9.
The insolubility of nitrile substrates in aqueous reaction mixture decreases the enzymatic reaction rate. We studied the interaction of fourteen water miscible organic solvents with immobilized nitrile hydrolyzing biocatalyst. Correlation of nitrilase function with physico-chemical properties of the solvents has allowed us to predict the enzyme behavior in such non-conventional media. Addition of organic solvent up to a critical concentration leads to an enhancement in reaction rate, however, any further increase beyond the critical concentration in the latter leads to the decrease in catalytic efficiency of the enzyme, probably due to protein denaturation. The solvent dielectric constant (epsilon) showed a linear correlation with the critical concentration of the solvent used and the extent of nitrile hydrolysis. Unlike alcohols, the reaction rate in case of aprotic solvents could be linearly correlated to solvent log P. Further, kinetic analysis confirmed that the affinity of the enzyme for its substrate (K (m)) was highly dependent upon the aprotic solvent used. Finally, the prospect of solvent engineering also permitted the control of enzyme enantioselectivity by regulating enantiomer traffic at the active site. 相似文献
10.
The fungal cyanide hydratases form a functionally specialized subset of the nitrilases which catalyze the hydrolysis of cyanide
to formamide with high specificity. These hold great promise for the bioremediation of cyanide wastes. The low resolution
(3.0 nm) three-dimensional reconstruction of negatively stained recombinant cyanide hydratase fibers from the saprophytic
fungus Neurospora crassa by iterative helical real space reconstruction reveals that enzyme fibers display left-handed D1 S5.4 symmetry with a helical rise of 1.36 nm. This arrangement differs from previously characterized microbial nitrilases which
demonstrate a structure built along similar principles but with a reduced helical twist. The cyanide hydratase assembly is
stabilized by two dyadic interactions between dimers across the one-start helical groove. Docking of a homology-derived atomic
model into the experimentally determined negative stain envelope suggests the location of charged residues which may form
salt bridges and stabilize the helix. 相似文献