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1.
The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity. 相似文献
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3.
《Process Biochemistry》2014,49(3):445-450
A cyanide hydratase from Aspergillus niger K10 was expressed in Escherichia coli and purified. Apart from HCN, it transformed some nitriles, preferentially 2-cyanopyridine and fumaronitrile. Vmax and Km for HCN were ca. 6.8 mmol min−1 mg−1 protein and 109 mM, respectively. Vmax for fumaronitrile and 2-cyanopyridine was two to three orders of magnitude lower than for HCN (ca. 18.8 and 10.3 μmol min−1 mg−1, respectively) but Km was also lower (ca. 14.7 and 3.7 mM, respectively). Both cyanide hydratase and nitrilase activities were abolished in truncated enzyme variants missing 18–34 C-terminal aa residues. The enzyme exhibited the highest activity at 45 °C and pH 8–9; it was unstable at over 35 °C and at below pH 5.5. The operational stability of the whole-cell catalyst was examined in continuous stirred membrane reactors with 70-mL working volume. The catalyst exhibited a half-life of 5.6 h at 28 °C. A reactor loaded with an excess of the catalyst was used to degrade 25 mM KCN. A conversion rate of over 80% was maintained for 3 days. 相似文献
4.
《Process Biochemistry》2014,49(4):655-659
An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37 °C, 100 mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1 M 3-cyanopyridine to nicotinic acid within 5 h at a cell mass concentration (fresh weight) of 500 mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use. 相似文献
5.
《Process Biochemistry》2010,45(7):1115-1120
Nitrilase activity in Fusarium solani IMI196840 (approx. 1500 U l−1 of culture broth) was induced by 2-cyanopyridine. The enzyme was purified by a factor of 20.3 at a yield of 26.9%. According to gel filtration, the holoenzyme was an approx. 550-kDa homooligomer consisting of subunits with a molecular weight of approximately 40 kDa, as determined by SDS-PAGE. Mass spectrometry analysis of the tryptic fragments suggested a high similarity of this enzyme to the hypothetical CN hydrolases from Aspergillus oryzae, Gibberella zeae, Gibberella moniliformis and Nectria haematococca. Circular dichroism and fluorescence spectra indicated that secondary structure content and overall tertiary structure, respectively, were almost identical in nitrilases from F. solani IMI196840 and F. solani O1. The melting temperatures of the enzymes were 49.3 °C and 47.8 °C, respectively. The best substrates for the purified nitrilase from F. solani IMI196840 were benzonitrile and 4-cyanopyridine, which were hydrolyzed at the rates of 144 and 312 U mg−1 protein, respectively, under the optimum conditions of pH 8 and 45 °C. The enzyme was highly chemoselective, producing ≤2% amides as by-products. 相似文献
6.
《Journal of Molecular Catalysis .B, Enzymatic》2011,70(3-4):127-132
An extracellular lipase gene ln1 from thermophilic fungus Thermomyces lanuginosus HSAUP0380006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase lgy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SDS-PAGE. The enzyme was stable at 60 °C and kept 65% enzyme activity after 30 min incubation at 70 °C. It kept half-activity after incubated for 40 min at 80 °C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca2+ and inhibited by Fe2+, Zn2+, K+, and Ag+. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications. 相似文献
7.
《Process Biochemistry》2014,49(12):2141-2148
A nitrilase gene from Acidovorax facilis ZJB09122 was cloned and expressed in Escherichia coli BL21 (DE3). To improve the activity of this nitrilase, a key amino acid Phe168 was selected and mutated by site-directed mutagenesis, based on the homology modeling and previously described “hot spot” mutation. After mutation and screening, a mutant (Mut-F168V) with higher activity and stability was obtained. The nitrilase activity of Mut-F168V to hydrolyze 1-cyanocyclohexylacetonitrile was 39.52-fold compared with wild type A. facilis nitrilase (Wt-Acf-Nit). The values of Km and Vmax of Mut-F168V were markedly decreased to 1.89-fold and increased to 50.34-fold as compared to Wt-Acf-Nit, respectively. The biotransformation study showed that 1.0 M of 1-cyanocyclohexylacetonitrile could be regioselectively hydrolyzed to 1-(cyanocyclohexyl) acetic acid with 90% yield. The yield of 1-(cyanocyclohexyl) acetic acid by Mut-F168V was 66.19-fold compared to Wt-Acf-Nit after 1 h at the concentration of 1.0 M 1-cyanocyclohexylacetonitrile as substrate. The 1-(cyanocyclohexyl) acetic acid was subsequently isolated and characterized. The mutant (Mut-F168V) appears promising for potential applications for the industrial production of 1-(cyanocyclohexyl) acetic acid. 相似文献
8.
《Process Biochemistry》2010,45(6):887-891
For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli. After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of (R)-(−)-mandelic acid after only 4 h of transformation reached 197 mM with an enantiomeric excess (eep) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure (R)-(−)-mandelic acid. 相似文献
9.
《Process Biochemistry》2014,49(9):1422-1428
A β-xylosidase gene (xylA4) was identified in the genome sequence of thermoacidophilic Alicyclobacillus sp. A4. The deduced amino acid sequence was highly homologous with the β-xylosidases of family 52 of the glycoside hydrolases (GH). The full-length gene consisted of 2097 bp and encoded 698 amino acids without a signal peptide. The gene product was successfully expressed in Escherichia coli with an activity of 564.9 U/mL. Recombinant XylA4 was purified by Ni2+-NTA affinity chromatography with a molecular mass of 78.5 kDa. The enzyme showed optimal activity at pH 6.0 and 65 °C, and remained stable over the pH range of 5.0–9.0. The thermostability of XylA4 is noteworthy, retaining almost all of the activity after 1 h incubation at 65 °C. Using p-nitrophenyl-β-d-xylopyranoside (pNPX) as the substrate, XylA4 had the highest specific activity (261.1 U/mg) and catalytic efficiency (601.5/mM/s) known so far for GH52 xylosidases. The enzyme displayed high tolerance to xylose, with a Ki value of approximately 88.7 mM. It also had synergy with xylanase XynBE18 from Paenibacillus sp. E18 in xylan degradation, releasing more xylose (up to 1.43 folds) than XynBE18 alone. Therefore, this thermostable xylose-tolerant β-xylosidase may have a great application potential in many industrial fields. 相似文献
10.
Alessio Innocenti Worraanong Leewattanapasuk Gheorghe Manole Andrea Scozzafava Fritz A. Mühlschlegel Claudiu T. Supuran 《Bioorganic & medicinal chemistry letters》2010,20(5):1701-1704
The protein encoded by the NCE103 gene of Candida glabrata, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as CgCA, was investigated for its activation with amines and amino acids. CgCA was weakly activated by amino acids such as l-/d-His, l-Phe, l-DOPA, and l-Trp and by histamine or dopamine (KAs of 21.2–37 μM) but more effectively activated by d-Phe, d-DOPA, d-Trp as well as serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (KAs of 10.1–16.7 μM). The best activators were l-/d-Tyr, with activation constants of 7.1–9.5 μM. This study may bring a better understanding of the catalytic/activation mechanisms of β-CAs from pathogenic fungi. 相似文献
11.
Semra Isik Feray Kockar Meltem Aydin Oktay Arslan Ozen Ozensoy Guler Alessio Innocenti Andrea Scozzafava Claudiu T. Supuran 《Bioorganic & medicinal chemistry letters》2009,19(6):1662-1665
The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, was investigated for its activation with amines and amino acids. scCA was poorly activated by amino acids such as l-/d-His, Phe, DOPA, Trp (KAs of 82–90 μM) and more effectively activated by amines such as histamine, dopamine, serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (KAs of 10.2–21.3 μM). The best activator was l-adrenaline, with an activation constant of 0.95 μM. This study may help to better understand the catalytic/activation mechanisms of the β-CAs and eventually to design modulators of CA activity for similar enzymes present in pathogenic fungi, such as Candida albicans and Cryptococcus neoformans. 相似文献
12.
The photoreaction and adsorption properties on surfaces, thermal decomposition, chemical transformation, and other properties of the formamide molecule are widely used to understand the origins of the formation of biological molecules (nucleosides, amino acids, DNA, monolayers, etc.) needed for life. The titanium oxide (TiO2) surface can act both as a template on which the accumulation of adsorbed molecules like formamide occurs through the concentration effect, and as a catalytic material that lowers the activation energy needed for the formation of intermediate products. In this paper, a formamide–water solution interacting with TiO2 (anatase) surface is simulated using the molecular dynamics method. The structural, diffusion and density properties of formamide–water mixture on TiO2 are established for a wide temperature range from T = 250 K up to T = 400 K. 相似文献
13.
Tarek Mohamed Wesseem Osman Gary Tin Praveen P.N. Rao 《Bioorganic & medicinal chemistry letters》2013,23(15):4336-4341
The commonly used beverage and psychostimulant caffeine is known to inhibit human acetylcholinesterase enzyme. This pharmacological activity of caffeine is partly responsible for its cognition enhancing properties. However, the exact mechanisms of its binding to human cholinesterases (acetyl and butyrylcholinesterase; hAChE and hBuChE) are not well known. In this study, we investigated the cholinesterase inhibition by the xanthine derivatives caffeine, pentoxifylline, and propentofylline. Among them, propentofylline was the most potent AChE inhibitor (hAChE IC50 = 6.40 μM). The hAChE inhibitory potency was of the order: caffeine (hAChE IC50 = 7.25 μM) < pentoxifylline (hAChE IC50 = 6.60 μM) ? propentofylline (hAChE IC50 = 6.40 μM). These compounds were less potent relative to the reference agent donepezil (hAChE IC50 = 0.04 μM). Moreover, they all exhibited selective inhibition of hAChE with no inhibition of hBuChE (IC50 > 50 μM) relative to the reference agent donepezil (hBuChE IC50 = 13.60 μM). Molecular modeling investigations indicate that caffeine binds primarily in the catalytic site (Ser203, Glu334 and His447) region of hAChE whereas pentoxifylline and propentofylline are able to bind to both the catalytic site and peripheral anionic site due to their increased bulk/size, thereby exhibiting superior AChE inhibition relative to caffeine. In contrast, their lack of hBuChE inhibition is due to a larger binding site and lack of key aromatic amino acids. In summary, our study has important implications in the development of novel caffeine derivatives as selective AChE inhibitors with potential application as cognitive enhancers and to treat various forms of dementia. 相似文献
14.
K. Nazari A. Mahmoudi M. Khosraneh Z. Haghighian A.A. Moosavi-Movahedi 《Journal of Molecular Catalysis .B, Enzymatic》2009,56(1):61-69
Kinetics of microperoxidase-11 (MP-11) as a heme–peptide enzyme model in oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied in the presence of amino acids, taking into account the inactivation of MP-11 during reaction by its suicide substrate, H2O2. Reliability of the kinetic equation was evaluated by non-linear mathematical fitting. Fitting of experimental data into a new integrated kinetic relation showed a close match between the kinetic model and the experimental data. Indeed, it was found that the mechanism of suicide-peroxide inactivation of MP-11 in the presence of amino acids is different from MP-11 and/or horseradish peroxidase. In this mechanism, amino acids compete with hydrogen peroxide for the sixth co-ordination position of iron atom in the heme group through a competitive inhibition mechanism.The proposed model can successfully determine the kinetic parameters including inactivation by hydrogen peroxide as well as the inhibitory rate constants by the amino acid inhibitor.Kinetic parameters of inactivation including the initial activity of MP-11, α0, the apparent inactivation rate constant, ki and the apparent inhibition rate constant for cysteine, kI were obtained 0.282 ± 0.006 min?1, 0.497 ± 0.013 min?1 and 1.374 ± 0.007 min?1 at [H2O2] = 1.0 mM, 27 °C, phosphate buffer 5.0 mM, pH 7.0. Results showed that inactivation and inhibition of microperoxidase as a peroxidase model enzyme occurred simultaneously even at low concentrations of hydrogen peroxide (0.4 mM). This kinetic analysis based on the suicide-substrate inactivation of microperoxidase-11, provides a tool and model for studying peroxidase models in the presence of reversible inhibitors. The introduced inhibition procedure can be used in designing activity tunable and specific protected enzyme models in the hidden and reversibly inhibited forms, which do not undergo inactivation. 相似文献
15.
Acetohydroxyacid synthase (AHAS) is the key enzyme in branched chain amino acid biosynthesis pathway. The enzyme activity and properties of a highly thermostable AHAS from the hyperthermophilic bacterium Thermotoga maritima is being reported. The catalytic and regulatory subunits of AHAS from T. maritima were over-expressed in Escherichia coli. The recombinant subunits were purified using a simplified procedure including a heat-treatment step followed by chromatography. A discontinuous colorimetric assay method was optimized and used to determine the kinetic parameters. AHAS activity was determined to be present in several Thermotogales including T. maritima. The catalytic subunit of T. maritima AHAS was purified approximately 30-fold, with an AHAS activity of approximately 160±27 U/mg and native molecular mass of 156±6 kDa. The regulatory subunit was purified to homogeneity and showed no catalytic activity as expected. The optimum pH and temperature for AHAS activity were 7.0 and 85 °C, respectively. The apparent Km and Vmax for pyruvate were 16.4±2 mM and 246±7 U/mg, respectively. Reconstitution of the catalytic and regulatory subunits led to increased AHAS activity. This is the first report on characterization of an isoleucine, leucine, and valine operon (ilv operon) enzyme from a hyperthermophilic microorganism and may contribute to our understanding of the physiological pathways in Thermotogales. The enzyme represents the most active and thermostable AHAS reported so far. 相似文献
16.
Coniferyl alcohol is one of the major precursors of lignin; the most abundant aromatic compound and a natural resource currently receiving attention because of the value-added metabolites resulting from its degradation. Growth study of Streptomyces albogriseolus KF977548 (strain AOB) isolated from decaying wood residues in a tropical estuarine ecosystem was carried out using coniferyl alcohol as a sole carbon source. Cell growth and metabolite production were monitored at 24 h interval by dry weight measurements and HPLC, LC–MS-DAD analyses. Biochemical and PCR assays were carried out to detect the major catabolic enzymes of interest. Strain AOB utilized coniferyl alcohol completely within 72 h (μ = 0.204 h−1, Td = 3.4 h). Laccase and peroxidase were released into the growth medium up to 0.099 and 98 μmol/mL respectively. Protocatechuate 3, 4-dioxygenase and demethylase were detected in the genome whilst ortho-adipate pathway was clearly indicated. Growth on coniferyl alcohol or caffeic acid as mono substrates resulted in the production of secondary metabolites identified by HPLC–MS as 1-caffeoylquinic and 3,4,5-tricaffeoylquinic acids, known as chlorogenic acids, in the culture medium. The microbial production of chlorogenic acids from a lignin-related substrate base by strain AOB could arouse a plausible biotechnological process. 相似文献
17.
Xiaolin Pei Hongyu Zhang Lijun Meng Gang Xu Lirong Yang Jianping Wu 《Process Biochemistry》2013,48(12):1921-1927
A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans was cloned and expressed in Escherichia coli BL21 (DE3). A downstream gene adjacent to the β-subunit was necessary for the functional expression of the recombinant NHase. The structural gene order of the Co-type NHase was α-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. The NHase exhibited adequate thermal stability, with a half-life of 1.5 h at 50 °C. The NHase efficiently hydrated 3-cyanopyridine to produce nicotinamide. In a 1-L reaction mixture, 3.6 mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings, exhibiting a productivity of 187 g nicotinamide/g dry cell weight/h. An industrial auto-induction medium was applied to produce the recombinant NHase in 10-L fermenter. A glycerol-limited feeding method was performed, and a final activity of 2170 U/mL culture was achieved. These results suggested that the recombinant NHase was efficiently cloned and produced in E. coli. 相似文献
18.
《Process Biochemistry》2014,49(1):69-76
Alkaline pectate lyases (PLs) play an important role in mild and eco-friendly bioscouring pretreatment processes in the textile industry. However, to date, only a few PLs can be applied in industrial-scale production, and many of them exhibit high production cost, low activity, and/or do not meet the treatment requirements. In this study, an alkaline PL gene was cloned from the metagenomic DNA of alkaline environment soils. The gene pelB consisted of 1263 nucleotides and encoded a mature protein (PelB) of 399 amino acids, which was expressed in Escherichia coli. The maximum catalytic activity of the enzyme exhibited a bimodal distribution at pH 8.1 and 9.8 and an optimal temperature of 55 °C. The Km and Vmax values of PelB were 1.78 g/L and 1084.8 μmol/(L min) at 45 °C, respectively. Substrate specificity analysis demonstrated the high cleavage capability of PelB on a broad range of substrates of natural methylated pectin. Based on the degradation products, PelB was considered to be an endo-acting lyase. Using high-cell-density cultivation in 7-L bioreactor, the highest PL activity (1816.2 U/mL) was achieved. Thus, the recombinant PelB, with promising properties for use in bioscouring in the textile pretreatment process, should be a potential enzyme for industrial applications. 相似文献
19.
In the present work nanoparticles (NPs) of pepsin were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on low-density polyethylene (PE) films, or on polycarbonate (PC) plates, or on microscope glass slides. The pepsin NPs coated on the solid surfaces have been characterized by HRSEM, TEM, FTIR, XPS and DLS. The amount of enzyme introduced on the substrates, the leaching properties, and the catalytic activity of the immobilized enzyme on the three surfaces are compared. Catalytic activities of pepsin deposited onto the three solid surfaces as well as free pepsin, without sonication, and free pepsin NPs were compared at various pH levels and temperatures using a hemoglobin assay. Compared to native pepsin, pepsin coated onto PE showed the best catalytic activity in all the examined parameters. Pepsin immobilized on glass exhibited better activity than the native enzyme, especially at high temperatures. Enzyme activity of pepsin immobilized on PC was no better than native enzyme activity at all temperatures at pH 2, and only over a narrow pH range at 37 °C was the activity improved over the native enzyme. A remarkable observation is that immobilized pepsin on all the surfaces was still active to some extent even at pH 7, while free pepsin was completely inactive. The kinetic parameters, Km and Vmax were also calculated and compared for all the samples. Relative to the free enzyme, pepsin coated PE showed the greatest improvement in kinetic parameters (Km = 15 g/L, Vmax = 719 U/mg versus Km = 12.6 g/L and Vmax = 787 U/mg, respectively), whereas pepsin coated on PC exhibited the most unfavorable kinetic parameters (Km = 18 g/L, Vmax = 685 U/mg). The values for the anchored enzyme-glass were Km = 19 g/L, Vmax = 763 U/mg. 相似文献
20.
Alessio Innocenti Rebecca A. Hall Andrea Scozzafava Fritz A. Mühlschlegel Claudiu T. Supuran 《Bioorganic & medicinal chemistry》2010,18(3):1034-1037
The proteins encoded by the Nce103 genes of Candida albicans and Cryptococcus neoformans are catalytically active β-carbonic anhydrases (CAs, EC 4.2.1.1) playing various roles in the life cycle of these fungal pathogens, such as CO2 sensing, regulation of capsule biosynthesis, filamentation, and adaptation of the organism to various pH and CO2 conditions in various niches where the fungi grow. Here, we report the first activation study of these two enzymes, CaNce103 and Can2, respectively, with amines and amino acids. The C. albicans enzyme, CaNce103 was activated by amino acids such as l-/d-His, l-d-Trp, l-Tyr with KAs in the range of 19.5–46 μM. More effective activators were some amines such as histamine, dopamine, 2-aminoethyl-piperazine, and l-adrenaline (KAs of 13.2–18.5 μM). The best CaNce103 activators were l- and d-Dopa, with KAs of 0.96–2.5 μM. The C. neoformans enzyme, Can2, showed much lower propensity to be activated by all these amino acids and amines, which had activation constants in the range of 28.7–47.2 μM. The best Can2 activator was l-Trp. This study may help to better understand the catalytic/activation mechanisms of the β-CAs and eventually to design CA activity modulators of such widespread enzymes in pathogenic fungi. 相似文献