RESUMEN
p-Hydroxybenzoate hydroxylase (PHBH) catalyzes the ortho-hydroxylation of 4-hydroxybenzoate (4-HB) to protocatechuate (PCA). PHBHs are commonly known as homodimers, and the prediction of pyridine nucleotide binding and specificity remains an ongoing focus in this field. Therefore, our study aimed to determine the dimerization interface in AspPHBH from Arthrobacter sp. PAMC25564 and identify the canonical pyridine nucleotide-binding residues, along with coenzyme specificity, through site-directed mutagenesis. The results confirm a functional dimeric assembly from a tetramer that appeared in the crystallographic asymmetric unit identical to that established in previous studies. Furthermore, AspPHBH exhibits coenzyme versatility, utilizing both NADH and NADPH, with a preference for NADH. Rational engineering experiments demonstrated that targeted mutations in coenzyme surrounding residues profoundly impact NADPH binding, leading to nearly abrogated enzymatic activity compared to that of NADH. R50, R273, and S166 emerged as significant residues for NAD(P)H binding, having a near-fatal impact on NADPH binding compared to NADH. Likewise, the E44 residue plays a critical role in determining coenzyme specificity. Overall, our findings contribute to the fundamental understanding of the determinants of PHBH's active dimeric conformation, coenzyme binding and specificity holding promise for biotechnological advancements.
Asunto(s)
4-Hidroxibenzoato-3-Monooxigenasa , Arthrobacter , Multimerización de Proteína , Arthrobacter/enzimología , 4-Hidroxibenzoato-3-Monooxigenasa/metabolismo , 4-Hidroxibenzoato-3-Monooxigenasa/química , NADP/metabolismo , Modelos Moleculares , Coenzimas/metabolismo , Especificidad por Sustrato , NAD/metabolismo , Conformación Proteica , Mutagénesis Sitio-Dirigida , Unión Proteica , Sitios de Unión , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , ParabenosRESUMEN
The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some xenobiotics, several enzymatic steps are needed to obtain nontoxic products. Another challenge is the low durability and stability of many native enzymes in their purified form. Herein, we coupled peptide-based encapsulation of bacterial phosphotriesterase with soil-originated bacteria, Arthrobacter sp. 4Hß as an efficient system capable of biodegradation of paraoxon, a neurotoxin pesticide. Specifically, recombinantly expressed and purified methyl parathion hydrolase (MPH), with high hydrolytic activity toward paraoxon, was encapsulated within peptide nanofibrils, resulting in increased shelf life and retaining â¼50% activity after 132 days since purification. Next, the addition of Arthrobacter sp. 4Hß, capable of degrading para-nitrophenol (PNP), the hydrolysis product of paraoxon, which is still toxic, resulted in nondetectable levels of PNP. These results present an efficient one-pot system that can be further developed as an environmentally friendly solution, coupling purified enzymes and native bacteria, for pesticide bioremediation. We further suggest that this system can be tailored for different xenobiotics by encapsulating the rate-limiting key enzymes followed by their combination with environmental bacteria that can use the enzymatic step products for full degradation without the need to engineer synthetic bacteria.
Asunto(s)
Biodegradación Ambiental , Paraoxon , Hidrolasas de Triéster Fosfórico , Paraoxon/metabolismo , Paraoxon/química , Hidrolasas de Triéster Fosfórico/metabolismo , Hidrolasas de Triéster Fosfórico/química , Arthrobacter/enzimología , Péptidos/química , Péptidos/metabolismo , Nitrofenoles/metabolismo , Nitrofenoles/química , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Hidrólisis , Plaguicidas/metabolismo , Plaguicidas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificaciónRESUMEN
N-Methylisothiazolinone (MIT) is a thiol group modifier and antimicrobial agent. Arthrobacter sarcosine oxidase (SoxA), a diagnostic enzyme for assaying creatinine, loses its activity upon the addition of MIT, and its inactivation mechanism remains unclear. In this study, SoxA was chemically modified using MIT (mo-SoxA), and its structural and chemical properties were characterized. Spectral analysis data, oxygen consumption rates, and reactions were compared between intact SoxA and mo-SoxA. These demonstrate that the oxidative half-reaction toward oxygen is inhibited by MIT modification. The oxidase activity of mo-SoxA was approximately 2.1% of that of intact SoxA, and its dehydrogenase activity was approximately 4.2 times higher. The C-to-S mutants revealed that cooperative modification of 2 specific cysteine residues caused a drastic change in the enzyme reaction mode. Based on the modeled tertiary structures, the putative entrance for oxygen uptake is predicted to be blocked by the chemical modification of the 2 cysteine residues.
Asunto(s)
Arthrobacter , Oxígeno , Sarcosina-Oxidasa , Tiazoles , Arthrobacter/enzimología , Cisteína/química , Cisteína/metabolismo , Cinética , Modelos Moleculares , Oxidación-Reducción , Oxígeno/metabolismo , Oxígeno/química , Sarcosina-Oxidasa/metabolismo , Sarcosina-Oxidasa/química , Sarcosina-Oxidasa/genética , Tiazoles/farmacologíaRESUMEN
Transaminases have shown the ability to catalyze the amination of a series of aliphatic and (hetero)aromatic α,α-difluorinated ketones with high stereoselectivity, thus providing the corresponding ß,ß-difluoroamines in high isolated yields (55-82%) and excellent enantiomeric excess (>99%). It was also observed that these activated substrates could be quantitatively transformed by employing a small molar excess of the amine donor since this amination process was thermodynamically favored. Selected transformations could be scaled up to 500 mg, showing the robustness of this methodology.
Asunto(s)
Aminas/síntesis química , Hidrocarburos Fluorados/química , Cetonas/química , Transaminasas/química , Aminación , Arthrobacter/enzimología , Proteínas Bacterianas/química , Biocatálisis , Chromobacterium/enzimología , Estructura Molecular , EstereoisomerismoRESUMEN
Recent advances in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers have paved the way for determining radiation-damage-free protein structures under nonfreezing conditions. However, the large-scale preparation of high-quality microcrystals of uniform size is a prerequisite for SFX, and this has been a barrier to its widespread application. Here, a convenient method for preparing high-quality microcrystals of a bacterial quinoprotein enzyme, copper amine oxidase from Arthrobacter globiformis, is reported. The method consists of the mechanical crushing of large crystals (5-15â mm3), seeding the crushed crystals into the enzyme solution and standing for 1â h at an ambient temperature of â¼26°C, leading to the rapid formation of microcrystals with a uniform size of 3-5â µm. The microcrystals diffracted X-rays to a resolution beyond 2.0â Å in SFX measurements at the SPring-8 Angstrom Compact Free Electron Laser facility. The damage-free structure determined at 2.2â Å resolution was essentially identical to that determined previously by cryogenic crystallography using synchrotron X-ray radiation.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/química , Arthrobacter/enzimología , Sincrotrones/instrumentación , Secuencia de Aminoácidos , Cristalografía por Rayos X , Rayos Láser , Modelos Moleculares , Conformación Proteica , TemperaturaRESUMEN
BACKGROUND: In unfavourable environment, such as nutrient limitation, some bacteria encased themselves into a three dimensional polymer matrix called biofilm. The majority of microbial infections in human are biofilm related, including chronic lung, wound, and ear infections. The matrix of biofilm which consists of extracellular polymeric substances (EPS) causes bacterial colonization on medical implanted device in patients, such as catheter and lead to patient's death. Biofilm infections are harder to treat due to increasing antibiotic resistance compared to planktonic microbial cells and escalating the antibiotic concentration may result into in vivo toxicity for the patients. Special compounds which are non-microbicidal that could inhibit or destroy biofilm formation are called antibiofilm compounds, for example enzymes, anti-quorum sensing, and anti-adhesins. Arthrobacter sp. CW01 produced antibiofilm compound known as amylase. This time our preliminary study proved that the antibiofilm compound was not only amylase, but also protease. Therefore, this research aimed to optimize the production of antibiofilm agents using amylase and protease inducing media. The five types of production media used in this research were brain heart infusion (BHI) (Oxoid), BHI with starch (BHIS), casein with starch (CS), yeast extract with starch (YS), and casein-yeast extract with starch (CYS). Biofilm eradication and inhibition activities were assayed against Pseudomonas aeruginosa (ATCC 27,853) and Staphylococcus aureus (ATCC 25,923). RESULTS: The results showed that different production media influenced the antibiofilm activity. Addition of starch, casein and yeast extract increased the production of amylase and protease significantly. Higher amylase activity would gradually increase the antibiofilm activity until it reached the certain optimum point. It was shown that crude extracts which contained amylase only (BHI, BHIS and YS) had the optimum eradication activity against P. aeruginosa and S. aureus biofilm around 60-70 %. Meanwhile, CS and CYS crude extracts which contained both amylase and protease increased the biofilm eradication activity against both pathogens, which were around 70-90 %. CONCLUSIONS: It was concluded that the combination of amylase and protease was more effective as antibiofilm agents against P. aeruginosa and S. aureus rather than amylase only.
Asunto(s)
Amilasas/biosíntesis , Antibacterianos/farmacología , Arthrobacter/efectos de los fármacos , Biopelículas/efectos de los fármacos , Caseínas/farmacología , Péptido Hidrolasas/biosíntesis , Almidón/farmacología , Levaduras/química , Antibacterianos/biosíntesis , Arthrobacter/enzimología , Arthrobacter/metabolismo , Medios de Cultivo/química , Medios de Cultivo/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacosRESUMEN
Over the last few years, there has been a dramatic increase in the number of reports related to Arthrobacter sp. lipase (ABL:MTCC No. 5125) catalyzed kinetic resolution performed in biphasic media. A strain displaying esterase/lipase activity and designated as ABL was isolated, during the course of a screening program at Indian Institute of Integrative Medicine, Jammu. Considerable research has shown that reactions catalyzed by ABL are more selective than many commercial lipases. Since new applications of this lipase are emerging, there is a great need to provide all the relevant information exclusively. This review article is an attempt to cover all the relevant reports based on isolation, purification, immobilization, and application of ABL in the biopharmaceutical sector.
Asunto(s)
Arthrobacter/enzimología , Lipasa/metabolismo , Biocatálisis , Enzimas Inmovilizadas/metabolismo , Cinética , EstereoisomerismoRESUMEN
Optically active ß-amino alcohols are very useful chiral intermediates frequently used in the preparation of pharmaceutically active substances. Here, a novel cyclohexylamine oxidase (ArCHAO) was identified from the genome sequence of Arthrobacter sp. TYUT010-15 with the R-stereoselective deamination activity of ß-amino alcohol. ArCHAO was cloned and successfully expressed in E. coli BL21, purified and characterized. Substrate-specific analysis revealed that ArCHAO has high activity (4.15 to 6.34â U mg-1 protein) and excellent enantioselectivity toward the tested ß-amino alcohols. By using purified ArCHAO, a wide range of racemic ß-amino alcohols were resolved, (S)-ß-amino alcohols were obtained in >99 % ee. Deracemization of racemic ß-amino alcohols was conducted by ArCHAO-catalyzed enantioselective deamination and transaminase-catalyzed enantioselective amination to afford (S)-ß-amino alcohols in excellent conversion (78-94 %) and enantiomeric excess (>99 %). Preparative-scale deracemization was carried out with 50â mM (6.859â g L-1 ) racemic 2-amino-2-phenylethanol, (S)-2-amino-2-phenylethanol was obtained in 75 % isolated yield and >99 % ee.
Asunto(s)
Amino Alcoholes/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Transaminasas/metabolismo , Amino Alcoholes/química , Arthrobacter/enzimología , Biocatálisis , Estructura Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Estereoisomerismo , Transaminasas/genéticaRESUMEN
Choline oxidase catalyzes the four-electron, two-step, flavin-mediated oxidation of choline to glycine betaine. The enzyme is important both for medical and biotechnological reasons, because glycine betaine is one among a limited number of compatible solutes used by cells to counteract osmotic pressure. From a fundamental standpoint, choline oxidase has emerged as one of the paradigm enzymes for the oxidation of alcohols catalyzed by flavoproteins. Mechanistic, structural, and computational studies have elucidated the mechanism of action of the enzyme from Arthrobacter globiformis at the molecular level. Both choline and oxygen access to the active site cavity are gated and tightly controlled. Amino acid residues involved in substrate binding, and their contribution, have been identified. The mechanism of choline oxidation, with a hydride transfer reaction, an asynchronous transition state, the formation and stabilization of an alkoxide transient species, and a quantum mechanical mode of reaction, has been elucidated. The importance of nonpolar side chains for oxygen localization and of the positive charge harbored on the substrate for activation of oxygen for reaction with the reduced flavin have been recognized. Interesting phenomena, like the formation of a metastable photoinduced flavin-protein adduct, the reversible formation of a bicovalent flavoprotein, and the trapping of the enzyme in inactive conformations, have been described. This review summarizes the current status of our understanding on the structure-function-dynamics of choline oxidase.
Asunto(s)
Oxidorreductasas de Alcohol/química , Arthrobacter/enzimología , Proteínas Bacterianas/química , Colina , Catálisis , Cinética , OxígenoRESUMEN
Arthrobacter sp. KFS-1 previously isolated from a dump site was used to produce keratinase in basal medium. The physico-chemical conditions were optimized to enhance the keratinase production, and biochemical properties of the enzyme were also evaluated. Arthrobacter sp. KFS-1 optimally produced keratinase in a basal medium that contained 1.0 g/L xylose, 2.5-5.0 g/L chicken feather; with initial pH, incubation temperature and agitation speed of 6.0, 30 °C and 200 rpm, respectively. Maximum keratinase activity of 1559.09 ± 29.57 U/mL was achieved at 96 h of fermentation; while optimal thiol concentration of 665.13 ± 38.73 µM was obtained at 144 h. Furthermore, the enzyme was optimally active at pH 8.0 and 60 °C. The enzyme activity was inhibited by ethylene diamine tetraacetic acid and 1,10-phenanthroline, but not affected by phenylmethylsulfonyl floride. In addition, the crude enzyme retained 55%, 63%, 80%, 81% and 90% of the original activity after respective pretreatment with some commercial detergents (Maq, Omo, Surf, Sunlight and Ariel). Moreso, the enzyme showed remarkable stability in the presence of reducing agents, surfactants, and organic solvents. Arthrobacter sp. KFS-1 significantly produced keratinase which exhibited excellent stability in presence of chemical agents and commercial laundry detergents; hence, suggesting its industrial application potentials especially in detergent formulation.
Asunto(s)
Arthrobacter/enzimología , Detergentes/química , Péptido Hidrolasas/biosíntesis , Péptido Hidrolasas/química , Álcalis , Arthrobacter/genética , Arthrobacter/crecimiento & desarrollo , Pruebas de Enzimas , Estabilidad de Enzimas , Fermentación , Concentración de Iones de Hidrógeno , Iones/química , Péptido Hidrolasas/aislamiento & purificación , Compuestos de Sulfhidrilo/análisis , TemperaturaRESUMEN
Toward a sustainable synthesis of value-added chemicals, the method of CO2 utilization attracts great interest in chemical process engineering. Biotechnological CO2 fixation is a promising technology; however, efficient methods that can fix carbon dioxide are still limited. Instead, some parts of microbial decarboxylases allow the introduction of carboxy group into phenolic compounds using bicarbonate ion as a C1 building block. Here, we identified a unique decarboxylase from Arthrobacter sp. K8 that acts on resorcinol derivatives. A high-throughput colorimetric decarboxylase assay facilitated gene cloning of orsellinic acid decarboxylase from genomic DNA library of strain K8. Sequence analysis revealed that the orsellinic acid decarboxylase belonged to amidohydrolase 2 family, but shared low amino acid sequence identity with those of related decarboxylases. Enzymatic characterization unveiled that the decarboxylase introduces a carboxy group in a highly regio-selective manner. We applied the decarboxylase to enzymatic carboxylation of resorcinol derivatives. Using Escherichia coli expressing the decarboxylase gene as a whole cell biocatalyst, orsellinic acid, 2,4-dihydroxybenzoic acid, and 4-methoxysalicylic acid were produced in the presence of saturated bicarbonate. These findings could provide new insights into the production of useful phenolic acids from resorcinol derivatives.
Asunto(s)
Arthrobacter/enzimología , Arthrobacter/genética , Carboxiliasas/química , Carboxiliasas/genética , Clonación Molecular , Resorcinoles/química , Resorcinoles/metabolismo , Secuencia de Aminoácidos , Arthrobacter/aislamiento & purificación , Escherichia coli/genética , Hidroxibenzoatos , Cinética , Fenoles/metabolismo , Análisis de Secuencia , Suelo , Microbiología del Suelo , Especificidad por SustratoRESUMEN
3-Ketosteroid Δ1-dehydrogenase (KsdD) is the key enzyme responsible for Δ1-dehydrogenation, which is one of the most valuable reactions for steroid catabolism. Arthrobacter simplex has been widely used in the industry due to its superior bioconversion efficiency, but KsdD information is not yet fully clear. Here, five KsdD homologues were identified in A. simplex CGMCC 14539. Bioinformatic analysis indicated their distinct properties and structures. Each KsdD was functionally confirmed by transcriptional response, overexpression, and heterologous expression. The substantial difference in substrate profiles might be related to the enzyme loop structure. Two promising enzymes (KsdD3 and KsdD5) were purified and characterized, exhibiting strong organic solvent tolerance and clear preference for 4-ene-3-oxosteroids. KsdD5 seemed to be more versatile due to good activity on substrates with or without a substituent at C11 and high optimal temperature and also possessed unique residues. It is the first time that KsdDs have been comprehensively disclosed in the A. simplex industrial strain.
Asunto(s)
Arthrobacter/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arthrobacter/química , Arthrobacter/genética , Bacterias/química , Bacterias/clasificación , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/genética , Dominio Catalítico , Oxidorreductasas/genética , Filogenia , Alineación de SecuenciaRESUMEN
ß-Galactosidase from Arthrobacter sp. 32cB (ArthßDG) is a cold-adapted enzyme able to catalyze hydrolysis of ß-d-galactosides and transglycosylation reaction, where galactosyl moiety is being transferred onto an acceptor larger than a water molecule. Mutants of ArthßDG: D207A and E517Q were designed to determine the significance of specific residues and to enable formation of complexes with lactulose and sucrose and to shed light onto the structural basis of the transglycosylation reaction. The catalytic assays proved loss of function mutation E517 into glutamine and a significant drop of activity for mutation of D207 into alanine. Solving crystal structures of two new mutants, and new complex structures of previously presented mutant E441Q enables description of introduced changes within active site of enzyme and determining the importance of mutated residues for active site size and character. Furthermore, usage of mutants with diminished and abolished enzymatic activity enabled solving six complex structures with galactose, lactulose or sucrose bounds. As a result, not only the galactose binding sites were mapped on the enzyme's surface but also the mode of lactulose, product of transglycosylation reaction, and binding within the enzyme's active site were determined and the glucopyranose binding site in the distal of active site was discovered. The latter two especially show structural details of transglycosylation, providing valuable information that may be used for engineering of ArthßDG or other analogous galactosidases belonging to GH2 family.
Asunto(s)
Arthrobacter/enzimología , Proteínas Bacterianas/química , beta-Galactosidasa/química , Sustitución de Aminoácidos , Arthrobacter/genética , Dominio Catalítico , Mutación Missense , beta-Galactosidasa/genéticaRESUMEN
The 165,137 bp plasmid pAO1 of Paenarthrobacter nicotinovorans carries the genes of a nicotine catabolic pathway. The genes are organized into several gene modules responsible for the catabolism of L- and D-nicotine to nicotine blue, alpha-ketoglutarate and succinate. Various modules of these genes have been shown to be present in gram-positive (Gram?) soil bacteria. The presence of the identical pAO1 nic-genes on the 288,370 bp plasmid pZXY21 of Arthrobacter sp. ZXY2 (96 percent to 100 percent at the nucleotide level) permitted the identification of the limits of this DNA fragment. At the 5' end of the nic-genes are located the ORFs of two predicted integrases of the tyrosine recombinase family with conserved R, H, R and Y catalytic residues and that of a small transposase with a predicted leucine zipper motive. They are related to Tn554A, Tn554B and Tn554C of Staphylococcus aureus and suggest that the entire nic-genes DNA fragment represents a large catabolic transposon. Surprisingly the nic-genes on pZXY21 were found to be interspersed by mobile elements encoding transposases of various IS families. Insertion of these IS elements disrupts nicotine degradation and divide the nic-genes DNA into potentially new transposons. This finding may illustrate how nicotine catabolic genes can be mobilized and spread by horizontal gene transfer to other soil bacteria.
Asunto(s)
Arthrobacter/enzimología , Arthrobacter/genética , Proteínas Bacterianas/genética , Elementos Transponibles de ADN , Nicotina/metabolismo , Arthrobacter/metabolismo , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos , Transferencia de Gen Horizontal , Genes Bacterianos , Integrasas/genética , Micrococcaceae/genética , Plásmidos , Microbiología del SueloRESUMEN
As a prebiotics, lactosucrose plays an important role in maintaining human gastrointestinal homeostasis. In this study, a thermostable enzyme from Arthrobacter sp. 10138 was screened from six ß-fructofuranosidase-producing strains for the lactosucrose production and the coding gene was heterologously expressed in Escherichia coli for efficient expression. Recombinant ß-fructofuranosidase was purified and biochemically characterized by MALDI-TOFMS spectrometry. The transfructosylation product by this recombinant enzyme was determined to be lactosucrose rather than other oligosaccharides or polysaccharides by HPLC and LC-MS. Efficient extracellular secretion of ß-fructofuranosidase was achieved by the optimization of signal peptide and induction conditions. It was found that with the signal peptide torT, the highest extracellular activity reached 111.01 U/mL, which was 38.4-fold higher than that with the OmpA signal peptide. Under the optimal conditions (pH 6.0, temperature 50°C, enzyme amount 40 µg/ml, sucrose 150 g/L and lactose 150 g/L), 109 g/L lactosucrose was produced with a molar conversion ratio of 49.3%. Here the thermostable ß-fructofuranosidase from Arthrobacter sp. 10138 can be used for efficient synthesis of lactosucrose, and this provides a good startpoint for the industrial production of lactosucrose in the future.
Asunto(s)
Arthrobacter/enzimología , Trisacáridos/metabolismo , beta-Fructofuranosidasa/metabolismo , Biocatálisis , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Lactosa/metabolismo , Sacarosa/metabolismoRESUMEN
Potato juice is a byproduct of starch processing currently used as feed. However, potato proteins are an untapped source of high-protein food for human nutrition if harmful constituents notably glycoalkaloids (GAs) are detoxified. The two principle GAs found in potato are α-chaconine and α-solanine, both consisting of a solanidine aglycone with a carbohydrate side chain. The first step in the detoxification of these compounds is the removal of the trisaccharide. Whole-genome sequencing of a bacterial isolate, Arthrobacter sp. S41, capable of completely degrading α-chaconine and α-solanine, revealed the presence of a gene cluster possibly involved in the deglycosylation of GAs. Functional characterization confirmed the enzymatic activity of the gene cluster involved in the complete deglycosylation of both α-chaconine and α-solanine. The novel enzymes described here may find value in the bioconversion of feed proteins to food proteins suitable for human nutrition.
Asunto(s)
Arthrobacter/metabolismo , Proteínas Bacterianas/metabolismo , Familia de Multigenes , Solanina/análogos & derivados , Solanum tuberosum/toxicidad , Arthrobacter/clasificación , Arthrobacter/enzimología , Arthrobacter/genética , Proteínas Bacterianas/genética , Biotransformación , Glicosilación , Filogenia , Solanina/química , Solanina/metabolismo , Solanina/toxicidad , Solanum tuberosum/metabolismoRESUMEN
Reliable kinetic parameters of enzymes are of paramount importance for a precise understanding of catalytic performance, which is essential for enzyme engineering and process optimization. Here, we developed a simple and convenient method to determine intrinsic kinetic parameters of R-selective ω-transaminases (ω-TAs) with a minimal set of kinetic data. Using (R)-α-methylbenzylamine ((R)-α-MBA) and pyruvate as a substrate pair, two R-selective ω-TAs from Arthrobacter sp. and Aspergillus fumigatus were subjected to kinetic measurements. In contrast to S-selective ω-TAs, both R-selective ω-TAs were observed to be devoid of substrate inhibition by pyruvate. Double reciprocal plot analysis was carried out with two sets of kinetic data obtained at varying concentrations of (R)-α-MBA under a fixed concentration of pyruvate and vice versa, leading to the determination of three intrinsic kinetic parameters, i.e., one kcat and two KM values, using three regression constants. The validity of the kinetic parameters was verified by a self-consistency test using a regression constant left out in the kinetic parameter determination, showing that deviations of calculated regression constants from the experimental ones were less than 15%. Because the kinetic parameters for (R)-α-MBA and pyruvate are not apparent but intrinsic, a cosubstrate substitution method enabled rapid determination of intrinsic parameters for a new substrate pair using just one set of kinetic data. Eventually, computational modeling of kinetic resolution of rac-α-MBA was carried out and showed a good agreement with experimental reaction progresses.
Asunto(s)
Arthrobacter/enzimología , Aspergillus fumigatus/enzimología , Proteínas Bacterianas/química , Proteínas Fúngicas/química , Fenetilaminas/química , Transaminasas/química , Cinética , Ácido Pirúvico/químicaRESUMEN
ArthßDG is a dimeric, cold-adapted ß-d-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its ArthßDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The ArthßDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: ArthßDG_E441Q/LACs and ArthßDG/IPTG (ligand bound in shallow mode) and structures of complexes ArthßDG_E441Q/LACd, ArthßDG/ONPG (ligands bound in deep mode), and galactose ArthßDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 ßDGs, in ArthßDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.
Asunto(s)
Arthrobacter/enzimología , Arthrobacter/metabolismo , beta-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Arthrobacter/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Frío , Hidrólisis , beta-Galactosidasa/genéticaRESUMEN
Glycosaminoglycans (GAGs) and their low-molecular weight derivates have received considerable interest in terms of their potential clinical applications, and display a wide variety of pharmacological and pharmacokinetic properties. Structurally distinct GAG chains can be prepared by enzymatic depolymerization. A variety of bacterial chondroitin sulfate (CS) lyases have been identified, and have been widely used as catalysts in this process. Here, we identified a putative chondroitin AC exolyase gene, AschnAC, from an Arthrobacter sp. strain found in a CS manufacturing workshop. We expressed the enzyme, AsChnAC, recombinantly in Escherichia coli, then purified and characterized it in vitro. The enzyme indeed displayed exolytic cleavage activity toward HA and various CSs. Removing the putative N-terminal secretion signal peptide of AsChnAC improved its expression level in E. coli while maintaining chondroitin AC exolyase activity. This novel catalyst exhibited its optimal activity in the absence of added metal ions. AsChnAC has potential applications in preparation of low-molecular weight GAGs, making it an attractive catalyst for further investigation.
Asunto(s)
Arthrobacter/enzimología , Condroitín Liasas/genética , Condroitín Liasas/metabolismo , Arthrobacter/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Clonación Molecular , Escherichia coli/genética , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Peso Molecular , Proteínas Recombinantes/metabolismoRESUMEN
A novel strategy was used to produce inulin fructotransferase from Arthrobacter aurescens (Aa-IFTase) embedded in curdlan-based mesoporous silica microspheres (CMSiM-Aa-IFTase). The CMSiM-Aa-IFTase was constructed by co-entrapping cross-linked Aa-IFTase aggregates and curdlan into biomemitic silica, and the curdlan was subsequently removed by digestion with endo-ß-1,3-glucanase. During this process, the curdlan served as an agent to introduce pores in silica microspheres. The resulting CMSiM-Aa-IFTase showed higher stability and activity than free Aa-IFTase and mCLEAs-Aa-IFTase (modified cross-linked enzyme aggregates with Aa-IFTase). Furthermore, the CMSiM-Aa-IFTase displayed good reusability and excellent storage stability. The excellent catalytic performances were due to the combinational structure from the cross-linked enzyme aggregates and hard shell of mesoporous silica microspheres, which might decrease the negative interaction between support and enzyme, and improve the mechanical properties. The CMSiM-Aa-IFTase was applicable for efficient production of Difructose Anhydride III (DFA III), and this approach should be highly valuable for preparing various mesoporous composites for catalysis.