The UGT74L2 of Andrographis paniculata catalyzes phloretin to produce trilobatin and its enzymatic study / 药学学报

The last essential enzyme in the biosynthetic pathway of trilobatin, phloretin-4'-O glycosyltransferase (P4'-OGT), catalyzes the conversion of trilobatin to phloretin in vitro. However, only a few P4'-OGTs have been found in plants. This study used Malus domestica phloretin-4'-O glycosyltransferase (MdPh-4'-OGT) as a query to identify and clone two UDP-glucuronosyltransferase (UGT) genes, designated UGT74L2 and UGT74L3, from the transcriptome of Andrographis paniculata. According to a phylogenetic tree analysis, UGT74L2 and UGT74L3 belonged to the UGT74 family, which has been linked to several activities in other species. The in vitro enzymatic reaction demonstrated that UGT74L2 could particularly catalyze the formation of trilobatin from phloretin, but UGT74L3 had no effects. By using Ni-NTA affinity chromatography to extract the soluble UGT74L2 recombinant protein, the enzymatic kinetics of the activity was investigated using phloretin as the substrate. The results showed that the optimal temperature and pH for UGT74L2 enzymatic reaction were 40 ℃ and 8.0 (Tris-HCl system), respectively. Three metal ions (Ca2+, Mn2+ and Co2+) showed inhibitory effect on the activity of UGT74L2, while Mg2+ could improve the activity of UGT74L2. Other tested metal ions have no significant effect on UGT74L2. The results of enzymatic kinetic parameters that the Km value was 29.84 μmol·L-1, the kcat was 0.02 s-1, and the kcat·Km-1 was 572.6 mol-1·s-1. By homology modeling, molecular docking and mutation experiments, we found that multiple amino acids residues around the substrate binding pocket play quite an important role during catalytic process, In summary, we identified a novel P4'-OGT gene from medicinal plant Andrographis paniculata and provided a new efficient catalyst to synthesize trilobatin. Meanwhile, this study provides a reference for mining new efficient glycosylation modules from plants.

Establishment and application of activity determining methods for different subtypes of xanthine oxidoreductase / 药学学报

Xanthine oxidoreductase (XOR), the key enzyme catalyzing purine to produce uric acid, including two subtypes, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), respectively, in vivo. Usually, XDH and XO can transform to each other. In this study, based on the principle that the subtype XO or XDH uses different electron acceptors, the methods for the measuring the activities of bovine milk XOR (pure enzyme) and its subtypes were established. The optimal concentrations of substrate xanthine (50 μmol·L-1) and electron acceptor NAD+ (50 μmol·L-1), pH value (7.80) were investigated. The ranges of the XOR, XO, XDH activity which could be determined were 0.97-17.5 U·L-1, 1-9 U·L-1, and 66-1 191 mU·L-1, respectively. Furthermore, the methods for determining the activities of XOR and its subtypes in mouse liver were established. The preparation of liver samples, the optimal concentrations of xanthine (100 μmol·L-1) and NAD+ (100 μmol·L-1) were researched. And the activity ranges of XOR, XO and XDH in mouse liver which could be determined were 0.67-3.98, 0.19-1.08, and 0.52-3.55 U·gprot-1, respectively. With the methods above, the effects of classic XOR inhibitor allopurinal (Allo) on XOR, XO and XDH from both milk and mouse liver were determined. All animal experiments have been approved by the Animal Experimental Center, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College (00003346). This study established new methods for the determination of XOR and its subtypes activity in pure enzyme system and in mouse liver, respectively, which were accurate and convenient. It laid the experimental foundation for exploring the different pathophysiological effects of XOR in the body and developing new XOR inhibitors.

LXYL-P1-2 immobilized on magnetic nanoparticles and its potential application in paclitaxel production

BACKGROUND: LXYL-P1-2 is the first reported glycoside hydrolase that can catalyze the transformation of 7-b-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT) by removing the D-xylosyl group at the C7 position. Successful synthesis of paclitaxel by one-pot method combining the LXYL-P1-2 and 10- deacetylbaccatin III-10-b-O-acetyltransferase (DBAT) using XDT as a precursor, making LXYL-P1-2 a highly promising enzyme for the industrial production of paclitaxel. The aim of this study was to investigate the catalytic potential of LXYL-P1-2 stabilized on magnetic nanoparticles, the surface of which was modified by Ni2+-immobilized cross-linked Fe3O4@Histidine. RESULTS: The diameter of matrix was 20­40 nm. The Km value of the immobilized LXYL-P1-2 catalyzing XDT (0.145 mM) was lower than that of the free enzyme (0.452 mM), and the kcat/Km value of immobilized enzyme (12.952 mM s 1 ) was higher than the free form (8.622 mM s 1 ). The immobilized form maintained 50% of its original activity after 15 cycles of reuse. In addition, the stability of immobilized LXYL-P1-2, maintained 84.67% of its initial activity, improved in comparison with free form after 30 d storage at 4 C. CONCLUSIONS: This investigation not only provides an effective procedure for biocatalytic production of DT, but also gives an insight into the application of magnetic material immobilization technology.

Biological evaluation of 2,3-dioxoindolin-N-phenylacetamide derivatives as potent CDC25B and PTP1B phosphatase inhibitors

A series of 2,3-dioxoindolin-N-phenylacetamide derivatives was evaluated for inhibitory activity against CDC25B and PTP1B enzymes. Most of the derivatives showed inhibitory activity against CDC25B (IC50 = 3.2-23.2 µg/mL) and PTP1B (IC50 = 2.9-21.4 µg/mL). Compound 2h showed the most inhibitory activity in vitro with IC50 values of 3.2 and 2.9 µg/mL against CDC25B and PTP1B, respectively, compared with the reference drugs Na3VO4 (IC50 = 2.7 µg/mL) and oleanolic acid (IC50 = 2.3 µg/mL). The results of selectivity experiments showed that the 2,3-dioxoindolin-N-phenylacetamide derivatives were selective inhibitors against CDC25B and PTP1B. Enzyme kinetic experiments demonstrated that compound 2h was a specific inhibitor with the typical characteristics of a mixed inhibitor. In cytotoxic activity assays compound 2h had potent activity against A549, HeLa, and HCT116 cell lines. In addition, compound 2h showed potent tumor inhibitory activity in a colo205 xenograft model in vivo.

L-DOPA extradiol dioxigenase de amanita muscaria: revisão da sequência codificadora, expressão heteróloga, caracterização funcional e contextualização filogenética / Amanita muscaria L-DOPA extradiol dioxygenase: coding sequence review, heterologous expression, functional characterization, and phylogenetic context

Extradiol dioxigenases são enzimas que catalisam a clivagem oxidativa de ligações C-C entre grupos hidroxila fenólicos adjacentes utilizando catecóis como substratos. Esta classe de enzimas é bem caracterizada em bactérias, onde catalisam a degradação de compostos aromáticos. Na maioria das plantas Caryophyllales, como a beterraba, primavera e a maravilha, L-3,4-diidroxifenilalanina (L-DOPA) extradiol dioxigenases (DODAs) catalisam a clivagem oxidativa de L-DOPA na posição 4,5 gerando o ácido betalâmico, aldeído precursor das betalaínas, uma classe de pigmentos naturais que substitui as antocianinas na pigmentação dessas espécies. Alguns fungos basidiomicetos também produzem betalaínas, como o agário-das-moscas (Amanita muscaria). Nesse organismo, DODA é capaz de catalisar uma clivagem adicional na posição 2,3 da L-DOPA, formando muscaflavina, um isômero do ácido betalâmico que dá origem a uma outra classe de pigmentos naturais: as higroaurinas. Desde a caracterização do gene dodA, o qual codifica para a DODA de A. muscaria (AMAMU), não existem relatos na literatura que explorem a promiscuidade catalítica desta enzima, sua relação com outras linhagens de DODAs e a síntese quimioenzimática de betalaínas a partir desta enzima. Dessa forma, buscamos contextualizar as relações filogenéticas e funcionais entre AMAMU e diferentes linhagens de DODAs, bem como estabelecer um método que viabilize a clonagem, expressão heteróloga e caracterização funcional destaenzima. As análises filogenéticas revelaram que AMAMU possui uma evolução convergente com DODAs de plantas e bactérias e que, apesar de AMAMU ser funcionalmente homóloga à DODA da bactéria Escherichia coli, esta última apresenta homologia com DODAs de plantas. Logo, não há uma relação direta entre a sequência primária de DODAs e sua função. Nós também demonstramos que não há uma relação entre a expressão de transcritos de BvDODA1, e de seu parálogo BvDODA2, e a diferença de pigmentação entre variedades de beterrabas amarelas e vermelhas. A clonagem da sequência codificadora (CDS) publicada para o gene dodA de A. muscaria resultou na retenção do primeiro íntron, o que impedia a sua expressão. Então, uma nova CDS de 558 nucleotídeos foi proposta para este gene, a qual inclui um códon de início da tradução que se mantém na fase de leitura e codifica para uma proteína de 185 resíduos, 43 a menos que AMAMU. A expressão desta CDS resultou na proteína recombinante AmDODA, capaz de catalisar a síntese de ácido betalâmico e muscaflavina a partir de L-DOPA e D-DOPA. AmDODA possui um tamanho aproximado de 22 kDa, com um pH ótimo de atividade de 8,5 e uma constante de Michaelis (KM) de 3,7 ± 0,9 mmol L-1 e de velocidade máxima (Vmax) de 3,3 ± 0,4 µ mol min-1 mg-1. Sua utilização foi demonstrada na síntese quimioenzimática de betalaínas-modelo com potencial aplicação como sondas para microscopia confocal de fluorescência de dois fótons. Neste contexto, esta Tese explora os aspectos moleculares, bioquímicos e biológicos da DODA do fungo A. muscaria e traz importantes contribuições acerca da pigmentação por betalaínas na natureza

Extradiol dioxigenases are enzymes that catalyze the oxidative cleavage of C-C bonds between adjacent phenolic hydroxyl groups using catechols as substrates. This class of enzymes is well characterized in bacteria, where they catalyze the degradation of aromatic compounds. In most plants of the Order Caryophyllales, such as beet, paperflower and four o'clock flower, L-3,4-dihydroxyphenylalanine (L-DOPA) extradiol dioxygenases (DODAs) catalyze the oxidative 4,5-cleavage of L-DOPA generating the betalamic acid, an aldehyde precursor of the betalains, a class of natural pigments that replaces anthocyanins in the pigmentation of these species. Some basidiomycete fungi also produce betalains, such as the fly agaric (Amanita muscaria). In this organism, DODA is able to catalyze an additional 2,3-cleavage of L-DOPA, yielding muscaflavine, an isomer of betalamic acid that gives rise to another class of natural pigments: the hygroaurins. Since the characterization of the dodA gene, which encodes the A. muscaria DODA (AMAMU), there are no reports in the literature that explore the catalytic promiscuity of this enzyme, its relation to other DODAs and the chemoenzymatic synthesis of betalains from this enzyme. Thus, we seek to contextualize the phylogenetic and functional relationships between AMAMU and different DODA lineages, as well as to establish a method that enable the cloning, heterologous expression and functional characterization of this enzyme. Phylogenetic analysis revealed that AMAMU has a convergent evolutionwith plant and bacterial DODAs and that although AMAMU is functionally homologous to the DODA of the Escherichia coli bacteria, this latter is homologous to the plant DODAs. Therefore, there is no direct relationship between the primary sequence of DODAs and their function. We have also shown that there is no relationship between the expression of BvDODA1 transcripts, and its BvDODA2 paralogue, and the pigment difference between yellow and red beet varieties. Cloning of the published coding sequence (CDS) for the dodA gene of A. muscaria resulted in the retention of the first intron, which prevented its expression. Then, a new CDS of 558 nucleotides was proposed for this gene, which includes a translation start codon that remains in the open reading frame and encodes for a protein 185 residues long, 43 less than AMAMU. Expression of this CDS resulted in the recombinant AmDODA protein, able to catalyze the synthesis of betalamic acid and muscaflavine from L-DOPA and D-DOPA. AmDODA has an approximate size of 22 kDa, with an optimum activity pH of 8.5 and a Michaelis constant (KM) of 3.7 ± 0.9 mmol L-1 and a maximum velocity (Vmax) of 3.3 ± 0.4 µmol min-1 mg-1. Its use was demonstrated in the chemoenzymatic synthesis of betalains-model with potential application as probes for confocal microscopy of two-photon fluorescence. In this context, this thesis explores the molecular, biochemical and biological aspects of the DODA of the fungus A. muscaria and brings important contributions about the pigmentation by betalains in nature

Progress and application of metabolic network model based on enzyme constraints / 生物工程学报

Genome-scale metabolic network models have been successfully applied to guide metabolic engineering. However, the conventional flux balance analysis only considers stoichiometry and reaction direction constraints, and the simulation results cannot accurately describe certain phenomena such as overflow metabolism and diauxie growth on two substrates. Recently, researchers proposed new constraint-based methods to simulate the cellular behavior under different conditions more precisely by introducing new constraints such as limited enzyme content and thermodynamics feasibility. Here we review several enzyme-constrained models, giving a comprehensive introduction on the biological basis and mathematical representation for the enzyme constraint, the optimization function, the impact on the calculated flux distribution and their application in identification of metabolic engineering targets. The main problems in these existing methods and the perspectives on this emerging research field are also discussed. By introducing new constraints, metabolic network models can simulate and predict cellular behavior under various environmental and genetic perturbations more accurately, and thus can provide more reliable guidance to strain engineering.

in vitro metabolism of daphnetin in rat liver S9 fractions / 药学学报

Daphnetin is quickly eliminated in rats after dosing, but the mechanism remains unclear. This study was aimed to investigate the in vitro metabolism of daphnetin using rat liver S9 fractions (RLS9). The metabolites formed in RLS9 were identified and the kinetic parameters for different metabolic pathways were determined. HPLC-DAD-MS analysis showed that daphnetin was biotransformed to six metabolites, which were identified as 7 or 8 mono-glucuronide and mono-sulfate, 8-methylate, and 7-suflo-8-methylate. Methylation and glucuronidation of daphnetin exhibited the Michaelis-Menten kinetic characteristics, whereas the substrate inhibition kinetic and the two-site kinetic were observed for 8-sulfate and 7-sulfate formations. Of the 3 conjugation pathways, the intrinsic clearance rate for sulfation was highest, followed by methylation and glucuronidation. By in vitro-in vivo extrapolation of the kinetic data measured in RLS9, the hepatic clearance were estimated to be 54.9 mL·min-1·kg-1 which is comparable to the system clearance (58.5 mL·min-1·kg-1) observed in rats. In conclusions, the liver might be the main site for daphnetin metabolism in rats. Sulfation, methylation and glucuronidation are important pathways of the hepatic metabolism of daphnetin in rats.

Enzyme kinetic analysis of Oncomelania hupensis exposed to active ingredient of Buddleja lindleyana (AIBL) / 中国血吸虫病防治杂志

Objective To analyze the enzyme kinetics of active ingredient of Buddleja lindleyana(AIBL)against Oncomela?nia hupensis,the intermediate host of Schistosoma japonicum. Methods O. hupensis snails were placed in 1 000 ml of 3.55 mg/L AIBL solution for 24,48 h and 72 h,respectively,and the enzyme kinetics of alanine aminotransferase(GPT)was deter?mined by Reitman?Frankel assay,lactate dehydrogenase(LDH)by the chemical inhibition lactic acid substrate method,alka?line phosphatase(AKP)by the disodium phenyl phosphate colorimetric method,acetylcholine esterase(AChE)and malate de?hydrogenas(MDH)by ELISA,and succinate dehydrogenase(SDH)by the phenazine methyl sulfate reaction method(PMS)in the soft tissues of O. hupensis before and after AIBL treatment. Results Following exposure to 3.55 mg/L AIBL solution for 24 h, the GPT,LDH,and AKP activities significantly improved in the soft tissues of O. hupensis,while the SDH and MDH activities were significantly lowered in the head?foot and liver. However,AIBL treatment did not cause significant effect on AChE activity in O. hupensis. Conclusions AIBL causes significant damages to O. hupensis liver and can efficiently act on anaerobic and aer?obic respiration loci,which will hinder energy metabolism,and cause inadequate energy supply in cells used for normal secre?tion,eventually leading to O. hupensis death.

Characterization of succinic semialdehyde dehydrogenase from Aspergillus niger.

The catabolism of fungal 4-aminobutyrate (GABA) occurs via succinic semialdehyde (SSA). Succinic semialdehyde dehydrogenase (SSADH) from the acidogenic fungus Aspergillus niger was purified from GABA grown mycelia to the highest specific activity of 277 nmol min-1 mg-1, using phenyl Sepharose and DEAE Sephacel chromatography. The purified enzyme was specific for its substrates SSA and NAD+. The substrate inhibition observed with SSA was uncompetitive with respect to NAD+. While product inhibition by succinate was not observed, NADH inhibited the enzyme competitively with respect to NAD+ and noncompetitively with respect to SSA. Dead-end inhibition by AMP and p-hydroxybenzaldehyde (pHB) was analyzed. The pHB inhibition was competitive with SSA and uncompetitive with NAD+; AMP competed with NAD+. Consistent with the kinetic data, a sequential, ordered Bi Bi mechanism is proposed for this enzyme.

Kinetic constraints and features imposed by the immobilization of enzymes onto solid matrices: A key to advanced biotransformation.

The kinetics of immobilized enzymes can not be analyzed by means of the simple Michaelis-Menten concept, which generally fails to describe the immobilized state due to both its probable barriers, and because the active concentration of the enzyme approaches, or even exceeds this of its substrate(s). In such cases, the various experimental data are usually treated by complex rate equations comprising too many parameters acquiring different natures and meanings, depending on both the properties of the immobilization state and the experimental conditions; thus, more likely, only apparent values of the Michaelis-Menten kinetic parameters can be estimated experimentally. Likewise, immobilization is often a key method in optimizing the operational performance of enzymes, in both laboratory and industrial scale, and affects considerably the kinetics in non-aqueous and non-conventional media due to several issues as the structural changes of the enzyme molecule, the heterogeneity of the system, and the partial or total absence of water. In this work a theoretical approach is described on the formulation of simplified rate equations, reflecting also the actual mass balances of the reactants, in the case where esterification synthetic reactions are catalyzed by immobilized lipases, in either a non-aqueous organic solvent or in a non-solvent system.

Rapid pharmacokinetic characteristic evaluation of scutellarin derivatives in vitro / 中国药理学通报

Aim To study the pharmacokinetic char-acteristics of serial compounds that took the scutellarin and scutellarein as lead compounds by using the model of in vitro liver microsomes, and to screen compounds whose medicinal properties were superior to scutellarin and scutellarein. Methods The content of candidate compounds at different times by incubation system of rat liver microsome was determined using UPLC-MS/MS method. Candidate compounds that contained opti-mum T1/2 and CLint were screened. Enzyme kinetics and conversions of candidate compounds were com-pared with those of scutellarin and scutellarein. Re-sults The T1/2 and CLint were optimum of W11 com-pared with those of scutellarin and scutellarein; the Vmax, Km and CLint of compound W11 were (10.25 ±2.59 ) μmol · min-1 · g-1 , ( 4.64 ±0.24 ) μmol · L-1 and ( 2.29 ±0.23 ) L · min-1 · g-1; the Vmax , Km and CLint of scutellarin were (45.95±9.50) μmol · min-1 · g-1 , ( 10.19 ± 1.66 ) μmol · L-1 and (4.48±0.20) L·min-1 ·g-1; W11 might be me-tabolized into scutellarin and M1 ( a compound with mo-lecular weight of 577 after demethylating ) . Conclu-sion The pharmacokinetic properties of candidate compound W11 are better than those of scutellarin, and it could release scutellarin.

Evaluation of in vitro Reduction of Hexavalent Chromium by Cell-Free Extract of Arthrobacter sp. SUK 1201.

Aims: This study aims at to evaluate the hexavalent chromium [Cr(VI)] reduction potential of crude cell-free extracts of chromium resistant and reducing bacterium Arthrobacter sp. SUK 1201 and determination of optimum conditions for Cr(VI) reduction for possible bioremediation of Cr pollutants. Place and Duration of Study: Chromium reduction studies with Arthrobacter sp. SUK 1201, was undertaken in the Microbiology Laboratory, Department of Botany, University of Calcutta, Kolkata during 2010-2012. Methodology: Cell-free extract was prepared from freshly grown cell mass of Arthrobacter sp. SUK 1201 following the standard procedure. Cell mass suspended in Tris-HCl was sonicated (120 KHz for 30 min), centrifuged (12,000×g at 4ºC for 10 min) and the supernatant (S12) was used as the cell- free extract (CFE). Chromate reductase activity of the CFE was assayed colorimetrically using 1, 5-diphenylcarbazide as the complexing reagent. Results: Chromate reductase activity of CFE of Arthrobacter sp. SUK 1201 was constitutive in nature and reduced Cr(VI) with decreasing efficiency as the concentration of Cr(VI) was increased. Its Km and Vmax were 263.45 M Cr(VI) and 17.5 U mg-1 protein respectively. Reduction of Cr(VI) was optimal at pH 7 and 32ºC but was extremely thermolabile. NADH was the most suitable electron donor, and the chromate reduction was enhanced by Cu(II) and Fe(III), but inhibited by Hg(II). Among the different inhibitors tested, 2, 4-dinitrophenol (DNP) restored nearly 96.4% reductase activity, while carbonyl cyanidem- chloro phenyl hydrazone (CCCP) was most inhibitory to the process. Conclusion: It has been established that the Cr(VI) reduction potential of the cell-free extract of Arthrobacter sp. SUK 1201 is promising and could be exploited in the bioremediation of toxic hexavalent chromium.

Molecular cloning, characterization and expression of lipoxygenase 2 (lox-2) isozyme from Indian soybean [Glycine max (L.) Merrill] cv. Pusa 16.

The consumption of soybean is limited worldwide, despite being highly nutritious and having versatile uses, due to the presence of grassy, beany and rancid off-flavour. The lipoxygenase-2 (LOX-2) is the key enzyme responsible for the production of volatiles released from the beans, which cause off-flavour in soy products. In this study, a 2.6-kb full-length lox2 gene (NCBI accession No. JQ929619.1) was isolated and cloned from soybean (Glycine max L. Merril) cv. Pusa 16. The cloned cDNA sequence of lox2 gene showed the complete open reading frame (ORF) of a putative protein, having 866 amino acids with start codon present at the foremost position and stop codon at the end. The theoretical pI of predicted protein was 6.22. A hydropathy profile calculated from the amino acid sequence resembled those of dicot LOXs, suggesting conservation of the secondary structure of these enzymes. The LOX-2 showed conserved six Histidine residues within a span of 520 to 590 amino acid position, a signature element for the enzyme activity. The lox2 gene was expressed using pET vector in prokaryotic expression system. The recombinant LOX-2 protein was purified after induction with IPTG (isopentyl thiogalactoside). A prominent band of 97 kDa was observed, when affinity purified fractions were analyzed by SDS-PAGE. The purified protein was characterized for the enzyme activity, substrate preference and Km. Inhibitor studies with natural antioxidant molecules present in soybean revealed α-tocopherol to be the most effective inhibitor of LOX-2.

Enzyme kinetics and molecular modeling studies of G6PDMahidol associated with acute hemolytic anemia.

G6PDMahidol enzyme is the most common variant in the Achang Chinese ethnic group and clinically manifests as class II. In this study, G6PDMahidol enzyme was characterized by molecular modeling to understand its kinetics. G6PDMahidol, G6PDG487A and G6PDWT proteins were heterologously expressed in the G6PD-deficient DF213 E. coli strain, purified and their steady-state kinetic parameters were determined. Compared with G6PDWT, the Km and Vmax of NADP+ with G6PDG487A were about 28-fold and 12-fold lower, respectively. The Ki values of dehydroepiandrosterone (DHEA), NADPH and ATP with G6PDG487A showed 29.5-fold, 2.36-fold reduction and 1.83-fold increase, respectively. A molecular modeling of G6PDG487A was performed based on the X-ray structure of human G6PD (PDB: 2BH9). It is suggested that Ser-163 might affect the stability of G6PDG487A -helix d and -strand E, besides the conformation of -strand D. In conclusion, the biochemical and structural properties of G6PDG487A and G6PDWT enzymes are significantly different, which may be responsible for clinical diversity of G6PD deficiencies.

The characteristics of enzyme kinetics of CTX-M-14 type extended-spectrum β-lactamase with Pro167 residue substitution / 中华微生物学和免疫学杂志

Objective To analyze and evaluate the characteristics of enzyme kinetics of CTX-M-14 type extended-spectrum β-lactamase(ESBL) with Pro167 residue substitution. Methods By molecular cloning and PCR techniques, CTX-M-14 gene was directionally cloned into plasmid pET28a( + ) from a clinical E. coli isolate and formed an expression vector to transform competent E. coli BL21 (DE3 ). Prol67 residue substitutions of P167G, P167Q, P167S and P167T were introduced to CTX-M-14 by site-directed muta-genesis based on overlap extension PCR with the former recombinant plasmid as PCR template, respectively.The wild-type CTX-M-14, recombinant CTX-M-14 protein and its variants were expressed and purified, then their steady-state kinetic parameters (Kcat, Km and Kcat/Km ) against β-lactam antibiotics were determined.Results The kinetic parameters of wild-type and recombinant CTX-M-14 had no statistically significant differences (P>0.1). The 1/Km, Kcat and Kcat/Km values of P167S variant against ceftazidime were 16-fold, 2.87-fold and 43.6-fold higher than those of recombinant CTX-M-14, respectively, and the Kcat/Km value of P167S variant against penicillin, ampicillin, cefazolin, cefuroxime, ceftriaxone, cefotaxime decreased( < 0.05). Compared with the kinetic parameters of recombinant CTX-M-14, the kinetic parameters of P167T variant against ceftazidime had no significant change, but the Kcat values of P167Q and P167G variants decreased dramatically(P<0.01). Conclusion There was no difference between the enzyme activities of wild-type and recombinant CTX-M-14. P167S variant could not only promote the enzyme affinity of CTX-M-14 to ceftazidime but also improve the conversion rate of enzyme-substrate complex in the ceftazidime hydrolysis. The comparison of the kinetic parameters of CTX-M-14 and its variants with Pro167 residue substitution showed that the increased activity of CTX-M ESBL variants against ceftazidime could not be simply explained with the enlarged cavity in active site that may be caused by the replacement of Pro167 residue by smaller amino acids.

Diffusional restrictions in glyoxyl-agarose immobilized penicillin G acylase of different particle size and protein loading / Restricciones para la difusión de penicilina G acilasa inmovilizada en glicosil agarosa

Particle size and enzyme protein loading are design parameters of enzyme immobilization affecting biocatalyst performance that can be varied within broad margins. Their effect on mass transfer limitations at different bulk penicillin G concentrations has been studied with glyoxyl agarose immobilized penicillin G acylase biocatalysts of average particle size of 5·10-5m and 10·10-4m at protein loadings from 15 to 130 mg/g gel. Internal diffusional restrictions were evaluated for such biocatalysts: Thiele modulus varied from 1.17 for the small particles at the lower protein load to 5.84 for the large particles at the higher protein load. Effectiveness factors at different bulk substrate concentrations were determined for all biocatalysts, values ranging from 0.78 for small particle size at 25 mM penicillin G to 0.15 for large particle size at 2 mM penicillin G. Enzyme protein loading had a strong impact on the effectiveness factors of immobilized penicillin G acylase, being it more pronounced in the case of large particle size biocatalysts. At conditions in which 6-aminopenicillanic acid is industrially produced, all biocatalysts tested were mass-transfer limited, being this information valuable for reactor design and performance evaluation.

Hyper-production of a thermotolerant β-xylosidase by a deoxy-D-glucose and cycloheximide resistant mutant derivative of Kluyveromyces marxianus PPY 125

Production of β-xylosidaseby a cycloheximide and 2-deoxy-D-glucose-resistant mutant of Kluyveromyces marxianus PPY125 was studied when cultured on growth media containing galactose, glucose, xylose, cellobiose, sucrose and lactose as carbon sources. Xylose, cellobiose, lactose and sucrose were the key substrates. Both K. marxianus PPY125 and its mutant (M 125) supported maximum β-xylosidase specific product yield (Y P/X) following growth on xylose. Basal level of activity was observed in non-induced cultures grown on glucose. The mutant produced 1.5 to 2-fold more β-xylosidase than that produced by the wild cells. Synthesis of β-xylosidase was regulated by an induction mechanism in both wild and mutant cells. Addition of glucose did not inhibit the synthesis of β-xylosidase in both parental and mutant cultures in the presence of corn steep liquor. Partially purified enzyme showed good stability when incubated at 60ΊC and was quite stable at pH 5.0-7.0. Thermodynamic studies revealed that the enzyme derived by the mutant M125 was more thermostable as evidenced by higher midpoint inactivation temperature, lower activation energy demand for β-xyloside hydrolysis, as well as lower enthalpy and entropy demand for reversible denaturation of enzyme.

Study on the Characteristics of Ceftazidime-resistant ?-Lactamase Produced by Escherichia Coli / 中国药房

OBJECTIVE:To study the characteristics of ceftazidime-resistant?-lactamase produced by Escherichia coli.METHODS:The types of2strains of drug fast?-lactamase produced by Escherichia coli were determined initially by K-B slip diffusion method and ampholine electrophoresis method;The plasmid was extracted by alkaline lysis and the PCR ampli?fication and sequencing were conducted;?-Lactamase was counter-extracted by saturated ammonium sulfate,filtrated by Sephadex G-75gel and purified by DE-52anion exchange chromatography;The molecular weight of which was determined by SDS-PAGE and the enzyme kinetics parameters of?-lactamas were determined by ultraviolet spectrophotometry.RE?SULTS:The2strains produced a super-broad spectrum?-lactamase(CTX-M-1V)with isoionic point at8.7and the molecular weight at29kDa,which can hydrolyze cefotaxim and aztreonam but imipenem and which was sensitive to sulbactam(IC 50 =94nmol/L)and tazobactam(IC 50 =5nmol/L).CONCLUSION:CTX-M-1V is a CTX-M type super-broad spectrum?-lactamase sensitive to suppressants.

Enzyme Kinetics of 6 Specific Substrates of Cytochrome P_(450) Enzymes / 中国药房

OBJECTIVE:To study the enzyme kinetics of six specific substrates of cytochrome P450 enzymes. METHODS: The transforming amount of the corresponding metabolites of six substrates was determined by LC-MS/MS. The incubation conditions of substrates in human liver microsomes were optimized and the parameters of enzyme kinetics were calculated based on substrate concentration-reaction velocity curve. RESULTS: The optimal incubation time was 20 min and the optimal enzyme concentration was 0.25 mg protein?mL-1. The Km(?mol?L-1) values of phenacetin, tolbutamide, omeprazole, dextromethorphan,chlorzoxazone and nifedipine were 21.6,163.4,10.4,4.6,41.9 and 9.4 respectively. The Vmax (nmol?min-1?mg protein-1) values were 2.08, 0.52, 1.02, 0.40, 2.36 and 1.00 respectively. CONCLUSION: The enzyme kinetics of six specific substrates of cytochrome P450 enzymes has been investigated in this study, and the study serves as a reference and support for the study and application of substrates in CYP.

Inhibition of glutathione-S-transferase by dihydromyricetin and its kinetics analysis in liver of mice / 中国药理学通报

Aim To study the inhibition of glutathione-S-transferase by dihydromyricetin and its kinetics analysis in liver of mice.Methods Mouse liver cytochyma enzyme was obtained by different velocity centrifugation,the mouse liver glutathione-S-transferase of michaelis constant(Km),maximum velocity(Vmax)and the inhibition of glutathione-S-transferase by dihydromyricetin of 50% inhibiting concentration(IC50),inhibition constant(Ki),the type of inhibition were calculated by Lineweaver-Burk and the low of semi-effect-probit.Results It was found that dihydromyricetin inhibited the glutathione-S-transferase activity with an IC50 of(121.14?13.66)?mol?L~-1.Kinetics analysis showed the Km was 0.1460 mmol?L~-1 and Vmax was 175.44 U?mg~-1 for reduced glutathione(GSH)substrate and 0.0937 mmol?L~-1(Km)and 212.77 U?mg~-1(Vmax)for 1-chloro-2,4,-dinitrobenzene(CDNB)substrate.Kinetics studies of dihydromyricetin on glutathione-S-transferase showed the inhibition was competitive with GSH and noncompetitive with CDNB,and the inhibition constant was 0.22 mmol?L~-1 with GSH and 0.54 mmol?L~1 with CDNB.Conclusion Dihydromyricetin can inhibit the glutathione-S-transferase activity in liver of mice.