Purification and Characterization of a Cephalosporin Esterase from Rhodosporidium toruloides (original) (raw)
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Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides
1999
Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co 2+ , Hg 2+ , Ag + , Mg 2+ and Ca 2+ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol −1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.
A process for bioconversion of cephalosporin C by Rhodotorula gracilis D-amino acid oxidase
Biotechnology Letters, 1995
A process for the production (in a stirred tank reactor) of glutaryl-7-ACA from cephalosporin C using immobilized D-amino acid oxidase is described. Results so obtained under optimal conditions (1.2 mg coupled enzyme/L, pH 8.5, 2 mM cephalosporin C) point to a system which shows high conversion efficiency and a remarkable operational stability. No exogenous H202 is requested to shift the reaction equilibrium toward glutaryl-7-ACA production, nor any side product is detected. The immobilized system productivity was 54 g/day/mg of enzyme. This process represents the first reported case of a reactor successfully developed with a DAAO for bioconversion of cephalosporin C.
Purification, Isolation, and Characterization of Esterase from Rhodococcus sp. LKE-021
Journal of Pure and Applied Microbiology, 2020
A thermophilic esterase isolated from Rhodococcus sp. LKE-021. This enzyme was purified with purification fold 60 from the crude extracts of enzyme and recovery of enzyme obtained approximately 21%. The specific activity of the lKe-021 esteraseis 795.1 U/mg. SDS-PAGE analysis determined the molecular weight of LKE-21 esteraseis around 32,000Da/32KDa. The enzyme activity of lKe-021 esterase exhibited over a wide range of temperature i.e. 30° to 80°C and the enzyme remained stable when incubated on 60° for 2h. This indicates that the isolated lKe-021 esterase is thermostable. The isolated enzyme exhibits activity on various pH ranges from 2.0 to 12.0 and the highest activity observed on 11.0 pH.The LKE-021 esterase was active after proteinase K treatment and shows over 75 % specific activity i.e. 50 U/µg Proteinase K.
Applied Microbiology and Biotechnology, 2001
An important problem during the production of cephalosporin C by Acremonium chrysogenum is the hydrolysis of cephalosporin C to deacetylcephalosporin C, since the latter compound has no commercial value and represents an unwanted side-product. Characterization of the enzymatic process that gives rise to deacetylcephalosporin C will help to avoid the accumulation of this side-product. An extracellular cephalosporin C acetylhydrolase (CPC-AH) from Acremonium chrysogenum C10 was purified to near homogeneity. This enzyme had a molecular mass of 31 kDa, a pI of 4.0, and showed relatively little affinity for cephalosporin C (K m 33.7 mM). We sequenced twenty amino acids at the amino-terminal end; a probe based on this sequence was then used to clone the cephalosporin acetylhydrolase (cahB) gene. cahB encodes a pre-protein of 383 amino acids with a deduced molecular mass of 38,228 Da. The sequenced 20 amino acids of the purified protein corresponded to amino acids 107-127 deduced from the cahB gene, suggesting that mature CPC-AH results from processing of the pre-protein after Gln-106. cahB is located on chromosome VIII of A. chrysogenum C10 and is not linked to the cephalosporin early or late gene clusters. It is expressed as a single 1.4-kb transcript after 72 h of cultivation. Expression declined in batch cultures after 120 h even though CPC-AH activity was observed until 144 h. The CPC-AH protein resembles other wide-spectrum substrate fungal esterases that are functionally related to serine proteases. The cahB gene does not seem to be related to the cephalosporin biosynthesis genes and encodes an esterase active on several substrates in addition to cephalosporin C. Materials and methods Strains and culture conditions Acremonium chrysogenum C10 (ATCC 48272), a high cephalosporin-C-producing strain provided by Panlabs (Demain 1983), was used in this study. To obtain spores, this strain was grown on
Biotechnology Progress, 2008
Lacking an efficient process to produce 7-aminocephalosporanic acid from cephalosporin C in a single step, D-amino acid oxidase (DAAO) is of foremost importance in the industrial, two-step process used for this purpose. We report a detailed study on the catalytic properties of the three available DAAOs, namely, a mammalian DAAO and two others from yeast (Rhodotorula gracilis and Trigonopsis variabilis). In comparing the kinetic parameters determined for the three DAAOs, with both cephalosporin C and D-alanine as substrate, the catalytic efficiency of the two enzymes from microorganism is at least 2 orders of magnitude higher than that of pig kidney DAAO. Furthermore, the mammalian enzyme is more sensitive to product inhibition (from hydrogen peroxide and glutaryl-7-aminocephalosporanic acid). Therefore, enzymes from microorganisms appear to be by far more suitable catalysts for bioconversion, although some different minor differences are present between them (e.g., a higher activity of the R. gracilis enzyme when the bioconversion is carried out at saturating oxygen concentration). The mammalian DAAO, even being a poor catalyst, is more stable with respect to temperature than the R. gracilis enzyme in the free form. In any case, for industrial purposes DAAO is used only in the immobilized form where a strong enzyme stabilization occurs.
Artificial Cells, Blood Substitutes, and Biotechnology, 2010
7-aminocephalosporanic acid (7-ACA) is the key intermediate for the synthesis of semisynthetic cephalosporin antibiotics and enzyme cephalosporin-C acylase (CPC acylase) plays an important role in the conversion of cephalosporin-C to 7-ACA. With an aim to increase the yield of 7-ACA production by Micrococcus luteus , a stepwise strategy, statistical medium was applied for optimizing the medium composition for the production of CPC acylase. Purifi ed enzyme was found to be of 80 kDa. The optimum pH and temperature for the production of 7-ACA were 7.6 and 340C, respectively. The Km and Vmax were estimated to be 9.43 mg/mL and 7.65 U/mL, respectively.
Journal of Fermentation and Bioengineering, 1994
We synthesized 7~-(6-bromohexanoylamido)cephalosporanic acid (6-BH-TACA), a substrate analogue of an acylase from Pseudomonas N176 (N|76 acylase) to determine the substrate binding site of the acylase. The enzyme was inactivated by incubation with 6-BH-7ACA in a time-dependent manner. A double reciprocal plot of the pseudo-first-order rate constant (kobs) against the 6-BH-7ACA concentration gave a straight line (kmx=0.113 min -1, KI=0.51 mM). A plot of log kobs against log [6-BH-TACA] was linear with a slope of 0.87. Inactivation of the enzyme with 6-BH-7ACA was inhibited by addition of 7-aminocephalosporanic acid and glutaric acid. These data indicate that 6-BH-7ACA functions as an affinity label reagent and the inactivation by 6-BH-7ACA is due to the modification of a single residue located in the neighborhood of the substrate binding region of the acylase. The digest of the inactivated enzyme with lysylendopeptidase was fractionated by reversed phase high-performance liquid chromatography (HPLC). One fragment was eluted with a different retention time from the corresponding fragment of the intact enzyme. From additional achymotryptic digestion followed by amino acid sequence analysis, Tyr 27° was determined as the site labelled by 6-BH-7ACA. Replacing Tyr 27° with a Phe residue by site-directed mutagenesis caused a decrease in the enzyme capability (kcat/Km). While the Km of the mutant acylase increased slightly, the kcat decreased to about 50~ of that of the wild-type. These results indicate that although labelled Tyr 27° is not essential, it does play an important role in the enzymatic activity.
Applied Microbiology and Biotechnology, 2007
The acylase from Arthrobacter viscosus was immobilized, studied in the enzymatic synthesis of some cephalosporins by kinetically controlled N-acylation (kcNa) of different cephem nuclei, and compared with the penicillin G acylase (PGA) from Escherichia coli. The reaction outcomes were dependent on the acylase microbial source and on the type of immobilization support. Generally, both enzymes, when immobilized onto hydrophilic resins such as glyoxyl-agarose (activated with aldehyde groups), displayed higher synthetic performances in comparison with hydrophobic acrylic epoxy-supports like Eupergit C. The kcNa of 7-amino cephalosporanic acid catalyzed by A. viscosus immobilized on glyoxyl-agarose afforded a quantitative conversion in 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ3-cephem-4-carboxylic acid, a useful intermediate for the synthesis of Cefamandole and Cefonicid. Similar results were obtained in the synthesis of these cephalosporins by direct acylation of the corresponding 3′-functionalized nucleus. In these reactions, A. viscosus displayed higher synthetic performances than the PGA from E. coli.
Advanced Synthesis & Catalysis, 2008
2-Oxoadipoyl-7-ACA is an intermediate in the conversion of cephalosporin C (CPC) to 7aminocephalosporanic acid (7-ACA) when using a new route involving d-amino acid oxidase, catalase and glutaryl acylase. A key point in the reaction design is to avoid the accumulation of hydrogen peroxide in the reaction medium as the yields of 7-ACA decrease in the presence of this compound due to its low stability. Looking for an enzyme with improved activity towards 2-oxoadipoyl-7-ACA, different mutants of glutaryl acylase from Pseudomonas SY-77 with an improved activity towards adipoyl-7-ACA were evaluated. The best results on 2-oxoadipoyl-7-ACA hydrolysis were found with the double mutant Y178F + F375H, which showed a K cat increase of 6.5-fold and a K m decrease of 3-fold compared to the wild-type (wt) enzyme. When this enzyme was tested in the tri-enzymatic system to convert CPC into 7-ACA, this mutant permitted us to reach more than an 80 % yield of 7-ACA using a 3-fold mass excess compared to DAAO; while the wt enzyme gave only a 40 % yield. Therefore, the application of this new mutant to the one-pot conversion of CPC to 7-ACA gives very good result in terms of efficiency, yield and rate of the process.