Substrate Report for: 7-Aminocephalosporanic-acid

OBJECTIVES: To obtain a new acetyl esterase from Paenibacillus sp. XW-6-66 and apply the enzyme to 7-aminocephalosporanic acid (7-ACA) deacetylation. RESULTS: The acetyl esterase AesZY was identified from Paenibacillus sp. XW-6-66, and its enzymatic properties were investigated. With the putative catalytic triad Ser114-Asp203-His235, AesZY belongs to the Acetyl esterase (Aes) family which is included in the alpha/beta hydrolase superfamily and contains the consensus Gly-X-Ser-X-Gly motif. The maximum activity of AesZY was detected at pH 8.0 and 40 degrees C. AesZY was stable at different pH values ranging from 5.0 to 12.0, and was tolerant to several metal ions. Furthermore, the deacetylation activity of AesZY toward 7-ACA was approximately 7.5 U/mg, and the Kcat/Km value was 2.04 s(-1) mM(-1). CONCLUSIONS: Our results demonstrate the characterization of a new acetyl esterase belonging to the Aes family with potential biotechnological applications.

The carbohydrate esterase family 7 (CE7) belonging to the alpha/beta hydrolase superfamily contains a structurally conserved loop extension element relative to the canonical alpha/beta hydrolase fold. This element called the beta-interface loop contributes 20-30% of the total buried surface area at intersubunit interfaces of the functional hexameric state. To test whether this loop is an enabling region for the structure and function of the oligomeric assembly, we designed a truncation variant of the thermostable CE7 acetyl esterase from Thermotoga maritima (TmAcE). Although deletion of 26 out of 40 residues in the loop had little impact on the hexamer formation, the variant exhibited altered dynamics of the oligomeric assembly and a loss of thermal stability. Furthermore, the mutant lacked catalytic activity. Crystal structures of the variant and a new crystal form of the wild type protein determined at 2.75A and 1.76A, respectively, provide a rationale for the properties of the variant. The hexameric assembly in the variant is identical to that of the wild type and differed only in the lack of buried surface area interactions at the original intersubunit interfaces. This is accompanied by disorder in an extended region of the truncated loop that consequently induces disorder in the neighboring oxyanion hole loop. Overall, the results suggest that the beta-interface loop in CE7 enzymes is dispensable for the oligomeric assembly. Rather, the loop extension event was evolutionarily selected to regulate activity, conformational flexibility and thermal stability.

A cephalosporin deacetylating acetyl xylan esterase was cloned from the genomic DNA of Bacillus subtilis CICC 20034 and functionally expressed in Escherichia coli. Its gene contained an open reading frame of 957 bp encoding 318 amino acids with a calculated mass of 35,607 Da, and it displayed significant identity to acetyl xylan esterases from Bacillus sp. 916, B. subtilis 168, and Bacillus pumilus Cect5072. The enzyme was a native homohexamer but a trimer under the condition of 1% sodium dodecyl sulfate (SDS); both forms were active and could transit to each other by incubating in or removing SDS. The enzyme belongs to carbohydrate esterase family 7 and had a double specificity on both the acetylated oligosaccharide and cephalosporin C (CPC) and 7-aminocephalosporanic acid (7-ACA). The activity of this purified enzyme toward CPC and 7-ACA was highest among all the acetyl xylan esterase from CE family 7, which were 484 and 888 U/mg, respectively, and endowed itself with great industrial interest on semi-synthetic beta-lactam antibiotics. The optimum pH of the purified enzyme was 8.0, and the optimum temperature was 50 degrees C, and the enzyme had high thermal stability, broad range of pH tolerance, and extremely organic solvent tolerance.

Deacetyl-7-aminocephalosporanic acid (D-7-ACA) is required for producing of many semisynthetic beta-lactam antibiotics; therefore, enzymes capable of converting 7-aminocephalosporanic acid (7-ACA) to D-7-ACA present a valuable resource to the pharmaceutical industry. In the present study, a putative acetylesterase, EstZY, was identified and characterized from a thermophilic bacterium Alicyclobacillus tengchongensis. Sequence alignment showed that EstZY was an acetylesterase which belonged to carbohydrate esterase family 7 (CE7), with substrate preference for short-chain acyl esters p-NPC(2) to p-NPC(8). Maximum enzyme activity was recorded at pH 9.0 and 50 degreesC, where K(m) and V(max) were calculated as 1.9 +/- 0.23 mM and 258 +/- 18.5 microM min(-)(1), respectively. The residues Ser185, Asp274, and His303 were identified as the putative catalytic triad by homology modelling, site-directed mutagenesis and molecular docking. Moreover, EstZY can remove the acetyl group at C3' position of 7-ACA to form D-7-ACA; this is the first report of a 7-ACA deacetylase from CE7 family in A. tengchongensis and may represent a new enzyme with industrial values.

OBJECTIVES: To obtain a new acetyl esterase from Paenibacillus sp. XW-6-66 and apply the enzyme to 7-aminocephalosporanic acid (7-ACA) deacetylation. RESULTS: The acetyl esterase AesZY was identified from Paenibacillus sp. XW-6-66, and its enzymatic properties were investigated. With the putative catalytic triad Ser114-Asp203-His235, AesZY belongs to the Acetyl esterase (Aes) family which is included in the alpha/beta hydrolase superfamily and contains the consensus Gly-X-Ser-X-Gly motif. The maximum activity of AesZY was detected at pH 8.0 and 40 degrees C. AesZY was stable at different pH values ranging from 5.0 to 12.0, and was tolerant to several metal ions. Furthermore, the deacetylation activity of AesZY toward 7-ACA was approximately 7.5 U/mg, and the Kcat/Km value was 2.04 s(-1) mM(-1). CONCLUSIONS: Our results demonstrate the characterization of a new acetyl esterase belonging to the Aes family with potential biotechnological applications.

Cold-active acetyl xylan esterases allow for reduced bioreactor heating costs in bioenergy production. Here, we isolated and characterized a cold-active acetyl xylan esterase (PbAcE) from the psychrophilic soil microbe Paenibacillus sp. R4. The enzyme hydrolyzes glucose penta-acetate and xylan acetate, reversibly producing acetyl xylan from xylan, and it shows higher activity at 4 degrees C than at 25 degrees C. We solved the crystal structure of PbAcE at 2.1-A resolution to investigate its active site and the reason for its low-temperature activity. Structural analysis showed that PbAcE forms a hexamer with a central substrate binding tunnel, and the inter-subunit interactions are relatively weak compared with those of its mesophilic and thermophilic homologs. PbAcE also has a shorter loop and different residue composition in the beta4-alpha3 and beta5-alpha4 regions near the substrate binding site. Flexible subunit movements and different active site loop conformations may enable the strong low-temperature activity and broad substrate specificity of PbAcE. In addition, PbAcE was found to have strong activity against antibiotic compound substrates, such as cefotaxime and 7-amino cephalosporanic acid (7-ACA). In conclusion, the PbAcE structure and our biochemical results provide the first example of a cold-active acetyl xylan esterase and a starting template for structure-based protein engineering.

The carbohydrate esterase family 7 (CE7) enzymes catalyze the deacetylation of acetyl esters of a broad range of alcohols and is unique in its activity towards cephalosporin C. The CE7 fold contains a conserved N-terminal extension that distinguishes it from the canonical alpha/beta hydrolase fold. The hexameric quaternary structure indicates that the N-terminus may affect activity and specificity by controlling access of substrates to the buried active sites via an entrance tunnel. In this context, we characterized the catalytic parameters, conformation and thermal stability of two truncation variants lacking four and ten residues of the N-terminal region of the hyperthermostable Thermotoga maritima CE7 acetyl esterase (TmAcE). The truncations did not affect the secondary structure or the fold but modulated the oligomerization dynamics. A modest increase was observed in substrate specificity for acetylated xylose compared with acetylated glucose. A drastic reduction of ~30-40 degrees C in the optimum temperature for activity of the variants indicated lower thermal stability. The loss of hyperthermostability appears to be an indirect effect associated with an increase in the conformational flexibility of an otherwise rigid neighboring loop containing a catalytic triad residue. The results suggest that the N-terminal extension was evolutionarily selected to preserve the stability of the enzyme.

The carbohydrate esterase family 7 (CE7) belonging to the alpha/beta hydrolase superfamily contains a structurally conserved loop extension element relative to the canonical alpha/beta hydrolase fold. This element called the beta-interface loop contributes 20-30% of the total buried surface area at intersubunit interfaces of the functional hexameric state. To test whether this loop is an enabling region for the structure and function of the oligomeric assembly, we designed a truncation variant of the thermostable CE7 acetyl esterase from Thermotoga maritima (TmAcE). Although deletion of 26 out of 40 residues in the loop had little impact on the hexamer formation, the variant exhibited altered dynamics of the oligomeric assembly and a loss of thermal stability. Furthermore, the mutant lacked catalytic activity. Crystal structures of the variant and a new crystal form of the wild type protein determined at 2.75A and 1.76A, respectively, provide a rationale for the properties of the variant. The hexameric assembly in the variant is identical to that of the wild type and differed only in the lack of buried surface area interactions at the original intersubunit interfaces. This is accompanied by disorder in an extended region of the truncated loop that consequently induces disorder in the neighboring oxyanion hole loop. Overall, the results suggest that the beta-interface loop in CE7 enzymes is dispensable for the oligomeric assembly. Rather, the loop extension event was evolutionarily selected to regulate activity, conformational flexibility and thermal stability.

A cephalosporin deacetylating acetyl xylan esterase was cloned from the genomic DNA of Bacillus subtilis CICC 20034 and functionally expressed in Escherichia coli. Its gene contained an open reading frame of 957 bp encoding 318 amino acids with a calculated mass of 35,607 Da, and it displayed significant identity to acetyl xylan esterases from Bacillus sp. 916, B. subtilis 168, and Bacillus pumilus Cect5072. The enzyme was a native homohexamer but a trimer under the condition of 1% sodium dodecyl sulfate (SDS); both forms were active and could transit to each other by incubating in or removing SDS. The enzyme belongs to carbohydrate esterase family 7 and had a double specificity on both the acetylated oligosaccharide and cephalosporin C (CPC) and 7-aminocephalosporanic acid (7-ACA). The activity of this purified enzyme toward CPC and 7-ACA was highest among all the acetyl xylan esterase from CE family 7, which were 484 and 888 U/mg, respectively, and endowed itself with great industrial interest on semi-synthetic beta-lactam antibiotics. The optimum pH of the purified enzyme was 8.0, and the optimum temperature was 50 degrees C, and the enzyme had high thermal stability, broad range of pH tolerance, and extremely organic solvent tolerance.

A bromothymol blue-based colorimetric assay has been devised to screen for acetyl xylan esterase or cephalosporin C (CPC) deacetylase activities using 7-amino cephalosporanic acid (7-ACA), CPC, or acetylated xylan as substrate. These enzymes are not screened with their natural substrates because of the tedious procedures available previously. Acetyl xylan esterase from Bacillus pumilus CECT 5072 was cloned, expressed in Escherichia coli Rosetta (DE3), and characterized using this assay. Similar K(M) values for 7-ACA and CPC were obtained when compared with those described using HPLC methods. The assay is easy to perform and can be carried out in robotic high-throughput colorimetric devices normally used in directed evolution experiments. The assay allowed us to detect improvements in activity at a minimum of twofold with a very low coefficient of variance in 96-well plates. This method is significantly faster and more convenient to use than are known HPLC and pH-stat procedures.

Bacillus subtilis SHS0133 cephalosporin-C deacetylase (CAH) overexpressed in Escherichia coli was immobilized on an anion-exchange resin, KA-890, using glutaraldehyde. The activity yield of immobilized enzyme was approximately 55% of the free enzyme. The pH range for stability of the immobilized enzyme (pH 5-10) was broader than that for free enzyme. The K(m)(app) value of immobilized enzyme for 7-aminocephalosporanic acid (7-ACA) was similar to that of the free enzyme. This immobilized enzyme obeyed Michaelis-Menten kinetics similar to those of the free enzyme. A batch-type reactor with a water jacket was employed for deacetylation of 7-ACA using CAH immobilized on KA-890. Ten kilograms of 7-ACA were completely converted to deacetyl 7-ACA at pH 8.0 within 90 min. The reaction kinetics agreed well with a computer simulation model. Moreover, the immobilized enzyme exhibited only a slight loss of the initial activity even after repeated use (52 times ) over a period of 70 days. This reaction will thus be useful for the production of cephalosporin-type antibiotics.

The genes encoding acetyl xylan esterase 1 (axe1) and a beta-xylosidase (xylB) have been cloned and sequenced from Thermoanaerobacterium sp. strain JW/SL YS485. axe1 is located 22 nucleotides 3' of the xylB sequence. The identity of axe1 was confirmed by comparison of the deduced amino acid sequence to peptide sequence analysis data from purified acetyl xylan esterase 1. The xylB gene was identified by expression cloning and by sequence homology to known beta-xylosidases. Plasmids which independently expressed either acetyl xylan esterase 1 (pAct1BK) or beta-xylosidase (pXylo-1.1) were constructed in Escherichia coli. Plasmid pXylAct-1 contained both genes joined at a unique EcoRI site and expressed both activities. Substrate specificity, pH, and temperature optima were determined for partially purified recombinant acetyl xylan esterase 1 and for crude recombinant beta-xylosidase. Similarity searches showed that the axe1 and xylB genes were homologs of the ORF-1 and xynB genes, respectively, isolated from Thermoanaerobacterium saccharolyticum. Although the deduced sequence of the axe1 product had no significant amino acid sequence similarity to any reported acetyl xylan esterase sequence, it did have strong similarity to cephalosporin C deacetylase from Bacillus subtilis. Recombinant acetyl xylan esterase 1 was found to have thermostable deacetylase activity towards a number of acetylated substrates, including cephalosporin C and 7-aminocephalosporanic acid.

An esterase that deacetylates cephalosporins was recovered from the supernatant of a Bacillus subtilis culture. It was partially purified by ammonium sulfate fractionation and ultrafiltration. The enzyme had a temperature optimum between 40 and 50 C and a pH optimum of 7.0. The molecular weight was estimated by gel filtration to be 190,000. The enzyme was very stable and retained greater than 80% of its activity after storage in solution at 25 C for 1 month. The esterase exhibited Michaelis-Menton kinetics with the substrates 7-aminocephalosporanic acid (7-ACA) and 7-(thiophene-2-acetamido)cephalosporanic acid (cephalothin); the K(m) values were 2.8 X 10(-3) and 8.3 X 10(-3) M, respectively. The products of 7-ACA deacetylation were weak competitive inhibitors, and a K(i) value of 5.0 X 10(-2) M was determined for acetate and of 3.6 X 10-2 M for deacetyl-7-ACA. Weak product inhibition did not prevent the deacetylation reaction from going to completion. A 5-mg/ml solution of partially purified esterase completely hydrolyzed (greater than 99.5%) a 24-mg/ml solution of 7-ACA in 3 h. Because of the kinetic properties and excellent stability, this enzyme may be useful in an immobilized form to prepare large quantities of deacetylated cephalosporin derivatives.