Biochemical characterization of a glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas strain BL072.

Pseudomonas strain BL072 produces an acylase enzyme active in hydrolyzing glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid. This acylase was purified by column chromatography and gel electrophoresis. The native acylase was composed of two subunits of approximately 65 and 24 kDa, though some heterogeneity was seen in both the native acylase and its small subunit. The isoelectric point of the acylase is approximately 8.5, and it has Km of 1.6 mM for glutaryl desacetoxy aminocephalosporanic acid. The acylase hydrolyzes the desacetoxy and desacetyl derivatives of glutaryl-7-aminocephalosporanic acid at rates similar to that of glutaryl-7-aminocephalosporanic acid. Cephalosporin C was hydrolyzed at a reduced rate. The pH optimum was found to be 8.0, and an activation energy of 9 kcal/mol (ca. 38 kJ/mol) was observed. The acylase has transacylase activity 10 times that of its hydrolytic activity. Eupergit C-immobilized acylase had a half-life of greater than 400 h.     

Enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid. Part I: Cultivation of Pseudomonas syringae and partial purification and immobilization of 7-β-(4-carboxybutanamido) cephalosporanic acid acylase

The enzymatic transformation of 7-β-(4-carboxybutanamido)cephalosporanic acid (Gl-7-ACA) to 7-amino-cephalosporanic acid (7-ACA) is reported. The optimum conditions for cultivation of the producer strain Pseudomonas syringae, as well as the procedures for isolation, purification, and immobilization of the enzyme Gl-7-ACA acylase, are described. It is shown that when glutaraldehyde is used for immobilization of this enzyme, the yield of immobilization is low. After six hydrolyses of Gl-7-ACA to 7-ACA, the immobilized enzyme activity loss is less than 10%.     

Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus.

Penicillin G acylase was purified from the cultured filtrate of Arthrobacter viscosus 8895GU and was found to consist of two distinct subunits with apparent molecular weights of 24,000 (alpha) and 60,000 (beta). The partial N-terminal amino acid sequences of the alpha and beta subunits were determined with a protein gas phase sequencer, and a 29-base oligonucleotide corresponding to the partial amino acid sequence of the alpha subunit was synthesized. An Escherichia coli transformant having the penicillin G acylase gene was isolated from an A. viscosus gene library by hybridization with the 29-base probe. The resulting positive clone was further screened by the Serratia marcescens overlay technique. E. coli carrying a plasmid designated pHYM-1 was found to produce penicillin G acylase in the cells. This plasmid had an 8.0-kilobase pair DNA fragment inserted in the EcoRI site of pACYC184.     

Cloning and high expression of glutaryl 7-aminocephalosporanic acid acylase gene from Pseudomonas diminuta

The gene coding for the glutaryl 7-aminocephalosporanic acid (GL 7-ACA) acylase from Pseudomonas diminuta KAC-1 was cloned and expressed in Escherichia coli. The acylase gene was composed of 2160 base pairs and encoded a polypeptide of 720 amino acid residues. The E. coli BL21 carrying pET2, the plasmid construct for high expression of GL 7-ACA acylase gene, produced this enzyme at approx. 30% of the total proteins with 3.2 units activity mg protein^–1. Growth at temperature below 31 °C and deletion of signal peptide increased the processing of precursor acylase to active enzyme in the recombinant E. coli cells.     

Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus

Penicillin G acylase was purified from the cultured filtrate of Arthrobacter viscosus 8895GU and was found to consist of two distinct subunits with apparent molecular weights of 24,000 (alpha) and 60,000 (beta). The partial N-terminal amino acid sequences of the alpha and beta subunits were determined with a protein gas phase sequencer, and a 29-base oligonucleotide corresponding to the partial amino acid sequence of the alpha subunit was synthesized. An Escherichia coli transformant having the penicillin G acylase gene was isolated from an A. viscosus gene library by hybridization with the 29-base probe. The resulting positive clone was further screened by the Serratia marcescens overlay technique. E. coli carrying a plasmid designated pHYM-1 was found to produce penicillin G acylase in the cells. This plasmid had an 8.0-kilobase pair DNA fragment inserted in the EcoRI site of pACYC184.     

Screening and characterization of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp.

Summary Three screening methods were used to isolate GL-7-ACA acylase-producing strains. Three positive isolates were identified with Pseudomonas nitroreducens CCRC 11041 possessing the highest activity, against GL-7-ACA and GL-7-ADCA. No activity was detected when Ceph C or succinyl-7-ACA was used as substrate; glutaric acid was found to be inhibitory. CCRC 11041 could produce maximal GL-7-ACA acylase activity when cultivated on meat extract medium II. The enzyme had a pH optimum of 5.0 and a temperature optimum of 42°C.     

Molecular cloning of bile acid 7-dehydroxylase from Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is a human intestinal bacterium which contains an inducible bile acid 7-dehydroxylase. Two-dimensional polyacrylamide gel electrophoresis showed that at least four new polypeptides were synthesized after exposure of growing cells to sodium cholate. One of these, of molecular weight 27,000 (PP-27), was implicated in 7-dehydroxylase catalysis. PP-27 was purified to greater than 95% homogeneity by DEAE-cellulose chromatography, high-pressure liquid chromatographic gel filtration, high-pressure liquid chromatography-DEAE chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 33 amino acid residues of the N terminus of PP-27 were determined with a gas-phase sequencer, and a corresponding mixed oligonucleotide (17-mer) was synthesized. Southern blot analysis of EcoRI total digests of chromosomal DNA showed a 2.2-kilobase fragment which hybridized to the 32P-labeled 17-mer. This fragment was enriched for by size fractionation of an EcoRI total digest of genomic DNA, ligated into the bacterial plasmid pUC8, and used to transform Escherichia coli HB101. Transformants containing the putative 7-dehydroxylase gene were detected with the 32P-labeled 17-mer by colony hybridization techniques. The insert was 2.2 kilobases in length and contained the first 290 bases of the PP-27 gene. Preliminary nucleic acid sequence data correlate with the amino acid sequence. The entire gene was cloned on a 1,150-base-pair TaqI fragment. Western blot analysis of E. coli strains containing these plasmids indicated that PP-27 is expressed in E. coli but is not regulated by bile acids under the conditions used.     

Molecular cloning of a phytase gene (phy M) from Pseudomonas syringae MOK1

A phytase gene (phy M) was cloned from Pseudomonas syringae MOK1 by two steps of degenerate PCR and inverse PCR. This gene consists of 1,287 nucleotides and encodes a polypeptide of 428 amino acids with a deduced molecular mass of 46,652 kDa. Based on its amino acid sequence, the Phy M shares the active site RHGXRXP and HD sequence motifs, typically characterized by histidine acid phosphatases familly. Each phy M gene fragment encoding mature Phy M with its own signal sequence (pEPSS) and without (pEPSM) was subcloned into the E. coli BL21 (DE3) expression vector, pET22b (+). The enzyme activity in crude extracts of clone pEPSM was 2.514 Umg⁻¹ of protein, and about 10-fold higher than that of clone pEPSS.*These two authors have contributed equally to this work.     

Molecular cloning and sequencing of a pectinesterase gene from Pseudomonas solanacearum

Two pectinesterase-positive Escherichia coli clones, differing in expression levels, were isolated from a genomic library of Pseudomonas solanacearum. Both clones contained a common DNA fragment which included the pectinesterase-encoding region. The different expression levels found with the two clones could be ascribed to different positioning of the pectinesterase gene with respect to a vector promoter. Restriction analysis, subcloning, and further exonuclease deletion mapping revealed that the genetic information for pectinesterase was located within a 1.3 kb fragment. A protein of 41 to 42 kDa was expressed from this fragment. Nucleotide sequence analysis of the respective region disclosed an open reading frame of 1188 bp. The deduced polypeptide had a calculated molecular mass of 41,004 Da, which is consistent with the determined size of the pectinesterase protein. The predicted amino acid sequence showed significant homology to pectinesterases from Erwinia chrysanthemi and tomato. In cultures of E. coli clones up to 30% of total pectinesterase activity was transported into the medium. However, no significant pectinesterase activity could be detected in the periplasm.     

Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph

A genomic library containing HindIII partial digests of Pseudomonas sp. strain AM1 DNA was constructed in the broad-host-range cosmid pVK100. PCT57, a Pseudomonas sp. strain AM1 methanol mutant deficient in malyl coenzyme A lyase activity, was complemented to a methanol-positive phenotype by mobilization of the pVK100 library into PCT57 recipients with the ColE1/RK2 mobilizing plasmid pRK2013. Six different complemented isolates all contained a recombinant plasmid carrying the same 19.6-kilobase-pair Pseudomonas sp. strain AM1 DNA insert. Subcloning and complementation analysis demonstrated that the gene deficient in PCT57 (mcl-1) was located in a 1.6-kilobase-pair region within a 7.4-kilobase-pair EcoRI-HindIII fragment.     

Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis.

Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.     

Molecular cloning and expression of the beta-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni.

The structural gene (hsd) of the Pseudomonas testosteroni encoding the 17 beta-hydroxysteroid dehydrogenase has been cloned using the cosmid vector pVK102. Escherichia coli carrying recombinant clones of hsd, isolated by immunological screening, were able to express the biologically active enzyme, as measured by the conversion of testosterone into androstenedione. Subcloning experiments, restriction and deletion analysis, and site-directed insertion mutagenesis showed that the hsd gene is located within a 1.3-kb HindIII-PstI restriction fragment. A 26.5-kDa protein encoded by a recombinant plasmid containing this Ps. testosteroni DNA restriction fragment was detected by SDS-PAGE analysis of in vitro [35S]methionine-labeled polypeptides.     

Oxidative modification of a cephalosporin C acylase from Pseudomonas strain N176 and site-directed mutagenesis of the gene.

A cephalosporanic acid acylase from Pseudomonas strain N176 catalyzes hydrolysis of both glutarylcephalosporanic acid and cephalosporin C to 7-amino-cephalosporanic acid. Chemical modification of the enzyme with acidic hydrogen peroxide was performed to investigate residues which play important roles in enzymatic activity. The activity of the enzyme was reduced to 76% of the original by oxidation. From protein chemical analysis combined with site-directed point mutagenesis, modification of Met-164 was found to correspond to the reduction in activity. To study the effect of Met-164 on the enzymatic character, we prepared mutant acylases in which Met-164 was replaced with several other amino acids and obtained the following data: (i) there existed a trend of mutation to noncharged hydrophilic residues, resulting in an increase of activity against glutarylcephalosporanic acid; (ii) the mutation of Met-164 to Gly and Ala resulted in the lowering of both Km values and the optimal pHs against glutarylcephalosporanic acid; (iii) the mutation to Leu enhanced cephalosporin C acylase activity; and (iv) the mutation to Gln improved the k(cat) value for glutarylcephalosporanic acid. In particular, the mutation to Gln resulted in a high rate of conversion of glutarylcephalosporanic acid to 7-amino-cephalosporanic acid under conditions similar to those of a bioreactor system. These results may indicate that Met-164 is located in or near the cephalosporin compound binding pocket on the enzyme.     

Molecular cloning and biological characterization of the recA gene from Pseudomonas syringae.

We have identified a recombinant plasmid, pCUV8, from a cosmid library of Pseudomonas syringae genomic DNA which contains a functional analog of the Escherichia coli recA gene. The plasmid was initially identified by its ability to restore UV resistance to E. coli HB101. Quantitative analysis demonstrated that it restored both recombination proficiency and UV resistance to an E. coli recA deletion mutant. By these criteria, pCUV8 appears to contain the P. syringae recA gene. Several pathogenic and epiphytic strains of P. syringae, but not E. coli, showed sequence homology to pCUV8 under normal stringency.     

Nucleotide sequence and expression in Escherichia coli of the cephalosporin acylase gene of a Pseudomonas strain.

The gene encoding cephalosporin acylase, which hydrolyzes 7-beta-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) to 7-aminocephalosporanic acid (7ACA) and glutaric acid, was cloned from a Pseudomonas sp. strain V22 and expressed in Escherichia coli, in a two-cistron system, and the enzyme was purified and characterized. The purified enzyme was composed of two non-identical subunits, their molecular weights were estimated by SDS-PAGE to be 40,000 and 22,000, and had a pI of 4.6. The amino acid sequence of the enzyme, deduced from the nucleotide sequence, showed high similarity (97%) with that of a previously reported acyI-encoded cephalosporin acylase. Cephalosporin acylase also resembles the bacterial gamma-glutamyl transpeptidases (GGTs) with respect to their molecular organization and amino acid sequence, but differs from them with respect to catalytic and immunological properties. Purified enzyme exhibited not only cephalosporin acylase activity, but also GGT activity. The Km values of the enzyme for GL-7ACA and L-gamma-glutamyl-p-nitroanilide were 6.1 and 3.8 mM, respectively. Cephalosporin acylase was not recognized by antibodies prepared against bacterial GGTs.     

Tryptophan 7-halogenase from Pseudomonas aureofaciens ACN strain: gene cloning and sequencing and the enzyme expression

The gene of tryptophan 7-halogenase was isolated from the Pseudomonas aureofaciens ACN strain producing pyrrolnitrin, a chlorocontaining antibiotic, and sequenced. A high homology degree (over 95%) was established for the genes and the corresponding halogenases from P. aureofaciens ACN and P. fluorescens BL915. The tryptophan 7-halogenase gene was amplified by PCR, and the corresponding enzyme was expressed in Escherichia coli cells using the pBSII SK+ vector.     

Involvement of arginine and tryptophan residues in catalytic activity of glutaryl 7-aminocephalosporanic acid acylase from Pseudomonas sp. strain GK16

The glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase from Pseudomonas sp. strain GK16 is an (alphabeta)2 heterotetramer of two non-identical subunits that are cleaved autoproteolytically from an enzymatically inactive precursor polypeptide. The newly formed N-terminal serine of the beta subunit plays an essential role as a nucleophile in enzyme activity. Chemical modification studies on the recombinant enzyme purified from Escherichia coli revealed the involvement of a single arginine and tryptophan residue, per alphabeta heterodimer of the enzyme, in the catalytic activity of the enzyme. Glutaric acid, 7-aminocephalosporanic acid (7-ACA) (competitive inhibitors) and GL-7-ACA (substrate) could not protect the enzyme against phenylglyoxal-mediated inactivation, whereas except for glutaric acid protection was observed in case of N-bromosuccinimide-mediated inactivation of the enzyme. Kinetic parameters of partially inactivated enzyme samples suggested that while arginine is involved in catalysis, tryptophan is involved in substrate binding.     

Molecular cloning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain B13.

The genes specifying the utilization of 3-chlorobenzoate by Pseudomonas sp. strain B13 WR1 have been cloned by using a broad-host-range cosmid cloning system. Analysis of the catabolic products of the enzymatic reactions encoded by two hybrid cosmids, pMW65 and pMW90, by thin-layer and high-performance liquid chromatography demonstrated that both encoded the genes for the complete catabolism of 3-chlorobenzoate. Physical analysis of one of the cosmid derivatives, pMW65, by restriction endonuclease mapping and subcloning demonstrated that the pathway genes are encoded on a fragment no larger than 11 kilobases.     

Cyclohexadienyl dehydratase from Pseudomonas aeruginosa. Molecular cloning of the gene and characterization of the gene product.

The gene encoding cyclohexadienyl dehydratase (denoted pheC) was cloned from Pseudomonas aeruginosa by functional complementation of a pheA auxotroph of Escherichia coli. The gene was highly expressed in E. coli due to the use of the high-copy number vector pUC18. The P. aeruginosa cyclohexadienyl dehydratase expressed in E. coli was purified to electrophoretic homogeneity. The latter enzyme exhibited identical physical and biochemical properties as those obtained for cyclohexadienyl dehydratase purified from P. aeruginosa. The activity ratios of prephenate dehydratase to arogenate dehydratase remained constant (about 3.3-fold) throughout purification, thus demonstrating a single protein having broad substrate specificity. The cyclohexadienyl dehydratase exhibited Km values of 0.42 mM for prephenate and 0.22 mM for L-arogenate, respectively. The pheC gene was 807 base pairs in length, encoding a protein with a calculated molecular mass of 30,480 daltons. This compares with a molecular mass value of 29.5 kDa determined for the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since the native molecular mass determined by gel filtration was 72 kDa, the enzyme probably is a homodimer. Comparison of the deduced amino acid sequence of pheC from P. aeruginosa with those of the prephenate dehydratases of Corynebacterium glutamicum, Bacillus subtilis, E. coli, and Pseudomonas stutzeri by standard pairwise alignments did not establish obvious homology. However, a more detailed analysis revealed a conserved motif (containing a threonine residue known to be essential for catalysis) that was shared by all of the dehydratase proteins.