Identification of precursor peptide of aculeacin A acylase as a protein with proteolytic activity

Abstract A protein with the proteolytic activity was isolated from culture filtrate of the aculeacin A acylase producing strain, Actinoplanes utahensis NRRL12052. The purified protein showed a single band of molecular mass of 87 kDa in SDS-PAGE and gel filtration using HPLC, and reacted with anti-aculeacin A acylase antiserum. The 87-kDa protein was degraded to two peptides of molecular mass of 60 kDa and 19 kDa by incubation at 37°C in the presence of 0.1% SDS and the former band also responded to the antiserum. These results indicate that the 87-kDa protein possessing the proteolytic activity is a precursor of aculeacin A acylase.     

Efficient production of aculeacin A acylase in recombinant Streptomyces strains

The productivities of aculeacin A acylase in various recombinant Streptomyces strains were examined. When the acylase gene was introduced into six species (S. lividans, S. albus, S. ambofaciens, S. parvulus, S. griseus and S. avermitilis) using Streptomyces multi-copy vector pIJ702, all strains produced the acylase extracellularly. The recombinant S. Griseus was the most efficient producer of the enzyme, producing 25-fold more than the original producer, Actinoplanes utahensis. On the other hand, the recombinant strains of S. lividans and S. avermitilis showed almost the same productivity as A. utahensis. The purified recombinant acylases from four strains, S. albus, S. ambofaciens, S. parvulus and S. griseus, were composed of two subunits; however, the molecular mass values of their small subunits were higher than that of the original acylase. Further, immunoblot analysis showed that the presumed precursor peptide and its degradation products were also detected in the low-producing strains, A. utahensis and S. avermitilis. These findings indicate that the productivity of the acylase was affected by proteolytic activity in the host strains. Correspondence to: S. mura     

Cloning and characterization of penicillin V acylase from Streptomyces mobaraensis

We report on the molecular cloning and characterization of penicillin V acylase (PVA) from an actinomycete, Streptomyces mobaraensis (Sm-PVA), which was originally isolated as an acylase that efficiently hydrolyzes the amide bond of various N-fatty-acyl-l-amino acids and N-fatty-acyl-peptides as well as capsaicin (8-methyl-N-vanillyl-6-nonenamide). In addition, the purified Sm-PVA hydrolyzed penicillin V with the highest activity (k(cat)) among the PVAs so far reported, penicillin G, and 2-nitro-5-phenoxyacetamide benzoic acid. The BLAST search revealed that the Sm-PVA precursor is composed of a polypeptide that is characteristic of enzymes belonging to the beta-lactam acylase family with four distinct segments; a signal sequence (43 amino acids), an alpha subunit (173 amino acids), a linker peptide (28 amino acids), and a beta subunit (570 amino acids). The mature, active Sm-PVA is a heterodimeric protein with alpha and beta subunits, in contrast to PVAs isolated from Bacillus sphaericus and B. subtilis, which have a homotetrameric structure. The amino acid sequence of Sm-PVA showed identities to PVA from S. lavendulae, N-acylhomoserine lactone-degrading acylase from Streptomyces sp., cyclic lipopeptide acylase from Streptomyces sp., and aculeacin A acylase from Actinoplanes utahensis with 68, 67, 67, and 41% identities, respectively.     

Newly discovered penicillin acylase activity of aculeacin A acylase from Actinoplanes utahensis

Aculeacin A acylase from Actinoplanes utahensis produced by Streptomyces lividans revealed acylase activities that are able to hydrolyze penicillin V and several natural aliphatic penicillins. Penicillin K was the best substrate, showing a catalytic efficiency of 34.79 mM(-1) s(-1). Furthermore, aculeacin A acylase was highly thermostable, with a midpoint transition temperature of 81.5 degrees C.     

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.     

Cloning and sequencing of the aculeacin A acylase-encoding gene from Actinoplanes utahensis and expression in Streptomyces lividans.

Aculeacin A acylase (AAC), produced by Actinoplanes utahensis, catalyzes the hydrolysis of the palmitoyl moiety of the antifungal antibiotic, aculeacin A. Using mixed oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequences of the two subunits of AAC, overlapping clones were identified in a cosmid library of A. utahensis DNA. After the sub-cloning of a 3.0-kb fragment into Streptomyces lividans, the recombinant produced AAC extracellularly. The nucleotide sequence of this fragment predicted an open reading frame of 2358 bp with GTG start and TGA stop codons. The deduced 786-aa sequence should correspond to a single polypeptide chain, indicating that this polypeptide is processed to the active form which is composed of the two subunits. Threefold more AAC was obtained from the S. lividans recombinant carrying the cloned gene than the original A. utahensis strain.     

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.     

Reactivation of heterodimer and individual subunits of penicillin acylase from E. coli after inactivation in urea

Individual subunits of penicillin acylase from E. coli were isolated by gel-filtration under denaturing conditions (8 M urea). Recovery of the catalytic activity of the penicillin acylase heterodimer was studied after removal of urea. In the case of the heterodimer, 40-60% of the initial activity was recovered, whereas the activity of individual subunits was not recovered. Combination of native enzyme subunits with subunits isolated from the enzyme pre-inactivated with the irreversible inhibitor phenylmethylsulfonyl fluoride resulted in heterodimers which were active only in the case of involvement of the beta-subunit of the active enzyme.     

Newly Discovered Penicillin Acylase Activity of Aculeacin A Acylase from Actinoplanes utahensis

Aculeacin A acylase from Actinoplanes utahensis produced by Streptomyces lividans revealed acylase activities that are able to hydrolyze penicillin V and several natural aliphatic penicillins. Penicillin K was the best substrate, showing a catalytic efficiency of 34.79 mM⁻¹ s⁻¹. Furthermore, aculeacin A acylase was highly thermostable, with a midpoint transition temperature of 81.5°C.     

The rat kidney acylase I, characterization and molecular cloning. Differences with other acylases I.

The soluble acylase I from rat kidney was purified to homogeneity using a five-step procedure. As the resulting protein was found to have a relative molecular mass of 125 kDa based on size-exclusion chromatography and 44 kDa based on SDS/PAGE, the native protein was taken to consist of three subunits. The amino-acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microsequencing (RHEFHALRAGFALDEGLA), was found to be very similar to the corresponding sequence of porcine kidney acylase I. However, as N-furyl-acryloyl-L-methionine, a synthetic substrate for porcine acylases, was not hydrolyzed by the rat enzyme, it was suggested that the polypeptide chain might differ in other respects from those of the other acylases I. A full length cDNA coding for the rat kidney acylase I was therefore isolated and found to contain a 1224-bp open reading frame encoding a protein consisting of 408 amino-acid residues, which corresponded to a calculated molecular mass of 45 847 Da per subunit. The deduced amino-acid sequence showed 93.6% and 87.2% identity with that of the human liver and porcine kidney, respectively.     

胞外青霉素酰化酶的纯化及部分理化性质

巨大芽孢杆菌产胞外青霉素酰化酶发酵液经硫酸铵分级抽提及ＳｅｐｈａｄｅｘＧ－１００、羟基磷灰石、ＤＥＡＥ纤维素ＤＥ５２等层析步骤,提纯了青霉素酰化酶,得到电泳均一的酶制剂。纯酶比活力约为２５Ｕ／ｍｇ蛋白,纯化４９倍,活力回收５８％,经ＰＡＧＥ及ＳＤＳ－ＰＡＧＥ测知该酶不含亚基,其分子量约为１４０ｋＤ。该酶最适ｐＨ为９．０,最适温度４７℃,用底物ＮＩＰＡＢ测活,其Ｋｍ值为６．２×１０~（－４）ｍｏｌ／Ｌ,Ｖｍ值为１．２４×１０４ｍｏｌ／Ｌ。此外还探讨了部分金属离子对该酶的影响。     

水稻氨基酰化酶的分离纯化与性质分析

氨基酰化酶（N－acylamino－acidamidohydrolase或acylaseⅠ,EC3．5．1．14）是专一水解N-酰基化L-氨基酸的蛋白酶．从水稻黄化苗得到的抽提液,经过硫酸铵分级沉淀、丙酮分级沉淀和阴离子交换层析三个步骤,纯化得到了该酶,比活达到100U／mg蛋白,在无还原剂存在的SDS-聚丙烯酰胺凝胶电泳上显单一条带,分子量为40kD．而凝胶层析分析表明活性分子的分子量约90kD,因此可推测它的活性分子由两个亚基通过非共价键作用组合而成．进一步研究此酶的性质,在所测的五种乙酰化氨基酸中,最适底物为N-乙酰-L-甲硫氨酸．该酶的最适温度为50℃,最适pH为7．0～8．0．Co2＋和Zn2＋能增强酶活性,但烷基化试剂对酶活性没有影响,表明酶活性中心不含活化的巯基或羟基基因     

Expression, purification and crystallization of penicillin G acylase from Escherichia coli ATCC 11105

The penicillin acylase gene (pac) from Escherichia coli ATCC 11105 was cloned into pUC 9 and the resulting vector (pUPA-9), when transformed into E. coli strain 5K, allowed the constitutive overproduction of mature penicillin acylase when grown at 28 degrees C. The enzyme was purified from the periplasmic fraction of E. coli pUPA-9 by hydrophobic interaction chromatography and anion exchange. Crystals of penicillin acylase were grown in batch using polyethylene glycol 8000 as a precipitant. The crystals (space group P1) diffracted to beyond 2.3 A.     

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.     

Chainia Penicillin V Acylase: Strain Characteristics, Enzyme Immobilization, and Kinetic Studies

Aerobic cultures of an actinomycete were found to produce penicillin V acylase (PVA) (PA, EC-3.5.1.11) extracellularly. The presence of L-2-3 diamino-propionic acid in cell wall and formation of sclerotia on culture media led to its identification as Chainia, a sclerotial Streptomyces. Partially purified acylase was adsorbed on kieselguhr and entrapped in polyacrylamide gel. The immobilized preparation proved effective with respect to retention of enzyme and enzyme activity even after 15 successful cycles. The pH optimum for crude enzyme was in the range of pH 7.5–8.0, and for the (NH4)₂ SO₄ fraction it was pH 8.5. The immobilized enzyme showed maximal activity at pH 9.5. The optimum temperature for acylase activity was at 55°C. The crude enzyme, ammonium sulfate fraction, and immobilized enzyme showed K _m value for penicillin V of 6.13 mM, 14.3 mM, and 17.1 mM, respectively. Received: 11 December 1997 / Accepted: 9 April 1998     

A novel strategy for the expression and purification of the DNA methyltransferase, M.AhdI

Biochemical and structural studies of the methylase from the type 1 1/2 R-M system AhdI require the ability to purify this multi-subunit enzyme in significant quantities in a soluble and active form. Several Escherichia coli expression systems were tested for their ability to produce the intact methylase but this could not be achieved in a simple co-expression system. Expression experiments were optimised to produce high yields of soluble M and S subunits as individual proteins. Temperature and conditions of induction proved to be the most useful factors and although purification of the S subunit was successful, an efficient strategy for the M subunit remained elusive. A novel strategy was developed in which individual subunits are expressed separately and the bacterial cells mixed before lysis. This method produced a high yield of the multi-subunit methylase when purified to homogeneity by means of heparin and size-exclusion chromatography. It was found to be essential, however, to remove tightly bound DNA by ammonium sulphate precipitation in 1 M NaCl. The intact methylase can now be consistently produced, avoiding the use of fusion proteins. The purified enzyme is stable over long time periods, unlike the individual subunits. This method may be of general application where the expression of multi-subunit proteins, or indeed their individual components, is problematic.     

巨大芽孢杆菌青霉素酰化酶在枯草杆菌中的表达及其分离纯化

青霉素酰化酶（ＰＧＡ）在医药工业起着重要的作用,它能够水解青霉素Ｇ产生６－氨基青霉烷酸（６－ＡＰＡ）和苯乙酸,６－ＡＰＡ是半合成青霉素的关键中间体．该酶广泛存在于各种微生物中如真菌和细菌中．国际上对Ｅ．ｃｏｌｉ、Ａｒｔｈｒｏｂａｃｔｅｒｖｉｓｃｏｓｕ...     

Purification, characterization, and genetic analysis of Mycobacterium tuberculosis urease, a potentially critical determinant of host-pathogen interaction.

Mycobacterium tuberculosis urease (urea amidohydrolase [EC 3.5.1.5]) was purified and shown to contain three subunits: two small subunits, each approximately 11,000 Da, and a large subunit of 62,000 Da. The N-terminal sequences of the three subunits were homologous to those of the A, B, and C subunits, respectively, of other bacterial ureases. M. tuberculosis urease was specific for urea, with a Km of 0.3 mM, and did not hydrolyze thiourea, hydroxyurea, arginine, or asparagine. The enzyme was active over a broad pH range (optimal activity at pH 7.2) and was remarkably stable against heating to 60 degrees C and resistant to denaturation with urea. The enzyme was not inhibited by 1 mM EDTA but was inhibited by N-ethylmaleimide, hydroxyurea, acetohydroxamate, and phenylphosphorodiamidate. Urease activity was readily detectable in M. tuberculosis growing in nitrogen-rich broth, but expression increased 10-fold upon nitrogen deprivation, which is consistent with a role for the enzyme in nitrogen acquisition by the bacterium. The gene cluster encoding urease was shown to have organizational similarities to urease gene clusters of other bacteria. The nucleotide sequence of the M. tuberculosis urease gene cluster revealed open reading frames corresponding to the urease A, B, and C subunits, as well as to the urease accessory molecules F and G.     

Malonyl coenzyme A synthetase. Purification and properties

Malonyl coenzyme A synthetase (EC 6.2.1.14) was induced in Pseudomonas fluorescens grown on malonate as a sole carbon source. This enzyme was purified, for the first time, over 30-fold by the combination of ammonium sulfate precipitation, Sephadex G-150 gel filtration, DEAE-Sephacel ion exchange chromatography, and hydroxylapatite chromatography. The purified enzyme, which had a specific activity of about 0.512 mumol/min/mg, appeared to be electrophoretically homogeneous. The molecular size of the enzyme was determined to be 98,000 Da which is composed of two 49,000-Da subunits. The optimum pH for the enzyme was 7.5. Malonyl coenzyme A synthetase requires ATP, CoA, and Mg2+ for the full enzyme activity. With succinate or acetate, the synthetic rate of CoA derivative was 40% of that observed with malonate. The malonyl coenzyme A synthetase showed typical Michaelis-Menten kinetics for the substrate, malonate, ATP, and coenzyme A, from which the Km values were calculated to be 3.8 X 10(-4) M, 2 X 10(-3) M, and 10(-4) M and Vmax values to be 0.117 mumol/min/mg, 0.111 mumol/min/mg, and 0.142 mumol/min/mg, respectively. The purified malonyl coenzyme A synthetase was immunogenic in the rabbit and Ouchterlony double diffusion analysis revealed a single precipitant line with the enzyme. The antiserum inhibited the enzyme activity and the extent of inhibition was dependent on the amount of the serum added.     

Expression and purification of penicillin G acylase enzymes from four different micro-organisms, and a comparative evaluation of their synthesis/hydrolysis ratios for cephalexin

Several genes for the enzyme penicillin G acylase, as isolated from four different micro-organisms (Alcaligenes facaelis, Escherichia coli, Kluyvera cryocrescens or Providencia rettgeri) were modified at their carboxy-termini to include His-tag fusions, then were expressed from the plasmid pET-24a(+) in E. coli JM109(DE3) cells. All fusion proteins were next purified to homogeneity in a single step by agar-based Co-IDA chromatography, and were then evaluated as catalysts for the synthesis of cephalexin by a kinetically controlled strategy. We find here that the penicillin G acylase enzyme from K. cryocrescens shows a higher intrinsic synthesis/hydrolysis ratio, when compared to three other enzymes from A. facaelis or P. rettgeri, or E. coli.