| Medically useful semisynthetic cephalosporin and penicillin antibiotics are made from precursor 7-aminocephalosporanic acid (7-ACA) and 6-amino penicillinic acid (6-APA). Beta-lactam antibiotics acylase, which was divided into cephalosporin acylase and penicilln acylase according to substrate specificity, converted the fermented cephalosporin and penicillin to 7-ACA and 6-APA. Beta-lactam antibiotics acylase was thus very important enzyme for producing semi-synthetic beta-lactam antibiotics. To facilitate the attempts of obtaining the microorganisms with higher CA activity from natural environments, two screening systems for cephalosporin acylase were established. The first system, which was based on cephalosporin acylases' recognition to a series of chromogenic model substrates with same or similar side chain of cephlosporin, can screen the acylase-producing-microorganisms from a large number of strains. The second one was a microbial method. In this method, the core part of cephalosporin was replaced by 6-amino penicillinic acid (6-APA) to generate two screening substrates, namely glutaryl-6-APA and adipoyl-6-APA and a commercial strain Serratia marcescen, sensitive to 6-APA and resistant to penicillin, was used as an indicator strain in an overlaid-agar screening procedure. Because of its specificity, simplicity and sensitivity, the method could be easily installed into a high-throughout system.A strain capable of producing cephalosporin acylase, penicilln G acylase and 5-adipoylamido-2-nitro-benzoic acid acylase was selected from thousands of candidates by the established methods. The genes encoding penicilln G acylase and 5-adipoylamido-2-nitro- benzoic acid acylase were cloned and the properities of this two acylases were studied.Cloned Flavobacterium sp.650 penicillin G acylase (PGA) gene was 2589bp. The penicillin G acylase is novel and has homology varied from 28-53% with other sourced PGAs. It is composed of two subunits,α-subunit andβ-subunit, whose molecular weights were 22kD and 62kD respectively. The enzyme properties of the acylase were investigated. It has lower Km against NIPAB and penicillin G, whichwere 2.7×10-5 M and 8.9×10-6M. Its kcat upon NIPAB and penicillin G were 68.84 s-1 and 72.69 s-1 respectively, higher than most of other PGAs. The acylase activity was inhibited strongly by PMSF and the Ki value is 0.1μM. The acylase activity is also inhibited by bromoacetyl-7-ACA, an inhibitor of the penicillin G acylase and cephalosporin acylase, and the Ki was 2.3mM. The ratio of synthetic specific activity and hydrolytic specific activity was determined as (1/60). The optimal temperature and pH for the hydrolytic activity were 600C and 8.0. Expression of the penicillin G acylase gene was constitutive and reperessed by phenylacetic acid (PAA), which was different from most of other penicillin G acylase genes. The temperature for synthesis of active enzyme was 280C, higher than that of other penicillin acylase genes. The properties of the penicillin G acylase implies that this penicillin acylase is more suitable for industrial application.Flavobacterium sp.650 5-adipoylamido-2-nitro-benzoic acid acylase has deacylation activity against both cephalosporin and several model substrates of cephalosporin such as 5-adipoylamido-2-nitro-benzoic acid(Ad-NABA),5- glutaryl- amido-2-nitrobenzoic acid(Gl-NABA), glutaryl-(-naphthylamine (GL-Nap),glutaryl- aniline(GL-Aniline),glutaryl p-nitroaniline (GL-NA) and adipoyl-p -nitroaniline (AD-NA). 5-Adipoyl amido-2-nitro benzoic acid acylase gene was cloned. The gene encodes a peptide of 53kD( 509 amino resudues). The deduced amino acid sequences of the acylase has homologies of 29% with amidase family protein from various microorganism and 36% with 6-amino hexanoate-cyclic-dimer hydrolase, which catalyzes the first step in the degradation of 6-amino hexanoic acid cyclic dimer, a by-product of nylon manufacture. It has only minor homologies(10%) with cephalosporin acylase derived from Pseudomonas V22 and SE83. The acylase was purified f...
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