Escherichia coli acylase

Y. L. Lee, H. N. Chang (1990) High cell density culture of a recombinant Escherichia coli producing penicillin acylase in a membrane cell recycle fermentor. Biotechnol. Bioeng., 36 330-337. 

M. Cole, Hydrolysis of Penicillins and Related Compounds by the Cell-Bound Penicillin Acylase of Escherichia coli , Biochem. J. 1969, 115, 733-739. 

Immobilization—stabilization of penicilhn G acylase from Escherichia coli. Appl. Biochem. Biotechnol., 26, 181-196. 

Another interesting example of resolution through formation of diastereo-mers is the isolation of four stereoisomers of 3-amino-2-methyl-3-trifluoro-methyl butanoic acid [55]. In this process, the chemical-enzymatic method by the combination of chemical and enzymatic reaction is a very convenient. At first, -phenylacetyl derivatives 61a and 61b were prepared in excellent isolated yields via the Schotten-Baumann procedure. After these materials were hydrolysed with penicillin acylase (EC 3.5.1.11) from Escherichia coli until attainment of 50% conversion, enzymatically unconverted -phenylacetyl derivatives 62 a and 62 b (organic layer) and amino acids 63 b and 63 d (aqueous layer) were separated. Acidic hydrolysis of unconverted materials produced other stereoisomers 63 a and 63 c in high optical pure form. 

Enantioselective enzymatic amide hydrolyses can also be applied for the preparation of optically active organosilicon compounds. The first example of this is the kinetic resolution of the racemic [l-(phenylacetamido)ethyl] silane rac-84 using immobilized penicillin G acylase (PGA E.C. 3.5.1.11) from Escherichia coli as the biocatalyst (Scheme 18)69. (R)-selective hydrolysis of rac-84 yielded the corresponding (l-aminoethyl)silane (R)-85 which was obtained on a preparative scale in 40% yield (relative to rac-84). The enantiomeric purity of the biotransformation product was 92% ee. This method has not yet been used for the synthesis of optically active silicon compounds with the silicon atom as the center of chirality. 

ECB deacylase is an 81-83-kDa heterodimer consisting of 63- and 18-20-kDa subunits. Penicillin G acylase from Escherichia coli is an 87-kDa heterodimer with 65- and 22-kDa subunits [32], For comparison, cephalosporin acylase from a Pseudomonas strain is an 83-kDa heterodimer consisting of 57- and 26-kDa subunits [33], The essential absence of any external catalytic requirement, cofactor stimulation, or product inhibition of ECB deacylase is also an intrinsic property of penicillin acylase [34], Based on the amino-terminal sequences of the two subunits of ECB deacylase, a 48% sequence similarity has been observed between the small subunit of ECB deacylase and a penicillin acylase [25]. This statistically significant albeit moderate sequence similarity from two short segments of the enzymes suggests an evolutionary relationship between ECB deacylase and peni-... 

C Kutzbach, E Rauenbusch. Preparation and general properties of crystalline penicillin acylase from Escherichia coli ATCC 11105. Hoppe-Seyler s Z Physiol Chem 354 45-53, 1974. 

Fig. 8.1. Screening for penicillin G acylase activity. A) Screening in agar plate formats using 6-nitro-3-(phenylacetamido)-benzoic acid (NIPAB) [106], Colonies secreting Penicillin G acylase activity stain a NIPAB-filter yellow. B) Screening in solution using phenylacetyl-MCA and periplasmic extracts without (open symbols) or with ) penicillin G acylases from Kluyvera citrophila, Proteus rettgeri and Escherichia coli respectively [65],...

In addition to anion and cation exchangers as enzyme carriers it has been demonstrated that mixed ion exchange supports can be used for binding enzymes with both acid and amino groups at pH values close to the isoelectric point, such as penidUin G acylase from Escherichia coli (Figure 2.4) ]49]. 

Yang YL, Yun DE, Guan YQ, Peng HL, Chen JM, He YS, Jiao RC. Cloning of GL-7-ACA acylase gene from Pseudomonas sp. 130 and its expression in Escherichia coli. Chin. J. Biotechnol. 1991 7(2) 93-104. 

Luo H, Yu H, Li Q, Shen Z. Cloning and co-expression of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli. Enz. Microb. Technol. 2004 35(6-7) 514-518. 

However, the range of types of amino acids that can be resolved in this way is much greater than just the natural substrates (i.e. peptides made up of the twenty coded amino acids), because methods to relax the specificity of the enzymes have been found, in some cases by using organic solvents for the reactions. Penicillin acylase from Escherichia coli and an aminoacylase from Streptovercillium olivoreti-... 

In spite of usefulness of the simplification obtained by decreasing the experimental substrate concentration, many studies are aimed at the investigation of kinetic properties of immobilized biocatalysts within broader concentration ranges. In a previous paper [29], cells of Escherichia coli with penicillin acylase activity were immobilized by entrapment in calcium pectate gel and tested on the transformation of penicillin G to 6-amino penicillanic acid. Figure 9 shows experimental data from a microcalorimetric investigation of the penicillin G transformation in steady state. As appreciable particle-mass transfer was expected, the mathematical model that includes particle-mass balance was used. 

Penicillins and cephalosporins are charaeterized by (3-lactam structures and are the antibiotics that have traditionally been those most eommonly used in the treatment of infeetions. Pharmaceutical companies have synthesized a variety of semisynthetic (3-lactam compounds for use as oral antibiotics, for example, ampicillin and amoxieillin. These penicillin derivatives are prepared by aeylation of 6-amino-penicillanie acid (6-APA) derived from penicillin G (benzyl penicillin) or penicillin V (phenoxymethyl penicillin). An immobilized penicillin amidase (penicillin acylase) from Escherichia coli or Bacillus megaterium is used to prepare the 6-APA in nearly quantitative yield (Fig. 4). This substance is used as the starting material for the produetion of a number of other penieillins. The immobilized enzyme can be reused more than... 

Factors affecting the synthesis of ampicillin by the cell-bound penicillin acylase of Escherichia coli have been studied . 

Cheng S, Wei D, Song Q (2006) Extraction of penicillin G acylase from Alcaligenes faecalis in recombinant Escherichia coli with cetyl-trimethylammoniumbromide. Biochem Eng J 32(1)56-60... 

Liu YC, Liao LC, Wu WT (2000) Cultivation of recombinant Escherichia coli to achieve high ceU density with a high level of penicillin G acylase. Proc Natl Sci Counc ROC(B) 24(4) 156-160 Luedeking R, Piret EL (1959) A kinetic study of the lactic acid fermentation. Batch process at controlled pH. J Biochem Microbiol Technol Eng 1 393 12 Ma JKC, Drake PMW, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4 794-805... 

Mao QM, Hearn MTW (1996) Optimization of affinity and ion-exchange chromatographic processes for the purification of proteins. Biotechnol Bioeng 52(2) 204-222 Marcos JC, Fonseca LP, Ramalho MT et al. (1999) Partial purification of penicillin acylase from Escherichia coli in poly(ethylene glycol)-sodium citrate aqueous two-phase systems). J Chro-matB 734(1) 15-22... 

Dixon M, Webb EC (1979) Enzymes, 3rd edn. Academic Press, New York, 1116 pp Eisenthal R, Cornish-Bowden A (1974) The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem J 139(3) 715-720 Ertan H, Kazan D, Erarslan (1997) Cross-hnked stabilization of Escherichia coli penicilhn G acy-lase against pH by dextran-dialdedhyde polymers. Biotechnol Tech 11 225-229 Ferreira JS, Straathof AJJ, Franco TT et al. (2004) Activity and stability of immobilized penicillin acylase at low pH values. J Mol Catal B Enzym 27 29-35 Guranda DT, Volovik TS, Svedas VK (2004) pH stability of penicillin acylase from Escherichia coli. Biochemistry (Moscow) 69(12) 1700-1705... 

Guisan JM, Alvaro G, Eernandez-Lafuente R (1990) ImmobUization-stabUization of peniciUin G acylase. An integrated approach. Ann NY Acad Sci 613 552-559 Guranda DT, Volovik TS, Svedas VK (2004) pH stabUity of peniciUin acylase from Escherichia coli. Biochem (Moscow) 69(12) 1700-1705... 

Kurochkina VB, Nys PS (2002) Kinetic and thermodynamic approach to design of processes for enzymatic synthesis of betalactams. Biocatal Biotransform 20(1) 35-41 Lee SB, Ryu DDY (1982) Reaction kinetics and mechanism of penicillin amidase a comparative study of computer simulation. Enzyme Microb Technol 4 35-38 Lin WJ, Kuo BY, Chou CP (2001) A biochemical engineering approach for enhancing production of recombinant penicillin acylase in Escherichia coli. Bioproc Biosys Eng 24 239-247 Lindsay JP, Clark DS, Dordick JS (2004) Combinatorial formulation of biocatalyst preparation for increased activity in organic solvents salt activation of penidllin amidase. Biotechnol Bioeng 85(5) 553-560... 

Meevootisom V, Saunders J (1987) Cloning and expression of penicillin acylase genes from overproducing strains of Escherichia coli and Bacillus megaterium. Appl Microbiol Biotechnol 25 372-378... 

Ozturk DC, Kazan D, Erarslan A (2002) Stabilization and flmctional properties of Escherichia coli peniciUinG acylase by covalent conjugation of anionic polysaccharide carboxymethyl cellulose. World J Microbiol Biotechnol 18 881-888... 

Production of modified penicillins is based predominantly on penicillin G (semi-synthetic penicillin). Penicillin G is split with carrier-linked acylase enzymes into phenylacetic acid and 6-aminopenicillanic acid examples of enzyme sources for the penicillinacylase are Escherichia coli or Bacillus megaterium. 6-Amino-penicillanic acid may be converted into a large number of highly-effective semisynthetic penicillin antibiotics, such as amoxycillin, by the introduction of suitable side-chains. 

The mild reaction conditions that can be employed in the cleavage of such amide linkages means, once again, that sensitive bonds can be preserved. This is relevant to the commercially important process which converts fermentated penicillins (e.g. penicillin G) into 6-aminopenicillanic acid (6-APA), a precursor of the semi-synthetic penicillin antibiotics. The enzyme used for this transformation is the acylase from the bacterium Escherichia coli (Scheme 3.11) (see also Chapter 6, Section 6.5). 

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