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Purification and properties of ap-nitrobenzyl esterase fromBacillus subtilis

  • Published:
Journal of Industrial Microbiology

Abstract

A procedure for purifying to homogeneity a microbially produced biocatalyst useful for deblocking intermediates in the manufacture of beta-lactam antibiotics is reported. In aqueous solution the purifiedp-nitrobenzyl (PNB) carboxy-esterase was soluble, monomeric (molecular weight: 54 000 by SDS-PAGE or by gel filtration) and exhibited an acidic pl, 4.1. The PNB carboxy-esterase catalyzed rapid ester hydrolysis for simple organic esters such as PNB-acetate, benzyl acetate and α-naphthyl acetate and catalyzed deblocking (ester hydrolysis) of beta-lactam antibiotic PNB esters such as cephalexin-PNB and loracarbef-PNB. TheN-terminal amino acid sequence and the amino acid composition are reported. A serine residue is involved in ester hydrolysis: the PNB carboxy esterase was inhibited by phenylmethylsulfonyl fluoride and diethylp-nitrophenyl phosphate; one mole of diisopropyl fluorophosphate titration was required per mole of PNB carboxy-esterase for complete inhibition. When the [3H]-diisopropyl fluorophosphate-treated biocatalyst was digested with Lys C and the resulting peptides separated by HPLC, a single [3H]-labeled peptide was obtained; its amino acid sequence is reported. Inhibition of the PNB carboxy esterase by diethyl pyrocarbonate suggests that a histidinyl residue (or residues) is (are) also involved in the catalytic site of the esterase.

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Abbreviations

β-ME:

β-mercaptoethanol

Cf:

cefaclor

Cf:

nucleus-PNB

(6R, 7R):

7-amino-3-chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester

Cp:

cephalexin

Cp-PNB:

p-nitrobenzyl carboxy-ester of cephalexin

DEPC:

diethyl, pyrocarbonate

DFP:

diisopropyl fluorophosphate

DMSO:

dimethyl sulfoxide

DNP:

diethylp-nitrophenyl phosphate

EDTA:

ethylenediaminetetraacetic acid

EGTA:

ethylene, glycol-bis(aminoethyl ether)

N,N,N′N′ :

tetracetic acid

Lc:

loracarbef

Lc-PNB:

p-nitrobenzyl carboxy-ester of loracarbef

Lc:

nucleus-PNB

(6R, 7S):

7-amino-3-chloro-8-oxo-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester

Lys C:

an endoproteinase specifically cleaving at C terminal lysine residues

MWr :

relative molecular weight

PAGE:

polyacrylamide gel electrophoresis

PMSF:

phenylmethylsulfonylfluoride

PNB:

p-nitrobenzyl

PNBCE:

p-nitrobenzyl carboxy-esterase

SDS:

sodium dodecyl sulfate

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, OAES, Oklahoma State University, 74078, Stillwater, OK

    Y-R Chen, S Usui & C-A Yu

  2. Eli Lilly and Company, 46285, Indianapolis, IN, USA

    S W Queener

Authors
  1. Y-R Chen
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  2. S Usui
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  3. S W Queener
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  4. C-A Yu
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Chen, YR., Usui, S., Queener, S.W. et al. Purification and properties of ap-nitrobenzyl esterase fromBacillus subtilis . Journal of Industrial Microbiology 15, 10–18 (1995). https://doi.org/10.1007/BF01570007

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  • DOI: https://doi.org/10.1007/BF01570007

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