Affinity purification of penicillin acylase on phenylacetic acid linked to Indion 48-R: Effect of spacer variation

S. K. Karyekar; M. V. Hegde

doi:10.1007/bf01875610

Immobilization of alginate-PAC on Sepabeads EC-HA support

Hemijska industrija ◽

10.2298/hemind110318041z ◽

2011 ◽

Vol 65 (4) ◽

pp. 431-437 ◽

Cited By ~ 1

Author(s):

Milena Zuza ◽

Nenad Milosavic ◽

Zorica Knezevic-Jugovic

Keyword(s):

Phenylacetic Acid ◽

Immobilized Enzyme ◽

Penicillin Acylase ◽

Covalent Immobilization ◽

Enzyme Inactivation ◽

Penicillin G ◽

Ph Profile ◽

Covalently Linked ◽

Hydrolysis Of ◽

Derivatives Of

Penicillin acylase (PAC) is an important industrial enzyme for the production of many ?-lactam antibiotics. It is capable of catalyzing the hydrolysis of penicillin G (Pen G) to generate phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA). In this paper, in order to prevent enzyme inactivation, an attempt of coupling enzyme modification and immobilization was presented. Chemical modification was promoted to introduce carbohydrate moiety into the PAC molecule, capable of being covalently linked to an amino support. This seems to provide a possibility to couple the enzyme without risking a reaction at the active site which might cause a loss of activity. PAC molecules were modified by cross-linking with polyaldehyde derivatives of alginate in order to add them new and useful functions. Immobilization of alginate-PAC on Sepabeads EC-HA was used as a model system in order to demonstrate the potential of this strategy. Optimal conditions for covalent immobilization of alginate-PAC from Escherichia coli on support Sepabeads EC-HA, were investigated. The immobilized enzyme was then characterized by evaluating the potential effects of immobilization on its thermal stability, temperature and pH profile in comparison with native non-modified PAC and modified non-immobilized PAC. The maximum amount of the alginate-PAC coupled on the dry support of 99 mg/g was satisfactory. Deactivation rate constants at 50 ?C for free PAC, alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA were 2,32; 50,65 and 1,68 h-1, respectively. Alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA had the same pH and temperature optimum as the native non-modified PAC.

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Repression of penicillin G acylase of Proteus rettgeri by tricarboxylic acid cycle intermediates

Journal of Bacteriology ◽

10.1128/jb.152.1.104-110.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 104-110

Author(s):

G O Daumy ◽

A S McColl ◽

D Apostolakos

Keyword(s):

Catabolite Repression ◽

Phenylacetic Acid ◽

Tricarboxylic Acid Cycle ◽

Dicarboxylic Acids ◽

Penicillin Acylase ◽

Tricarboxylic Acid ◽

Penicillin G ◽

Acid Cycle ◽

E Coli ◽

Tricarboxylic Acid Cycle Intermediates

The regulation of the penicillin acylase in proteus rettgeri ATCC 31052 was compared with that of the enzyme in Escherichia coli ATCC 9637. Unlike the E. coli acylase, the P. rettgeri enzyme was not induced by phenylacetic acid, nor was it subject to catabolite repression by glucose. The P. rettgeri acylase appears to be expressed constitutively but is subject to repression by the C4-dicarboxylic acids of the tricarboxylic acid cycle, succinate, fumarate, and malate.

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Penicillins and other acylamino compounds synthesized by the cell-bound penicillin acylase of Escherichia coli

Biochemical Journal ◽

10.1042/bj1150747 ◽

1969 ◽

Vol 115 (4) ◽

pp. 747-756 ◽

Cited By ~ 50

Author(s):

M. Cole

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Phenylacetic Acid ◽

Enzyme Reaction ◽

Reaction Rates ◽

Hydroxamic Acids ◽

Penicillin Acylase ◽

E Coli ◽

Hydroxyphenylacetic Acid ◽

Derivatives Of

1. The penicillin acylase of Eschericha coli N.C.I.B. 8743 is a reversible enzyme. Reaction rates for the two directions have been determined. 2. Measurements of the rates of enzymic synthesis of penicillins from 6-aminopenicillanic acid and various carboxylic acids revealed that p-hydroxyphenylacetic acid was the best substrate, followed by phenylacetic, 2-thienylacetic, substituted phenylacetic, 3-hexenoic and n-hexanoic acids. 3. The rate of synthesis of penicillin improved when amides or N-acylglycines were used; α-aminobenzylpenicillin and phenoxymethylpenicillin were only synthesized when using these more energy-rich compounds. 4. Phenyl-acetylglycine was the best substrate for the synthesis of benzylpenicillin compared with other derivatives of phenylacetic acid. 5. The enzyme was specific for acyl-l-amino acids, benzylpenicillin being synthesized from phenylacetyl-l-α-aminophenylacetic acid but not from phenylacetyl-d-α-aminophenylacetic acid. 6. α-Phenoxyethylpenicillin was synthesized from 6-aminopenicillanic acid and α-phenoxypropionylthioglycollic acid non-enzymically, but the rate was faster in the presence of the enzyme. 7. The E. coli acylase catalysed the acylation of hydroxylamine by acids or amides to give hydroxamic acids, the phenylacetyl group being the most suitable acyl group. The enzyme also catalysed other acyl-group transfers.

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Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645062 ◽

1990 ◽

Vol 63 (03) ◽

pp. 439-444 ◽

Cited By ~ 19

Author(s):

C Kuyas ◽

A Haeberli ◽

P Walder ◽

P W Straub

Keyword(s):

Gel Electrophoresis ◽

Affinity Purification ◽

Polyacrylamide Gel Electrophoresis ◽

Ease Of Use ◽

Terminal Sequence ◽

Purification Method ◽

Human Fibrinogen ◽

Isolation Methods ◽

One Step ◽

Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

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Isolation of Multiple Types of Plasminogen Activator Inhibitors from Vascular Smooth Muscle Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646626 ◽

1989 ◽

Vol 61 (03) ◽

pp. 517-521 ◽

Cited By ~ 15

Author(s):

Walter E Laug ◽

Ruedi Aebersold ◽

Ambrose Jong ◽

Willian Rideout ◽

Barbara L Bergman ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Affinity Purification ◽

Muscle Cells ◽

Exchange Chromatography ◽

Natural Resistance ◽

Protease Nexin ◽

Pai 1

SummaryLarge arteries have a natural resistance to tumor cell invasion thought to be due to the production of protease inhibitors. Vascular smooth muscle cells (VSMC) representing the major cellular part of arteries were isolated from human aortas and grown in tissue culture. These cells were found to produce large amounts of inhibitors of plasminogen activators (PA). Fractionation of VSMC-conditioned medium by heparin-affigel chromatography separated three immunologically and functionally distinct PA inhibitors (PAI), namely PAI-1, PAI-2 and protease-nexin I. The three inhibitors were characterized by functional assays and immunoblotting. PA inhibitor 2 (PAI-2) had little affinity for heparin, whereas PA inhibitor 1 (PAI-l) bound to heparin and was eluted from the column at NaCl concentrations of 0. 1 to 0.35 M. Protease-nexin I, eluted at NaCl concentrations of 0.5 M and higher. Most of the PAI-1 was present in the latent, inactive form. PAI-1 was further purified by ion exchange chromatography on a Mono-Q column. Partial sequencing of the purified PAI-1 confirmed its nature by matching completely with the sequence deduced from the cDNA nucleotide sequence of endothelial cell PAI-1. Thus, human VSMC produce all three presently known PAI and these can be separated in a single heparin affinity purification step.

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