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Intraparticle concentration gradients for substrate and acidic product in immobilized cephalosporin C amidase and their dependencies on carrier characteristics and reaction parameters
Author(s) -
Boniello Caterina,
Mayr Torsten,
Klimant Ingo,
Koenig Burghard,
Riethorst Waander,
Nidetzky Bernd
Publication year - 2010
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22694
Subject(s) - chemistry , cephalosporin c , particle size , substrate (aquarium) , phosphorescence , immobilized enzyme , chromatography , luminescence , analytical chemistry (journal) , hydrolysis , reagent , particle (ecology) , fluorescence , enzyme , organic chemistry , cephalosporin , biochemistry , oceanography , physics , optoelectronics , quantum mechanics , geology , antibiotics
Cephalosporin C amidase was covalently attached using a protein loading of 7.0–200 mg protein/g dry carrier on four epoxy‐activated Sepabeads differing in particle size and pore diameter. Initial‐rate kinetic analysis showed that for Sepabeads with small pore diameters (30–40 nm), the apparent K M of the amidase for hydrolysis of cephalosporin C at 37°C and pH 8.0 increased ∼3‐fold in response to increased particle size (∼120–400 µm) and increased amount of immobilized enzyme (7.0–70 mg protein/g dry carrier) while maximum specific activity (3.2 U/mg protein; 25% of free amidase) was affected only by particle size. In contrast, for Sepabeads with wide pores (150–250 nm), the K M was independent of the enzyme loading. Internal effectiveness factors calculated from observable Thiele modulus reflected the dependence of K M on geometrical parameters of the particles. A new method for determination of the overall intraparticle pH was developed based on luminescence lifetime measurements in the frequency domain. Sepabeads were doubly labeled using a lipophilic variant of the pH‐sensitive dye fluorescein, and Ru(II) tris(4,7‐diphenyl‐1,10‐phenantroline) whose phosphorescence properties are independent of pH. Luminescent lifetime measurements of doubly labeled particle suspensions showed superior signal‐to‐noise ratio compared to fluorescence intensity‐based measurements using singly labeled particles. The difference at apparent steady state (ΔpH) between bulk (external pH) and intraparticle pH (internal pH) was as large as ∼0.6 units. The ΔpH was dependent on substrate concentration, particle size, and pore diameter. Therefore, these results characterize the role of carrier characteristics and reaction parameters in the formation of concentration gradients for substrate and acidic product during hydrolysis of cephalosporin C by immobilized amidase. The strong pH dependence of the immobilized amidase underscores the importance of considering intraparticle pH gradients in the design of an efficient carrier‐bound biocatalyst. Biotechnol. Bioeng. 2010;106: 528–540. © 2010 Wiley Periodicals, Inc.