Flat‐sheet and hollow fiber membrane bioreactors: A study of the kinetics and active site conformational changes of immobilized papain including sorption studies of reaction constituents

J of Applied Polymer Sci

Nakasone

Nagano

et al. 2003

This study investigated the activity of trypsin that had been covalently immobilized onto acrylic acid (AA)-and methacrylic acid (MAA)-grafted polyethylene (PE) plates-PE-g-PAA and PE-g-PMAA-using a watersoluble carbodiimide as a coupling agent, as a function of the immobilized amount, the grafted amount, the pH value on immobilization, and the pH value and temperature at the activity measurement. The activity of trypsin immobilized on the PE-g-PAA plates at pH 6.0 decreased with an increase in the immobilized amount because of the crowding of trypsin molecules in the vicinity of the surfaces of the grafted PAA layers. The increase in the grafted amount resulted in a decrease in the activity of immobilized trypsin because of a decrease in the diffusivity of BANA molecules caused by the formation of dense grafted PAA layers for the PE-g-PAA plates and led to the increased activity because of the increase in the hydrophilicity of the whole grafted layers for the PE-g-PMAA plates. The activity of trypsin immobilized on the PE-g-PAA and PE-g-PMAA plates at pH 6 increased with an increase in the pH value, probably because of the expansion of trypsin-carrying grafted PAA and PMAA chains and the increased diffusivity of N␣-benzoyldl-arginine-nitroanilide hydrochloride molecules in the grafted layers. The optimum temperature of the activity of immobilized trypsin shifted to 50°C from 30°C for native trypsin. Immobilized trypsin was reusable without any denaturation and isolation at temperatures ranging from 20°C to 60°C and pH values ranging from 6 to 10. Trypsin immobilized on a PE-g-PAA plate had 95% of the remaining activity in relation to native trypsin at 30°C after preservation in a pH 7.8 buffer at 4°C over 6 months. These results made clear that alkaline and thermal stability, reusability, and storage stability can be much improved by the covalent coupling of trypsin on PE-g-PAA and PE-g-PMAA plates.

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Retention and reusability of trypsin activity by covalent immobilization onto grafted polyethylene plates

J of Applied Polymer Sci

Nakasone

Nagano

et al. 2003

“…The membrane matrix was composed of poly(ether)sulfone blended with polyacrolein to provide aldehyde functionality. The average pore size of the membrane was 0.45 m, and the thickness of the membrane was 152 m. The surface area of the membrane was determined by nitrogen gas adsorption studies using the Brunauer-Emmett-Teller pore (BET) isotherm, to be 9 m 2 /g (Ganapathi et al, 1995). The other membrane used for enzyme immobilization was purchased from Whatman (product # 6872-1703, Actidisk), and was composed of poly(vinyl chloride) (PVC) sheets with embedded aldehyde-functionalized silica (approximately 65% silica) particles to provide functionality for enzyme attachment.…”

Section: Immobilization Supportsmentioning

confidence: 99%

“…The rate of reaction was analyzed by measuring the amount of the product p-nitroaniline in the permeate. There is a significant amount of product sorbed to the membrane which, if not accounted for, can result in erroneous kinetic data (Ganapathi et al, 1995). Hence, the amount of product adsorbed on the membrane that is in equilibrium with the product in the reaction mixture was added up to give the total amount of product formed in the reaction.…”

Section: Wild-type Subtilisinmentioning

confidence: 99%

Site-directed and random immobilization of subtilisin on functionalized membranes: Activity determination in aqueous and organic media

Viswanath

Wang

Bachas

et al. 1998

Biotechnol. Bioeng.

Kinetic comparisons have been made between a randomly immobilized and a site‐specifically immobilized subtilisin BPN′ on microfiltration membranes of varying hydrophilicities in both aqueous and organic media. Site‐directed mutagenesis was employed to introduce a single cysteine into the amino acid sequence of subtilisin at a location away from the active site. Immobilization of this mutant enzyme was then carried out using the single cysteine residue to orient the active site of the enzyme away from the membrane surface. Kinetic comparison of the immobilized mutant enzyme with the randomly immobilized wild‐type enzyme in aqueous media showed an activity enhancement on both hydrophilic silica‐containing and hydrophobic poly(ether)sulfone membranes. Higher loading efficiencies were observed for the site‐directed enzyme on immobilization. Optimal enzyme loading values were calculated for the randomly immobilized enzyme. An enhancement of activity was also observed for the site‐directed immobilized systems using nearly anhydrous hexane as the solvent. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 608–616, 1998.

“…In addition, urease immobilized on the ePTFE‐ g ‐PHEMA films with lower grafted amounts had higher relative activity. As urease was also immobilized on grafted PHEMA chains in the inside of the grafted layer at higher grafted amount, diffusion of urea molecules into the grafted layers was suppressed, resulting in a decrease in the relative activity 19, 22, 43…”

Section: Resultsmentioning

confidence: 99%

Retention of activity of urease immobilized on grafted polymer films

Iizawa

J of Applied Polymer Sci

et al. 2006

Expanded poly(tetrafluoroethylene) (ePTFE) films grafted with 2-hydroxyethyl methacrylate (HEMA) and 2-hydroxyethyl acrylate (HEA) were applied to a polymer support for urease immobilization. The HEMA-and HEAgrafted ePTFE (ePTFE-g-PHEMA and ePTFE-g-PHEA) films prepared by the combined use of the plasma treatment and photografting possessed high water-absorptivities. Imidazole groups were introduced to grafted PHEMA and PHEA chains with 1,1 0 -carbonyldiimidazole (CDI) in acetonitrile. The activity of urease covalently immobilized to the ePTFEg-PHEMA and ePTFE-g-PHEA films in a pH 7.0 buffer at 48C had the maximum value at the optimum pH value of 7.5 for native urease. Urease immobilized on the ePTFE-g-PHEMA films with the extent of CDI bonding of about 20% had the maximum activity, and the repeatedly measured activity was kept almost constant. The relative activity of immobilized urease stayed almost constant in the range of the immobilized amounts between 10 and 30 mg/g for both grafted ePTFE films, and decreased at higher immobilized amounts because of the crowding of immobilized urease molecules in the grafted layers. The relative activity of immobilized urease had the maximum values at the grafted amounts of 1.2 and 1.7 mmol/g for the ePTFE-g-PHEMA and ePTFE-g-PHEA films, respectively, and the further increase in the grafted amount resulted in the decrease in the relative activity. The optimum temperature of the activity for immobilized urease was shifted from 30 to 508C for native urease by the covalent immobilization on both grafted ePTFE films and immobilized urease was repeatedly usable without a considerable decrease in the activity in the regions of the pH 6.0-9.0 and 10-608C.