Optimization of covalently coupling enzymes to polymeric membranes: EPR studies of papain

Abstract

SYNOPSISIn an effort to better understand bioreactor systems, papain (EC 3.4.22.2) was covalently immobilized onto vinyl alcohol/vinyl butyral copolymer (PMB) membrane by means of glutaraldehyde (GA) , 1,l'-carbonyldiimidazole (CDI), or 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP ) . Various kinetic and performance characteristics of the immobilized papain were evaluated. It was found that the characteristics of the membranebound papain depended on the immobilization methods. The CDI-and FMP-immobilized papain bioreactors showed better storage and thermal stability than did the GA-immobilized papain bioreactor, although the apparent Michaelis constant, IS,, of the GA-immobilized papain was closer to the free enzyme than to the corresponding CDI-and FMP-immobilized enzymes. In separate experiments, a 6-carbon spacer was inserted between the membrane surface and the covalently bound enzyme. It was found that the insertion of a spacer reduced the disturbance of the enzyme systems, resulting in K,,, values intermediate between the free and directly bound enzymes for all three immobilization methods. Electron paramagnetic resonance spectroscopy was also used to investigate the conformational change and the active site structure of papain. It was found that the active site SH group of papain immobilized with a 6-carbon spacer had faster motion than that of directly bound enzyme, but slower motion than that of the free enzyme. With both direct-coupling and with a spacer, the SH group motion at the active site of papain by CDI and FMP immobilizations was similar, but slower than the corresponding GA immobilization. The conformational changes of the active site of papain upon immobilization with and without a spacer were in agreement with the functional properties of the enzyme. There was a good correlation between the motion of spin-labeled cysteine in the active site of papain and kinetic properties of this protease: As motion slowed, K,,, increased and Vmax decreased. Of the immobilization procedures used, GA immobilization with a spacer yielded kinetic and structural characteristics most similar to the free enzyme while providing increased stability and reusability relative to the latter.

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

“…Investigations have dealt with immobilized horse liver dehydrogenase on sepharose and CNBr-sepharose supports.21*22 Thermal inactivation of immobilized aamylase, chymotrypsin and trypsin has also been studied by EPICz3 Activity measurements of papain, in conjunction with EPR, on PVB membranes showed a good relationship between the properties and structure of the immobilized enzyme. 6 In most cases of immobilized enzyme systems studied by EPR spectroscopy, two subpopulations of the bound protein have been shown to exist; the more restricted subpopulation has been reported as being the less activez4…”

Retention of activity of urease immobilized on grafted polymer films

“…The reaction time dependence of the degrees of CDI bonding was investigated at 25°C for the ePTFE‐ g ‐PHEMA films with the grafted amounts of 1.31, 5.37, and 12.52 mmol/g 35. Figure 4 shows the changes in the extent of CDI bonding with the reaction time for the ePTFE‐ g ‐PHEMA films of three different grafted amounts.…”

“…1). After the reaction, the ePTFE‐ g ‐PHEMA and ePTFE‐ g ‐PHEA films were washed sequentially with a decreasing amount of acetonitrile in water (100, 75, 50, and 25 vol % of acetonitrile) and finally with water 35. The extent of bonded CDI was determined by measuring the absorbances of the aliquots diluted 200‐fold with acetonitrile at 210 nm after the reaction.…”

Polyclonal‐antibody‐mediated insolubilization and stabilization of papain

“…The insolubilized preparations also exhibited higher resistance to denaturation induced by 4 M urea (Figure 3). In these experiments preparation B was also more stable than preparation A. Zhuang and Butterfield [6,39] have also studied the urea inactivation of free and membrane‐immobilized papain, and enhanced stability was observed in their studies.…”

“…Attention has now been paid to enzyme immobilization [1–3], which offers advantages over free enzymes in terms of choice of batch or continuous processes, rapid termination of reactions, controlled product formation, ease of enzyme removal from the reaction mixture and adaptability to various engineering designs. Furthermore, many approaches to the preparation of water‐insoluble enzymes have been explored in recent years [4–7].…”