Hydrolytic enzymes adsorption

Most of the soluble hydrolytic enzymes in common use in industry are formed extra-cellularly the wide range of intracellular microbial enzymes are virtually unexplored commercially. In order to exploit these enzymes, it is necessary to develop economic methods of purification. Reports of continuous methods for harvesting, breaking cells, and fractionating their protein represent major advances toward this end. Recently, enzymes as well as whole cells have been imobilized by adsorption, encapsulation, or inclusion in... 

Adsorption kinetic was investigated, and the influence of contact time between coconut fiber and lipase, at different enzyme concentrations from 0 U/ml (control without enzyme) to 90 U/ml, were evaluated. No hydrolytic activity was detected when the fiber without immobilized enzyme (control) was used as catalyst. Figure 1 pictures the influence of different initial concentrations of lipase in the supernatant ( 0 equal to 30, 60, or 90 U/ml) on the hydrol)ftic activity of immobilized CALB. The experimental data were subjected to statistical analysis (analysis of variance). At the probability level of p<0.05 (data not shown), it was observed that immobilized amount increases as time increased until 2 h (in... 

Fig. 1 Effect of contact time on the hydrolytic activity of lipase immobilized on coconut fiber by adsorption using initial enzyme concentrations of 30 U/ml (closed triangle), 60 U/ml (closed circle), or 90 U/ml (closed square)...

In subsequent experiments, to evaluate the influence of enzyme loading on the properties of the immobilized enzyme, contact time was set in 2 h, and enzyme concentration in the supernatant, during incubation, was changed from 30 to 500 U/ml (Fig. 2). It can be seen that hydrolytic activity of the immobilized enzyme enhances with increasing lipase concentration in the supernatant. However, the higher value of hydrolytic activity achieved by adsorption of CALB on coconut fiber (135.14 U/g) was still lower than the hydrolytic activity of Novozyme 435 (1,039 U/g, determined according to the methodology described in Assay of Hydrolytic Activity Methyl Butyrate Hydrolysis ), a commercial derivative. 

Fig. 2 Hydrolytic activity of immobilized enzjrme as a function of initial enzyme activity in the supernatant Lipase was immobilized on eoeonut fiber by adsorption after 2 h of contact time at room temperature...

Reusability of immobilized CALB was tested in subsequent cycles of methyl butyrate hydrolysis. It can be observed in Fig. 7 that CALB-7A retained less than 50% of its initial hydrolytic activity after the third cycle of reaction whereas Novozyme 435 retained almost 70% after the tenth cycle (Fig. 7). Other authors [36] observed that CALB immobilized on activated carbon retained more than 55% of its initial activity after the sixth cycle of methyl butyrate hydrolysis. The worse operational stability of CALB immobilized on coconut fiber, when compared to CALB immobilized on activated carbon and to Novozyme 435, may be due to enzyme desorption during reaction, induced by the hydrophobic substrate, and by the low enzyme load adsorbed. As discussed before, the driven forces of CALB adsorption on coconut fiber are electrostatic interactions that are weaker than hydrophobic interactions, which predominate on Novozyme 435 and CALB adsorbed on activated carbon. Furthermore, both aetivated carbon and the resin used in the preparation of Novozyme 435 are porous support with high superficial area available for enzyme immobilization, allowing obtaining of high enzyme load. Coconut fiber, on the other hand, does not have a porous structure, and it has a low surface area [27], making it difficult to achieve high enzyme loads. 

In the present work, green coconut fiber was successfiilly used to immobilize lipase B from C. antarctica by adsorption. During adsorption studies, it was observed that adsorption equilibrium was achieved afler a contact time of 2 h (in case of o=30 U/ml and Eo=(0 U/ml) or 6 h ( o=90 U/ml). Moreover, an improvement of hydrolytic activity of immobilized CALB is also observed with increasing concentrations of lipase offered to immobilization. This increase in activity is due to formation of multilayers, confirmed by thermal stability essays. Two plateaus of enzyme activity were observed when the pH of lipase solution during adsorption was varied in the range studied. This behavior is typical of an ionic support At 50 and 60 °C, the adsorbed enzyme was, respectively, 2- and 92-fold more stable than the soluble enzyme. At 60 °C, however, Novozyme 435 s stability was higher than that of CALB-7A. TUIer 10 h of incubation at 60 °C, Novozyme 435 retained more than 70% of its initial activity, whereas CALB-7A retained only 50%. Last but not least operational stabilities studies of butyl butyrate synthesis, compared to a commercial derivative, showed that C7U..B-7A is a suitable biocatalyst to be used in the synthesis of flavors. 

Sample handling, prepurification, and other operations prior to HPLC must be minimized to reduce the possibility of enzymic or hydrolytic breakdown of the peptides, and general losses by adsorption to surfaces. It is desirable to use a method which can cope directly with rather impure samples (e.g., crude brain extracts). 

These three functional domains (namely catalytic domain, linker domain and substrate-binding domain) are essential for the enzymatic degradation of water-insoluble polymers. Hence it has been proposed that the degradation of the polymer should proceed in three steps (1) adsorption of the enzyme to the polymer, (2) non-hydrolytic disruption of the structure of the polymer, and (3) hydrolysis. " ... 

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