Immobilization yield

Adsorption of the cells on the polyurethanes was more successful. The research focused on the two MDI-based prepolymers. In the inoculation phase, an immobilization yield of 70% was achieved. It should be noted that immobilization was observed in the large pores only. Hypol 5000 was thought to be the better of the two MDI-based prepolymers, but the reason was not hypothesized. While the production rates were lower than those of the free suspensions, the production was sufficient to be considered a viable technique. Table 5.5 presents the data. 

Although the specific antibodies can be directly attached onto a GEC surface by simple dry-adsorption (the simplest immobilization method and the easiest to automate), this procedure implies multisite attachment, and improved immobilization yields have been obtained with single-point attachment strategies. 

Therefore, the immobilization yield, or fraction of enzyme activity that was transferred to the support, Y would equal (1 - R). For these experiments, the uptake was determined via a point-by-point comparison of absorbance values from the "before" and "after" samples. 

In a different approach penicillin G amidase was coupled to Nylon 6, which was partially hydrolyzed and activated by conversion of the liberated amino groups with N-hydroxysuccinimide and dicyclohexylcarbodiimide [66]. Immobilization yields were fairly high, and preliminary data showed a good operational stability in a column reactor. 

Laval etal. (1984) bound LDH covalently to electrochemically pretreated carbon. The enzyme was fixed by carbodiimide coupling simultaneously with anodic oxidation of the electrode surface. The total amount of immobilized LDH was determined fluorimetrically after removal from the electrode and hydrolysis. The authors found that at a maximal enzyme loading of 13 pmol/cm2 six enzyme layers are formed. The immobilization yield was about 15%. The kinetic constants, pmax and. Km, were not affected by the immobilization. The obtained enzyme loading factor of 10-3 indicates that diffusion in the enzyme layer was of minor influence on the response of the sensor. The layer behaved like a kinetically controlled enzyme membrane, i.e., the NADH oxidation current was proportional to the substrate concentration only far below Km- With increasing enzyme loading the sensitivity for NADH decreased due to masking of the electrode surface. 

As shown in Equation 1, the capacity per cycle is directly proportional to the amount of antibody immobilized, the immobilization yield, the M.W. of the protein and the column volume and an exponential function of the number of cycles. The amount of antibody immobilized will usually be less than 10 gL l. Higher activation of the matrix required for greater than 10 gL l loading results in a decrease in the immobilization yield. The maximum immobilization yield is 1.0 (100%) while 0.8 (80%) is not difficult to obtain. The M.W. of the protein to be isolated is fixed. The only way to increase the capacity per cycle significantly is to increase the volume of the immunosorbent or increase the number of cycles prior to reaching 50% of initial capacity (cycle half-life). Increasing the volume of immunosorbent increases the amount of monoclonal antibody required. 

Ultimately, the cost of immunosorbent isolation will depend on the entire process and must be evaluated against alternative processes. Consider, as an example, the costs and decisions involved in the purification of urokinase. One course of drug therapy consists of 33 mg of urokinase (4,000,000 CTA units). At the hospital pharmacy the drug costs for one course of treatment are currently 3,000 (9), or 91,000/gram. There are approximately 76,000 patients in the U.S. that could be treated with urokinase therapy each year requiring an annual production of approximately 2,500 g. We have selected a monoclonal antibody that has allowed the purification of urokinase from urine, tissue culture media, and bacterial culture media in a single step with 85% retention of urokinase activity (6). This monoclonal antibody was immobilized at 2 gL l with an immobilization yield of 0.8 and a cycle half number of 300 cycles. The urokinase capacity for the first cycle would be 1.2 gL l of immunosorbent. 

Among the two hydrophobic supports, octadecyl-sepabeads presented the best result concerning YLL immobilized activity (15.5 U/g), as its activity was over twice higher than YLL immobilized onto octyl-agarose (7.0 U/g). The other immobilization parameters calculated (immobilization yield and activity retention) were also better for octadecyl sepabeads (Table 1). These results were probably related with the chemical nature of hydrophobic supports. When a very hydrophobic support like octadecyl-sepabeads is used, there might be an increment of the affinity between the support and the substrate that... 

Tabk 1 Immobilization yield (rj) and activity retention (R) of Y. lipolytica lipase on different supports and hydrolytic activity of the immobilized enzyme. 

On the other hand, the ionic adsorption of K lipolytica lipase on MANAE-agarose showed activity retention, immobilization yield and immobilized activity of 61%, 97% and 17 U/g, respectively (Table 1). The use of ionic adsorption has been very successfully used for lipase immobilization [24], Although, the above results are better than the YLL lipase immobilized by adsorption in polypropylene support [25], they showed that these immobilization procedures did not lead to lipase hyperactivation phenomenon, as it was described for hpases of different sources [17, 18], Hyperactivation of the lipase when immobilized on very hydrophobic octadecyl-sepabeads derivative probably occurs because it occurred with the open structure of the lipase that is also much more active than the corresponding closed structure even when undergoing multipoint covalent immobilization. In our study, this phenomenon was not observed probably due to the absence of the lid in this Y. lipolytica lipase [26],... 

Immobilized enzyme was obtained by adsorption, at room temperature, by contacting enzyme solution and support in 5 ml syringes, stirred using the apparatus presented in a previous work [27]. For each gram of dry support, 10 ml of lipase solution in 25 mM sodium phosphate bufler was used. After immobilization, the biocatalyst was separated by filtration, rinsed with phosphate bufler (10 ml), and dried at vacuum for 10 min. In this study, recovered activity was defined as the ratio of enzymatic activity of the immobilized enzyme and the total units of soluble lipase that disappeared from the supernatant during immobilization [19]. Immobilization yield was defined as the difference between enz5mie activity in the supernatant before and after immobilization divided by the enzyme activity in the sup>ematant before immobilization [29]. 

Recovered activity and immobilization yield were calculated, and results are listed in Table 1. It can be observed that recovered activity increased when the initial enzyme concentration in the supernatant (Eq) was increased to 60 and 90 U/ml, but it remained almost constant for the other concentrations studied. Immobilization yield, on the other hand, decreased when high concentrations of enzyme were used (60 to 150 U/ml, compared to 30-40 U/ml). When Ef,=9Q U/ml, the highest value of recovered activity was obtained, which suggests that protein molecules are probably immobilized at close proximity to each other, which may prevent deactivation caused by enzyme unfolding by covering the support surface. In other words, when enzyme load was increased, more enzyme molecules were immobilized and less area of the support is available for lipase to spread itself, which may prevent loss in activity [31]. 

Nevertheless, when higher concentrations of lipase in the supernatant were used, Eo= 150 U/ml, recovered activity decreased, and immobilization yield was enhanced (Table 1). According to the literature [4, 6, 9, 10], protein adsorption is not restricted to a monolayer on the support, and adsorption of secondary layers has been reported. Therefore, when Eo= 150 U/ml, probably a second layer of lipase was adsorbed on the first layer, leading to an improvement on immobilization yield, as more enzyme molecules were adsorbed. However, although more molecules were immobilized on coconut fiber, not all of them... 

Table 1 Influence of enzyme concentration on recovered activity and immobilization yield. ...

Concentration of enzyme (U/ml) Recovered activity (%) Immobilization yield (%)... 

Immobilization parameters (recovered activity and immobilization yield) were calculated for the biocatalyst with higher hydrolytic activities (adsorptions performed between pH 3 and 6), and results are presented in Fig. 4. It ean be observed that best results for recovered activity and immobilization yield were obtained at pH 4 and at 5 or 6, respectively. 

Although the amount of C. antarctica lipase type B adsorbed to coconut fiber was nearly independent on the pH of adsorption (between pH 3 and 6), immobilization yield and recovered activity were dependent on the pH of adsorption because interactions between the molecule and its environment influence the structure of a protein molecule, and these interactions are pH-dependent [8],... 

Fig. 4 Effect of pH enzyme solution on the recovered activity (open square) immobilization yield (closed circle) of lipase immobilization on coconut fiber by adsorption after 2 h of contact time at room temperature...

Ui The enzymatic charge offered for immobilization in U/g of support, U t the enzymatic charge theoretically immobilized in U/g of support, R1 the immobilization yield, defined as (C/rr/Ui)x 100, U i the measured immobilized enzyme activity, in U/g of biocatalyst, RA the activity recovered in the immobilized enzyme, defined as (Uei/[/it)=< 100, Note I gelation/encapsulation=45 °C, ethanolic/acid medium, 155 min, aging=18 h at 4 °C, drying=suck dried by vacuum, followed by a 24-h resting period in a desiccator All the results of this immobilization are the averages of duplicates. 

Three resins (Amberlite XAD-4, Duolite A-568 et A-7) were tested, at different pH values [8], The results obtained indicate that pH 4 is the pH of choice to immobilize the B-glucosidase to Duolite A-568. The immobilization yields were calculated with this resin (table 2) and the results showed that the B-glucosidase of Candida molischiana 35M5N was immobilized very efficiently on Duolite A-568 resin. The immobilization was rapid (Ih) and the activity retained after immobilization is 82% of that of the original soluble enzyme. 

A covalent bond is established between the functional groups in the activated carrier and the functional groups in the amino acid residues of the enzyme, like —OH, —SH, —NH2, and —COOH. Covalent immobilization has been thoroughly studied and detailed information on methods and procedures can be found in several publications devoted to it (Zaborsky 1973 Cao 2005b Guisan 2006). It is a rather complex method where the carrier is hardly recoverable after enzyme exhaustion, immobilization yield is rather low and the kinetic properties of the enzyme can be severely altered. However, operational stability is high and it is quite flexible, so that directed immobilization can be done to suit the particular characteristics of the process. 

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