Biocatalyst activity

The limitations preventing commercialization are mainly related to biocatalyst activity and specificity. Currently, increasing rate with respect to the biocatalyst is the main objective of the biocatalytic refining processes being developed. New applications of by-products could contribute to improving economic parameters. 

Apart from biocatalyst activity, several other parameters are important in development of a biodesulfurization process. These parameters include oil/water ratio, composition of aqueous phase used for biocatalyst suspension during desulfurization, biocatalyst loading, oil/water separation following completion of desulfurization, potential for biocatalyst recycle, recycle of aqueous phase to reduce fresh water usage and wastewater minimization, as well as secondary oil separation and purification operations. 

An important parameter that has to be considered during desulfurization as well as for subsequent biocatalyst separation and recycle is the impact of the oil phase on the biocatalyst activity and half-life. Additionally, the effect of the biocatalyst on forma-tion/breakage of the oil-water emulsions is also important. The latter will be discussed in Section 2.3.3. It becomes important for lower boiling feedstocks such as gasoline, which offers the most toxic solvent environment for the biodesulfurization catalyst. The effect of solvents on biocatalysts has been investigated in very few reports. A study by the Monot group reported effect of two solvents on several Rhodococcus strains [254], The strains contacted with the solvents and their desulfurization activity, growth, and... 

Improving functionality may involve a complex biocatalytic system including more than one biocatalyst, as it is the case in BDM reactions. Additionally to the BDS for instance, another biocatalyst active for BDM [395,406], which consists of a heme oxygenase or a Cytochrome reductase could be used to widen up the functionality. 

US5733773 [46] desulfurization of fossil fuel with flavoprotein. comprising DNA which encodes a flavoprotein and DNA of Rhodococcal origin which encodes a protein biocatalyst active for BDS. The DNA biomolecule is not a Rhodococcus genome. 

Blackwood, A.D., Curran, L.J., Moore, B.D. and Hailing, P.J. (1994) Organic phase buffers control biocatalyst activity independent of initial aqueous pH. Biochim. Biophys. Acta, 1206, 161-165. 

Functionalisation. Finally, microencapsulation may be used to develop new functions such as regulating biocatalyst activity by controlling the membrane permeability through pH changes. Microcapsules may also offer a marketing function such as giving specific metallic aspects to functional food to differentiate them from food and medication. 

To facilitate the identification of the best available biocatalysts for a specific application, a database of empirically determined substrate specificities should be consulted. The construction of such a database, which contains critical features such as operational parameters for biocatalyst utilization as well as substrate specificity, has recently been addressed.127 Coupled with a fundamental understanding of the kinetic and thermodynamic factors that affect biocatalyst activity, such databases are indispensable in the identification of a suitable biocatalyst and reasonable reaction conditions. 

An enzyme stability test in SC-CO2 was performed at different pressures. CALB was incubated overnight at certain conditions and afterwards its residual activity was determined. No direct influence of the pressure on its biocatalyst activity was found. 

The different properties of ILs, with regard to their polarity, hydrophobicity, and solvent miscibility behavior through combination with different anions, are the reason for the different biocatalyst activities. Good to excellent activity of CALB was observed with a decrease in polarity and hydrophobicity and a viscosity increase of the ILs. In [bmim][PF6] a conversion of (R)-l-phenylethanol into the ester of 48.9% and an ee of 95.6% were achieved after 5h and 100% of (R)-l-phenylethanol was converted into the enantiopure (R)-l-phenylethyl acetate after a 1-day reaction. Immobilized CALB exhibited excellent stability, activity, and selectivity towards the (R)-enantiomer of 1-phenylethanol in [bmim][PF6]. In some research bis(trifluoromethylsulfonyl)imide-based ILs have been regarded as very suitable media for biocatalysis [39, 46, 50]. On the contrary, in the present work, lower suitability of the same IL was demonstrated. Since immobilized CALB catalyzed both hydrolytic and transesterification reactions, its enantioselectivity for long reaction times was lower. 

It is worth noting that a low buffer concentration also resulted in higher activity with both supports [56]. The highest activity of the biocatalyst (15.2 U/g) was achieved with Sepabeads EC-EP5, using 0.3 M sodium carbonate (pH 9.0). With Sepabeads EC-EP3, a shghtly acidic pH (5.5) and low buffer concentration (0.3 M) yielded the highest biocatalyst activity (4.2 U/g). The incubation time was also analyzed a further increase from 24 to 72 h did not result in a significant improvement in biocatalyst activity. 

Although fructosyl-transfer enzymes have been immobilized by different techniques such as adsorption [71], entrapment [72], or covalent attachment [73], the biocatalyst activity per mass unit is not commonly reported. However, Chiang et al. [74] described a maximum activity of 77 U/g for A. niger 5-fructofuranosidase covalently attached to methacrylamide-based polymeric beads. More recently, fruc-tosyltransferase from A. aculeatus present in Pectinex Ultra SP-L was covalently bound to Eupergjt C [75] although the activity recovery was high, the activity per gram of biocatalyst was not reported. 

Another type of stability of immobilized biocatalysts is the retention of activity after periodic use in batch processes, as has been reported previously for penicillin acylase entrapped in polyacrylamide gel [40]. This option can be used to advantage for rapid monitoring of biocatalyst activity under conditions of industrial application. Apart from the measurement of activity as an indication of the necessity to replace the biocatalyst, the periodic analysis of the variation of kinetic properties permits greater insight into deviation from the optimal parameters. 

In spite of the clear advantages, membrane-coupled processes often turn out to be still more expensive, because of the costs of membrane and all additional hardware associated with a membrane operation. Membrane separations tend to become more favorable for processes where the selectivity is more important than the conversion, because it replaces other purification steps that might lower such selectivities, like in the production of the chiral diltiazem intermediate. Membrane fouling, mass transfer limitations, biocatalyst activity loss, and biocatalyst denaturation are other potential disadvantages related to it. 

Li et al. [152] fabricated electrospun PAN fiber mats immobilized with C. rugosa lipase by amidination. Enzyme molecules were covalently bound to the fiber mats and formed small protein aggregates. The immobilized lipase on the electrospun PAN fiber mats showed a good biocatalystic activity for soybean oil... 

As was described above in a number of MBR processes the membrane, in addition to performing the separation functions previously discussed, also acts as a host for the biocatalysts (whole cells or enzymes) which are immobilized in the membrane s pore structure. Concerns with such MBR configurations include membrane biofouling, mass transport limitations and biocatalyst activity loss and denaturation. In the two sections that follow we discuss further some of the key aspects of MBR for biochemical synthesis. We classify these reactors into two types, namely whole-cell and enzymatic MBR. 

Though MBR offer advantages over the more conventional bioreactors, they, themselves, are not completely free of problems. One such key problem, as previously noted, relates to changes in biocatalyst activity. This is a serious concern for whole-cell MBR, when the cells are immobilized in the membrane s pore structure. Diffusional limitations for nutri-... 

Thus, in order to produce a low-cost immobilized lipase, the objective of this work is to evaluate the immobilization of CALB by adsorption on green coconut fiber. For this purpose, the influence of contact time, lipase concentration, and pH of lipase solution during the adsorption step on the biocatalyst activity and stability was investigated. 

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