CALB Immobilization

CALB was immobilized on different hydrophobic support materials by physical adsorption. Table 3.1 shows the physical properties of the support materials. 

Carrier Material Particle Size (pm) Pore size or void volume Bulk Density (Kg/m3) 

1 Lewatit Poly methyl methacrylate copolymer 315-1000 14-17nm 650-800 

4 Accurel pellet MP100 Polypropylene 3000x3000 800-2 500nm 78 2% 115-145 

The support materials were vacuum dried for 24 hours. CALB enzyme solution was diluted in water. To the support material enough diluted enzyme was added to reach 55kLU g 1 (activity units per g carrier). The buffer used for immobilization was a 1.25 M potassium phosphate buffer pH = 8. The slurry was stirred for 20 hours using a mechanical laboratory overhead stirrer. The immobilization liquor was filter out using vacuum and the particles were rinsed two times with water. The immobilized enzyme was finally vacuum dried at 45 °C for 30 hours. The immobilized enzymes were used to synthesize PCL. The molecular weight of the synthesized polymer and PLU g 1 values were determining factors in selecting the best support material. 

---

Chemoenzymatic polymerizations have the potential to further increase macro-molecular complexity by overcoming these limitations. Their combination with other polymerization techniques can give access to such structures. Depending on the mutual compatibility, multistep reactions as well as cascade reactions have been reported for the synthesis of polymer architectures and will be reviewed in the first part of this article. A unique feature of enzymes is their selectivity, such as regio-, chemo-, and in particular enantioselectivity. This offers oppormnities to synthesize novel chiral polymers and polymer architectures when combined with chemical catalysis. This will be discussed in the second part of this article. Generally, we will focus on the developments of the last 5-8 years. Unless otherwise noted, the term enzyme or lipase in this chapter refers to Candida antarctica Lipase B (CALB) or Novozym 435 (CALB immobilized on macroporous resin). 

We subsequently found that CaLB could be used for both steps (Fig. 9.22). That a lipase was able to effectively catalyze an amide hydrolysis was an unexpected and pleasantly surprising result. Good results were obtained with CaLB immobilized as a CLEA (see later). 

A CLEA prepared from CaLB was recently shown to be an effective catalyst for the resolution of 1-phenylethanol and 1-tetralol in supercritical carbon dioxide in continuous operation [47]. Results were superior to those obtained with Nov 435 (CaLB immobilized on a macroporous acrylic resin) under the same... 

Table 2.2 Recycling of Novozym 435 (CALB immobilized on a hydrophobic carrier) in the kinetic resolution of 1c in toluene at 60°C.

Scheme 2.3 CALB immobilized on Celite R-633 catalyzes the enantioselective synthesis of aromatic cyanohydrins via a DKR.

In order to optimize and/or improve the behaviour of bioprocesses in ionic liquids/supercritical carbon dioxide biphasic systems, a better understanding of the chemical reaction and mass-transfer phenomena in these biphasic systans is required. In this context, de los Rfos et al. [38] analysed the chemical reaction and the mass-transfer phenomena in the synthesis of bntyl propionate from vinyl propionate and 1-butanol catalysed by CaLB immobilized on dynamic monbranes at... 

Abstract An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t /2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction. 

CALB immobilized by adsoiption on coconut fiber at pH 7 and room temperature... 

By comparing the remaining activity of the intermediate state , expressed by tt, for the two biocatalysts obtained at pH 7.0 and different initial enzyme activities in the supernatant, it is possible to evaluate the formation of lipase multilayers during adsorption. The intermediate of CALB-7A, CALB immobilized at pH 7 using Ea= 40 U/ml, has higher activity (ai=59.2%) than the intermediate of CALB-7B (oi = 14.3%), CALB immobilized at pH 7 using 0=280 U/ml, probably because of the formation of multilayers on CALB-7B. Due to this enzyme aggregation, lipase is weakly linked, and it is quickly deactivated [10], As it can be seen, CALB-7A is 4.6-fold more stable than CALB-7B. 

Table 2 Kinetics parameters of thermal deactivation, at 50 and 60 °C, of soluble CALB, CALB immobilized by adsorption on coconut fiber and Novozyme 435. ...

CALB-7A CALB immobilized by adsorption on coconut fiber at pH 7, using b=40 U/ml CALB-7B CALB immobilized by adsorption on coconut fiber at pH 7, by using o=280 U/ml CALB-4A CALB immobilized by adsorption on coconut fiber at pH 4, by using E(,=40 U/ml CALB-5A CALB immobilized by adsorption on coconut fiber at pH 5, by using o=40 U/ml. 

Based on the results obtained so far, CALB-7A, CALB immobilized on coconut fiber after 2 h of contact between the support and an enzyme solution containing Eo=40 U/ml, in... 

Thermal stabilities of CALB immobilized on coconut fiber were investigated at 50 and 60 °C, and results were compared to the stability of soluble CALB and Novozyme 435 (Fig. 5 and Table 2). It can be observed, by analyzing stabilization factors F) on Table 2, that immobilization of CALB on coconut fiber promoted an improvement on thermal stabilities, as CALB-7A is 2- and 92-fold more stable than soluble CALB at 50 and 60 °C, respectively. Other authors [19] obtained similar results when immobilizing the same enzyme on activated carbon by adsorption immobilized CALB was 2-fold more stable than soluble enzyme with ti/2=8 h to thermal stability studies at 50 °C. 

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. 

Table 1. Candida antartica Lipase B (CALB) immobilization on MM A resins of differing particle size enzyme loading, fraction of active lipase,...

The activity of CALB immobilized on resins 1 to 4 (see Table 1) was assessed by s-CL ring-opening polymerizations. All immobilized resins have similar CALB loading (5.1 to 5.7 %-by-wt) and water content (1.4 to 1.9%). As shown previously , different chemical shifts are observed for methylene protons (-OC//2-) of e-CL monomer, PCL internal repeat units and chain terminal -012-OH moities. Hence, by in situ NMR monitoring, monomer conversion and polymer number average molecular weight (A/ ) were determined. 

As Figure 3 illustrates, the polymerization rate strongly depends on the matrix particle size used for CALB immobilization. For example, at 80 minutes reaction time, as the particle size decreased from 560-710, 120, 75 and 35 pm, 8-CL %-conversion increased from 20 to 36,42 and 61%, respectively. 

A series of close-to-spherical styrene/DVB resins of varying particle size and pore diameter were employed as supports for non-covalent adsorptive attachment of CALB by hydrophobic interaction. The effect of matrix particle and pore size on CALB i) adsorption isotherms, ii) fraction of active sites, iii) distribution within supports, and iv) catalytic activity for s-CL ring-opening polymerizations and adipic acid/l,8-octanediol polycondensations is reported. Important differences in the above for CALB immobilized on methyl methacrylate and styrene/DVB resins were found. The lessons learned herein provide a basis to others that seek to design optimal immobilized enzyme catalysts for low molar mass and polymerization reactions. 

The activity of CALB immobilized on resins 1 to 5 (see Table 1) was assessed by s-CL ring-opening polymerizations. All immobilized resins have... 

Figure 3 illustrates that the polymerization rate is independent of resin diameter. During 30 min reactions, CALB immobilized on resins 1 to 4 gives turnover frequency (TOF) of e-CL of about 12 s" In contrast, our previous work of CALB immobilized on PMMA resins showed a large dependence of e-CL %-conversion on resin particle diameter. For example, in 30 minutes reaction time, as the particle size decreased from 560-710, 120, 75 and 35 pm, turnover frequency (TOF) of e-CL increased from 3.8 to 5.3, 7.5 and 11.2, respectively. However, by increasing the resin pore size from 300 (resin 4) to 1000 A (resin 5) for 35 pm beads, the TOF reached 28.2 s. As discussed above, increase in resin pore diameter also corresponds to an increase in %-area of beads at which CALB is found (37 to 88%). 

From the above results on CALB activity as a function of particle size for polystyrene and PMMA resins, we believe %-surface area occupied by CALB is a critical factor that can be used to improve immobilized CALB activity. Increased %-accessible surface area will increase the probability of collisions between substrates and CALB. As %-accessible surface area for CALB increased for PMMA resins a corresponding increase in polyester synthesis reaction rates was observed (see above). CALB immobilized on styrenic particles of variable size showed little differences in both %-accessible surface area and polyester synthesis catalyst activity. The potential benefit of decreasing bead particle size is to decrease diffusion constraints that lead to productive collisions between enzyme and substrate. However, for polystyrene resins, as particle size decreased, the percent of resin area in which reactions can occur does not change. In contrast, decreasing PMMA particle size dramatically... 

Among the various lactones, s-CL is the most extensively studied monomer. The ROP was induced by many lipases from different origin. Candida antarctica [lipase CA or CA lipase B (CALB) immobilized on an acrylic resin, produced commercially as Novozym 435] was found to be the most effective lipase for the ROP of e-CL. 

The 50% maximal theoretical yield of KR processes may be overruled by DKR to achieve 100% of the desired product if a fast racemization process for the substrate can be coupled to the KR in situ. A mixture of CaLB immobilized onto IL-coated particles and an acidic zeolite are allowed to carry out DKR in a one-pot under scCOj flow conditions (50 C, lOMPa) [133]. In this way, acylation of rac-23i with vinyl propionate in a DKR gave the propionate of (R)-23i in 92% yield and >99.9% ee. 

When the l-decyl-2-methyl-3-butyl imidazolium cation covalently attached onto the resin surface was used as the support for CALB immobilization, it could function as an active catalyst for methyl oleate synthesis [126], which is a typical biodiesel, by the methanolysis of triolein using tot-butanol as cosolvent with 95% yield. Further on, this catalyst was stable enough to be reused in scCOj using tert-butanol as cosolvent. The yield of methyl oleate was maintained at 85% after 45 cycles. The presence of tert-butanol as cosolvent was crucial to avoid deactivation caused by glycerol, which was the byproduct of biodiesel synthesis, through the blockage of the active sites. 

---