Transesterification activity

Fontes tt al. [224,225 addressed the acid—base effects of the zeolites on enzymes in nonaqueous media by looking at how these materials affected the catalytic activity of cross-linked subtilisin microcrystals in supercritical fluids (C02, ethane) and in polar and nonpolar organic solvents (acetonitrile, hexane) at controlled water activity (aw). They were interested in how immobilization of subtilisin on zeolite could affected its ionization state and hence their catalytic performances. Transesterification activity of substilisin supported on NaA zeolite is improved up to 10-fold and 100-fold when performed under low aw values in supercritical-C02 and supercritical-ethane respectively. The increase is also observed when increasing the amount of zeolite due not only to a dehydrating effect but also to a cation exchange process between the surface proton of the enzyme and the sodium ions of the zeolite. The resulting basic form of the enzyme enhances the catalytic activity. In organic solvent the activity was even more enhanced than in sc-hexane, 10-fold and 20-fold for acetonitrile and hexane, respectively, probably due to a difference in the solubility of the acid byproduct. 

Figure 1.12 Transesterification activity of PPL, CCL and MML in various organic solvents...

Khmelnitsky et al. were the first to observe the activating effects salt showed on enzymes in the nonaqueous environment [88]. As shown in Figure 3.7, the transesterification activity of the serine protease subtilisin Carlsberg in anhydrous solvents is strongly dependent on the KC1 content in a lyophilized enzyme preparation and increases sharply as the salt content is increased. This increase in activity was determined to be a result primarily of an increase in kcat and not a decrease in Km, as shown in (Table 3.4). 

The cutinase from Fusarium solani pisii maintained its transesterification activity in [BMIm][BF4], [OMIm][PF6] and [BMIm][PF6] (in order of increasing activity) at aw=0.2 [59]. Candida rugosa lipase (CrL), which is generally much less tolerant of anhydrous media than other microbial lipases, has successfully been used in anhydrous as well as water-saturated ionic liquids [60, 61, 62, 63, 64, 65]. 

Subtilisin generally shows poor transesterification activity in water-free ionic liquids, but Shah and Gupta [73] studied various approaches to maintaining its activity. The best results-up to 10000 times increase in the initial rate N-acetyl-phenylalanine ethyl ester in [BMIm] [PF6]-were obtained by precipitating and washing with 1-propanol. 

The lyophilization of enzymes from solutions containing salts or amphiphilic compounds is known to increase the activity in organic media by up to several orders of magnitude. Thus, the transesterification activity of a-chymotrypsin was increased 82-fold by co-lyophilization with pentaglyme [75]. The colyophilization of lipases and (poly ethylene)glycol (PEG) led to an enhanced transesterification activity in various ionic liquids [76, 77]. 

L. Ferreira, M. A. Ramos, M. H. Gil, and J. S. Dordick, Exquisite regioselectivity and increased transesterification activity of an immobilized Bacillus subtilis protease, Biotechnol. Prog. (2002) 986-993. 

It is generally stated that biocatalysis in organic solvents refers to those systems in which the enzymes are suspended (or, sometimes, dissolved) in neat organic solvents in the presence of enough aqueous buffer (less than 5%) to ensure enzymatic activity. However, in the case of hydrolases water is also a substrate and it might be critical to find the water activity (a ) value to which the synthetic reaction (e.g. ester formation) can be optimized. Vahvety et al. [5] found that, in some cases, the activity of Candida rugosa lipase immobihzed on different supports showed the same activity profile versus o but a different absolute rate. With hpase from Burkholderia cepacia (lipase BC), previously known as lipase from Pseudomonas cepacia, and Candida antarctica lipase B (CALB) it was found that the enzyme activity profile versus o and even more the specific activity were dependent on the way the enzyme was freeze dried or immobihzed [6, 7]. A comparison of the transesterification activity of different forms of hpase BC or CALB can be observed in Tables 5.1 and 5.2, respectively. 

It can be noted that the way in which the enzyme is prepared in the dry form for catalysis in organic solvent is responsible for striking differences (up to two orders of magnitude) in the enzyme-specific activity. Furthermore, it is worth mentioning that the transesterification activity of lipase from B. cepacia entrapped in sol gel (sol gel-AK-lipase BC) was 83% of the activity in water measured using tributyrin as a substrate [6]. Analogously, in the case of CALB lyophilized with methoxypoly(ethylene glycol) (CALB -i- PEG) the activity was 51% of the activity in water in the hydrolysis of vinyl acetate [7]. It is important to note that, for both... 

Table 5.1 Transesterification activity in carbon tetrachloride (Ow < 0.1) of the various lipase BC formulations.

Figure 5.1 Transesterification activity of (a) 0.025 or (b) 0.3 mg of lipase BC co-lyophilized with different amounts of sucrose ( ), trehalose (x) and mannitol ( ). Activity was tested in toluene by measuring the rate of 1-...

The storage stability of other lipases has been also analysed. For instance, the storage stability of PsL in hydrophobic ILs for a period of 20 days at room temperature, measured with the variation of the transesteriiication activity of this enzyme during transesterification of ethyl 3-phenylpropanoate with different alcohols, resulted in an increased yield of 62-98% in [bmim fNTfj ] and 45-98% in [bmim ] [PFg ], respectively, depending on the nature of alcohol used in the tiansesterifica-tion reaction. In these ionic liquids, the operational stability was also measured and found that the P.yL-IL mixture was recycled live times without any decrease in the transesterification activity [13]. 

Transesterification. Activation of 2,2,2-trihaloethyl esters by phosphines enables exchange of the alkoxy residues. 

Hsu AF, Jones KC, Foglia TA et al. (2004) Transesterification activity of lipases immobilized in a phyllosilicate sol-gel matrix. Biotechnol Lett 26 917-921 Hung TC, Giridhar R, Chiou SH et al. (2003) Binary immobilization of Candida rugosa lipase on chitosan. J Mol Catal B Enzym 26 69-78... 

The incubation of enzymes in ionic liquids can also lead to increased reactivity. Sheldon showed that CaLB could be heated as a suspension in [bmim][PF6] before use. Higher transesterification activity was observed than for the untreated enzyme, both when the enzyme was free (SP 525, 120% activity after 20 h incubation)... 

Water has also an effect on the polarity of the active site of enzymes, which has an effect on the kinetics of reactions, as will be shown later. Affleck and coworkers have measured the mobility of a spin label in the active site of subtilisin Carlsberg by electron spin resonance (28). They observed that the transesterification activity augments as the polarity increases, following addition of water. However, it is difficult to separate the effect of water on the fiexibility of the enzyme and on the polarity of the active site. 

Function 2 concerns the part of the titanate molecule immediately adjacent to the titanium center and that affects the specific effect, the resistance to hydrolysis, the transesterification activity, and the thermal stabiUty of titanates. This part of the molecule can be represented by the following groups ... 

Figure 9.16 Effect of the Amount of Grafted Triethylamine on the Transesterification Activity, Expressed in Terms of Glyceryl Tributyrate Conversion, on the Nitrogen-Grafted CNT Catalysts.

Mg-Al hydrotalcite catalysts were used for the transesterification of rape-seed oil with oil to methanol ratio of 1 6, at a temperature of 65° C with 1.5% catalyst in reaction time of 4 h. It was found that calcined hydrotalcite with Mg/Al molar ratio of 3.0 showed highest transesterification activity with a conversion of 90.5% and the catalyst could be separated easily [61]. Similar types of catalysts were employed for the transesterification of soybean oil with methanol. Oil to methanol ratio of 1 15 at reflux temperature for 9 h with 7.5% catalyst yielded 67% of biodiesel [62]. In a similar study for the transesterification of canola oil with methanol using Mg-Al hydrotalcite catalyst prepared by coprecipitation method, 71.9% conversion was observed. The conditions followed were 1 6 ratio of oil to MeOH 60° C for 9 h with 3% catalyst [63]. Cerium modified Mg-Al hydrotalcites were used for the transesterification of soybean oil with methanol, fith a molar ratio of 1 9 of oil to methanol, at a temperature of 67° C and 5% of catalyst and reaction time of 3 h, a biodiesel yield of 90% was obtained. It was observed that catalysts with Ce/Mg atomic ratio of >0.3 was able to produce biodiesel of 90% yield [64]. Potassium loaded-calcined Mg-Al hydrotalcite was used as a base catalyst for the transesterification of palm oil with methanol. Biodiesel yield of 86.8% was achieved with 7% catalyst at 100° C for 6 h using 1 30 molar ratios of oil to methanol. The blend with prepared biodiesel was tested for its impact on elastomer properties [65],... 

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