Solubilised enzymes

The scope and limitations of biocatalysis in non-conventional media are described. First, different kinds of non-conventional reaction media, such as organic solvents, supercritical fluids, gaseous media and solvent-free systems, are treated. Second, enzyme preparations suitable for use in these media are described. In several cases the enzyme is present as a solid phase but there are methods to solubilise enzymes in non-conventional media, as well. Third, important reaction parameters for biocatalysis in non-conventional media are discussed. The water content is of large importance in all non-conventional systems. The effects of the reaction medinm on enzyme activity, stabihty and on reaction yield are described. Finally, a few applications are briefly presented. 

Solubilised enzyme preparations are well suited for many ftmdamental studies, for example spectroscopic investigations reqtriring transparent solutiorrs. When the solubilised preparatiorrs are used as catalysts it is an advantage that mass trarrsfer limitations are normally absent, but product isolation and ertzyme recovery are usirally more difficult than with solid enzyme preparations. Methods used to separate the enzyme from the product solution include precipitation of the enzyme, and the use of membranes which retain the enzyme but not the product. 

Heifetz, A. Elbein, A.D. (1977) Biosynthesis of Man-GlcNAc-GlcNac-pyrophosphoryl-polyprenol by a Solubilised Enzyme from Aorta , Biochemical and Biophysical Research Communications, 75, 20-8... 

D-Lactate dehydrogenase (solubilised enzyme) formate dehydrogenase NH2 HCOj /NAD [ 1 , /v C02H — ACE inhibitors OH... 

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

The solubilisation of soy cell wall material (CWM) by the two rhamnogalacturonases (RGase A and RGase B) in combination with other pectinolytic enzymes were compared in order to identify enzymes for new soy processes or products. The experiments were carried out at pH 5.0, where both rhamnogalacturonases have about 25% of their maximum activity, and with high enzyme dosages to ensure that the maximum effects are obtained. 

As seen on Table IV RGase A is only able to solubilize 7% of the CWM, whereas addition of RGAE and galactanase increases the solubilisation to 17% and 44% respectively. The solubilisation of the CWM by RGase B is less dependent on the presence of the other pectino-lytic enzymes, as 37% are solubilized with RGase B alone. 

When the apple tissues were treated with enzyme preparation for liquefaction (Fig. 3), the cell-wall materials were solubilised with different yields, 95, 86, 66 and 59 % for zones B, C, D and A, respectively. The sequence was the same with the maceration treatment (use of polygalacturonase [PG] only) but the yields were lower. 

AE catalyses the cleavage of acetyl groups from different substrates. The enzyme activity was determined by measuring the release of acetic acid. The amount of acetic acid was measured spectrophotometrically using an acetic acid analysis kit (Boehringer, Mannheim). The activity of AE was measured in 0.6% sugar beet pectin solubilised in 25 mM Na-succinate pH 6.2 and incubated with enzyme fraction in total 500 nl assay. The samples were incubated at 40°C and aliquots were examined after 0, 1, 2 and 3 hours of incubation. The enzyme reaction was stopped by incubating the samples at lOO C for 5 min. Precipitated... 

The European Economic Community have shown considerable interest in releasing ferulic acid from low value agricultural waste residues such as SBP, with subsequent bioconversion of the free acid to vanillin. Previous studies with commercial enzyme preparations have shown a high degree of solubilisation of ferulic acid from SBP is possible, giving rise to a mixture of free and esterified forms [9,10], However, it is not known which... 

Fish. Initial reports to determine NP in fish referred to the non-specific analysis of 14C-labeled compounds administered in exposure experiments. Granmo and Kollberg [7] used a solubiliser employing pancreatic enzymes to extract 14C-labeled NP from exposed cod and analysed it by liquid scintillation counting. 

Immune complexes are solubilised in 50 xl of 2X Laem mli buffer, boiled and subjected to SDS-polyacrylamide gel electrophoresis or washed once with the enzyme assay buffer and used for enzymatic assays. 

The most often investigated enzymes in micro emulsions are lipases, because these enzymes are very stable and active in this medium [ 14]. Until now, most of the relevant interactions between the biomolecules and the reaction medium have been investigated. Many enzymes which are well investigated in aqueous reaction media can be solubilised in w/o-microemulsion, retaining their activity and stability, as shown in Table 1. 

In order to obtain a thermodynamically stable micro emulsion, the analysis of the phase behaviour is indispensable. With bovine serum albumin instead of an enzyme (because of the cost of the bio-catalyst) phase behaviour studies are shown in Fig. 2. A strong shift of the phase boundary is observed, yielding a system that solubilises much less water in the presence of the protein. In case of hydrophobic enzymes, the addition of dry lyophilised protein to an already prepared reverse micellar solution can also work well [53]. 

An auspicious new strategy, in order to perform biocatalysis with hydrophobic substrates in w/o-microemulsion, is the usage of whole cells instead of purified enzymes [3,124,141]. There exist only a few surfactant-oil systems, in which whole cells are stable and suitable for a segmentation. Mainly the biodegradable surfactant based on sorbitan (Tween and Span) seems to be well suited for the solubilisation of whole cells in organic reaction media [142,143]. 

Another important advantage with many non-conventional media is that they can solubilise hydrophobic compounds which are poorly soluble in water. Thereby the conversion of these important substrates is facilitated. Further advantages are that the risk of microbial contamination is much lower in non-conventional media, and under optimized conditions the stability of enzymes is often higher than in aqueous solutions. 

Figure 9.2 A schematic presentation of different types of enzyme preparations used in non conventional media, a enzyme powder, b enzyme crystals c enzyme on a porous support d covalently modified enzyme dissolved in the solvent e enzyme solubilised by surfactant ...

The methods described below have been used for enzyme solubilisation in organic media but they should be applicable to supercritical media and solvent-free systems, as well. 

Surfactants have been used to solubilise lipases in organic solvents (Okahata and Mori, 1997). One method starts with mixing aqueous solutions of the surfactant and the enzyme. The enzyme-surfactant complex precipitates and can subsequently be dissolved in organic media. Several surfactants have been tested and especially good results have been obtained with dialkyl glucosyl glutamates. In one case it was shown that the complex consisted of one enzyme molecule surrounded by approximately 150 surfactant molecules. 

The performance of immobilized biocatalyst (enzyme) reactors is influenced by enzyme inactivation during operation, mainly due to thermal denaturation, desorption of the biocatalyst from the solid support, disintegration or solubilisation of the support and microbial attack. 

Forget, P. (1971). Les nitrate-reductases bacteriennes. Solubilisation, purification, et properties de I enzyme A de Micrococcus denicrificans. Eur. ]. Biochem. 18, 442-450. 

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