A Hydrophilic and Hydrophobic Organic Solvent Mixture
Enhance Enzyme Stability in Organic Media
Pepito A. Comeque
Clinical Biochemistry / CLS 301
16 JAN 2014
Dr. Jared Thomas Rutledge A Hydrophilic and Hydrophobic Organic Solvent Mixture
Enhance Enzyme Stability in Organic Media The enzymatic catalysis in organic solvents present many potential reaction that are impossible in aqueous solution, including chiral synthesis and resolution, the alteration of fats and oils and the creation of biodegradable polymers. The altered selectivities, pH memory, and regio-, enantio- and stereoselectivity can be some of the advantages for using enzymes in non aqueous media. Numerous solvents have been utilized to try to reduce the influence of solvents on enzymes, such as the polarity, solvating ability, molecular geometry and hydrophobicity of the solvent. Some theories based on diffusional limitations, protein conformational change, and thermodynamics have also been suggested to describe protein-solvent relation and to raise the function of enzymes for synthetic organic chemists. From the most methodical study of solvent effects, it’s still not clear how the nature of solvent affect the stability of a protein. In most instances, hydrophobic solvents cause less inactivation of biocatalysts. Since most solvent seems to be hydrophilic and given that solvents with P values (between 0 and 2) can inert enzymes. According to Choi and Yoo (2012) “an agreement is required to take into consideration both enzyme activity and enzyme stability. The stability of the enzyme in particular should be considered as one of the most important factors for industrial applications” (p. 1132). A combination of hydrophilic and hydrophobic solvents can be used for determining enzyme with the management of enzyme stability in organic solvents. Aqueous organic solvent mixtures have been used solely to enhance the solubility and selectivity of substrates. This experiment explored enzyme stability and activity in hydrophilic/ hydrophobic solvent mixtures using subtilisin Carlsberg and horseradish peroxidase (HRP) as model enzymes.

The initial rate of N-Acetyl-L phenylalanine (NAPEE) transesterification by subtilisin Carlsberg was measured using GC with a HP5 silica gel capillary column (30 m) and flame ionization detector. The temperature of the injector and the detector was 275 C, and the program was isothermal for 15 min at 200 C. A total of 5 mg prepared subtilisin Carlsbe\rg was suspended in 10 ml organic solvent amended with 20 mM NAPEE, and the mixture was sonicated to disperse the enzyme particles for 20 s. The enzyme reactions were initiated by adding 0.1 M 1-propanol at 25 C. Samples were collected at predetermined intervals. The FID response was calibrated using a standard solution of N-acetyl-L-phenylalanine propyl ester. The activity of HRP was measured at 25C using pyrogallol and H2O2 as substrates (Kim et al.2000). Typical tests were performed by adding 100l l HRP solution to 2.9 ml 20 mM sodium phosphate buffer, pH 7.0, containing 0.3% H2O2 (w/w) and 0.3% pyrogallol (w/v). The reaction product was monitored at 420 nm. One unit of HRP activity was defined as the formation of 1 mg purpurogallin from pyrogallol in 20 s at pH 7.0 and 25 degree Celsius. For examining the effects of organic solvent mixtures on activity and stability, the enzyme powder was dissolved in the reaction buffer, and the enzyme solution was added to 50 ml Falcon tubes with caps. Each sample vial was lyophilize and suspended in various ratios of anhydrous solvents. Next, the vials were placed in an incubator at 25 degree cd, and the enzyme reactions were conducted after incubation at various time points. A model solvent system was initially chosen for investigating the effects of mixing hydrophilic and hydrophobic solvents on enzyme activity and stability in organic solvent systems. The initial rates of NAPEE propanolysis by subtilisin Carlsberg in different hydrophilic organic solvents containing 1% (v/v) water have been reported, and the catalytic activity of transesterification in acetone was higher than in other hydrophilic solvents (Wu et al. 2009).Because dioxane has no absorbance peak between 200 and 500 nm, it is possible to analyze enzyme structure by spectrometric methods in this solvent. Thus, dioxane was also selected as a model hydrophilic solvent, although the catalytic activity of subtilisin Carlsberg was relatively low in this solvent. In addition, dodecane (log P= 6.6) was selected as a model hydrophobic and water-immiscible solvent. Most of the hydrophobic solvents tested (log >P4) could not dissolve NAPEE well, and the reactivity was unde-tectable (data not shown

References
Choi, Y. S., & Yoo, Y. J. (2012). A hydrophilic and hydrophobic organic solvent mixture enhances enzyme stability in organic media. Biotechnology Letters, 34(6), 1131-5. doi:http://dx.doi.org/10.1007/s10529-012-0886-7