생촉매제로서의 격자화 효소 결정화:

단백질 분해효소와 유기용매에 대한 알콜탈수소 효소의 안정도

김계심, 이강민

전북대학교 분자생물학과

Cross-Linking Enzyme Crystals as Biocatalysts:

Stability of Horse Liver Alcohol Dehydrogenases

against Proteinase and Organic Solvent

Kye-Shim Kim, Kang-Min Lee

Department of Mol. Biology, University of Chunbuk, Chonju, 561-756, Korea

INTRODUCTION

The benefits of using  enzymes  in   organic synthesis and biosensor are generally well understood. The high selectivity and mild reaction  conditions  associated  with  enzymes have made  them very attractive for the synthesis of biologically active compounds, which are often difficult to be obtained  by chemical methods. Despite of the enormous potential of enzymatic catalysis, only a tiny portion of enzymes is used in the synthesis on industry such as the preparation of natural products, pharmaceuticals, fine  chemicals and food ingredients.

Although significant improvements have been achieved through the use of enzymes in microheterogeneous systems such as organic media(1), their applications in enzyme technology  were limited.  The process  may cause low activity of enzymes and instability in reaction media. Thus, a variety of methods(2) to stabilize enzymes have been developed. Recently, a new technique was introduced, the use of perfectly, to our knowledge, stable  cross linked enzyme crystals(3). We will show that enzyme CLEC has advantages as a biocatalyst for enzyme technology.

MATERIALS AND METHODS

General. □-Nicotinamide adenine dinucleotide(□-NAD) from yeast was purchased from Boehringer of 100% purity. E-cinnamaldehyde of 99% purity was from Aldrich. Other reagents were all of the highest quality available and were used without purification.

Enzyme Preparation.  Horse liver alcohol dehydrogenase  was obtained as a crystalline suspension from Boehringer. The crystals were centrifuged at 6000,8000 rpm, 4℃ and dissolved in 0.2M sodium chloride, 50mM TES buffer, pH 7.5. The enzyme solution was extensively dialyzed against 50mM TES buffer, pH 7.0, at 4℃ for overnight. The enzyme concentration was determined spectrophotometrically at 280□ using a 0.455 optical density for a 1□/□ enzyme solution. Enzyme manipulations were carried out at 4℃ and after crystal insolubilization at 20℃.

HPLC Method. High performance liquid chromatography on Tosho 8010 apparatus equipped with a C-18 column (Merck). The products were eluted with acetonitrile-water 1:1 mixture(v/v) at 0.5□/min and detected by UV absorption at 260□ . We crystallized the enzyme and prepared CLEC of HLADH by the method of Lee(4).

Manipulation of CLECs.  Crosslinked enzyme crystals(CLECs) were fully washed with buffer solution prior to use. The buffer above the CLECs was gently decanted or removed by suction. The washing process was done on the Amicon Centricon for next experiments.

Activity of HLADH-CLEC, against organic solvents. We studied the effect of acetonitrile and DMSO on the activity of CLECs. Organic solvent effect on CLEC was conducted in presence of increasing volume of organic solvent from 0 to 40%(v/v). As a comparision solvents effects on enzyme in solution was studied in same contents of organic solvents. We determined enzyme activity in solution of 50mM TES buffer, pH 7.5 in the presence of 10mM NAD+, 4M ethanol.

Stability test of HLADH-CLEC against proteases.  Three endo/ exogenous proteases were products of  Sigma, □-chymotrypsin from bovine pancrease, Pronase E from streptomyces griseus, Proteinase K from Tritirachium album. The protease solution were prepared (2□/□, in the case of Proteinase K, 4□/□). Optimum pH of them were 7.0-9.0, 6.0-7.5, 7.5-10.5, respectively. For degradation test, HLADH was prepared two types, solution form and CLEC form. The proteases to HLADH ratio was 1:5 and the reaction times were each 0, 6, 12, 24 hours on Amicon Centricon sized 10K(for solution from), 100K(for CLEC form). After cetrifugation at given rpm on guidline text, the filtered solutions were assayed by modified Lowry.

RESULT and DISCUSSION

Crytallization and cross-linking of horse liver alcohol dehydrogenase were referred to the preceding investigation by Lee.(4) Alcohol dehydrogenase in CLEC state was stronger than enzyme in solution. at 10% organic solvent, more than 50% of activity was lost in solution but almost of activity was retained in CLEC.(Fig. 1) CLEC was a little more resistant to DMSO than acetonitrile.  When concentration of ethanol was calculated more than 60% of activity was found in medium containing 65% organic solvents. Enzyme stability was tested in protease solutions, the remarkable feature observed with  CLECs may result from a crystal packing reenforced by covalent cross-links.

Figure 1. The activity of HLADH in solution and CLEC  against organic solvents.   HLADH in solution(□)  and in CLEC (□) in acetonitrile solution and  HLADH in solution (□)and in CLEC  in DMSO.

Two types of HLADH were used comparatively. Activities of soluble enzymes and CLECs were on the same level for control experiment. Remarkably, CLECs are stable in protease or organic solvents mixtures where enzymes denature. Different protease solutions were tested and results in that free enzyme retained only 40% of its activity, while CLECs retained >95% activity.(Fig 2).

[A]                                 [B]

Figure 2. The stability of HLADH against protease. HLADH (0.5□/□)  in solution[A] and in CLEC[B] was exposed   to various protease solutions, 0.1□/□ of  □-chymotrypsin (□) and  pronase E (□), 0.2□/□ of proteinase K (□).

Proteolysis resistance of CLECs is explained by the size of the solvent channels defined by the crystal lattice.(Fig3).

Figure 3.   Microscopic   pictures   of   two   types crystallized HLADH in protease solution. The enzymes were exposed in the  pronase E solution. 2□/□ pronase  solution was  added to each  enzymes,  20□ in soluble form and 40□ in CLEC form.

When  an enzyme molecule is in a crystal lattice, due to surrounded in  three dimensions by other protein molecules, it is much more difficult to unravel the  tertiary structure than when dissolved in solution(5). It may significantly enhance protein stability with regard to heat and other denaturants by preventing unfolding, aggregation or dissociation (6). In the case  of CLECs,  the rigidity of their structure makes possible to resist the protease and organic solvents attack. We, here, ensure the stability of HLADH-CLEC against protease and organic solvents with emphasizing that it has  a great potential as a biosensor. The CLEC technology may contribute to an acceleration of practical chemical synthesis and other areas.

REFERENCES

1. Davies, H. G., Green, R. H., Kelly, D. R., Roberts, S. M.: CRC Crit. Rec. Biotechnol, 10, 129-154(1990).

2. Dordick, J. S.:  Enzyme Microb. Technol, 11, 194-211(1989).

3. Jones. J. B.: Tetrahedron, 42, 539-553(1986).

4. Lee, K. M., Blaghen, M., Samama, J. P. and Biellmann, J. F.: Bioorganic Chemistry, 14, 202-210(1986).

5. Vilenchik, L. Z.,  Griffith, J. P.,  St. Clair, N.,  Navia, M.  A. and Margolin, A.  L.: J. Am. Chem. Soc, 120, 4290-4294(1998).

6. Lalonde, J.: CHEMTECH, 27, 38-45(1997).