The stabilizing effects of immobilization in D-amino acid oxidase from Trigonopsis variabilis.
BACKGROUND
Immobilization of Trigonopsis variabilis D-amino acid oxidase (TvDAO) on solid support is the key to a reasonably stable performance of this enzyme in the industrial process for the conversion of cephalosporin C as well as in other biocatalytic applications.


RESULTS
To provide a mechanistic basis for the stabilization of the carrier-bound oxidase we analyzed the stabilizing effects of immobilization in TvDAO exposed to the stress of elevated temperature and operational conditions. Two different strategies of immobilization were used: multi-point covalent binding to epoxy-activated Sepabeads EC-EP; and non-covalent oriented immobilization of the enzyme through affinity of its N-terminal Strep-tag to Strep-Tactin coated on insoluble particles. At 50 degrees C, the oriented immobilizate was not stabilized as compared to the free enzyme. The structure of TvDAO was stabilized via covalent attachment to Sepabeads EC-EP but concomitantly, binding of the FAD cofactor was weakened. FAD release from the enzyme into solution markedly reduced the positive effect of immobilization on the overall stability of TvDAO. Under conditions of substrate conversion in a bubble-aerated stirred tank reactor, both immobilization techniques as well as the addition of the surfactant Pluronic F-68 stabilized TvDAO by protecting the enzyme from the deleterious effect of gas-liquid interfaces. Immobilization of TvDAO on Sepabeads EC-EP however stabilized the enzyme beyond this effect and led to a biocatalyst that could be re-used in multiple cycles of substrate conversion.


CONCLUSIONS
Multi-point covalent attachment of TvDAO on an isoluble porous carrier provides stabilization against the denaturing effects of high temperature and exposure to a gas-liquid interface. Improvement of binding of the FAD cofactor, probably by using methods of protein engineering, would further enhance the stability of the immobilized enzyme.
Dib
Iskandar
I
Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria. iskandar.dib@gmx.at
Nidetzky
Bernd
B
D002384:Catalysis; D002511:Cephalosporins / Q000737:chemistry; D003605:D-Amino-Acid Oxidase / Q000737:chemistry*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D020442:Poloxamer / Q000737:chemistry; D015202:Protein Engineering; D004718:Saccharomycetales / Q000201:enzymology*; D013696:Temperature
D002511:Cephalosporins; D004800:Enzymes, Immobilized; D020442:Poloxamer; C025163:cephalosporin C; D003605:D-Amino-Acid Oxidase
Biotechnol Bioeng. 2008 Feb 1;99(2):251-60
17680679
Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3089-93
10716694
Appl Biochem Biotechnol. 2001 Aug;95(2):83-92
11694065
Biotechnol Prog. 2002 May-Jun;18(3):629-34
12052083
Anal Chem. 2008 Mar 1;80(5):1589-97
18229946
Eur J Biochem. 1997 Feb 15;244(1):220-5
9063467
Cell Mol Life Sci. 2000 Nov;57(12):1732-47
11130179
Biotechnol Bioeng. 2006 Jul 5;94(4):645-54
16538681
Biochemistry (Mosc). 2005 Jan;70(1):40-54
15701048
Biotechnol Prog. 2004 Mar-Apr;20(2):467-73
15058991
Biotechnol Prog. 2003 May-Jun;19(3):784-8
12790639
Appl Environ Microbiol. 2007 Jan;73(1):331-3
17056691
Appl Microbiol Biotechnol. 2006 May;70(6):683-9
16217653
Biochem Soc Trans. 2006 Apr;34(Pt 2):287-90
16545096
Appl Microbiol Biotechnol. 2008 Feb;78(1):1-16
18084756
J Biotechnol. 2004 May 27;110(2):201-7
15121338