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J. Biochem, 2003, Vol. 133, No. 1 123-131
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

Expression of a Synthetic Gene Coding for Ostrich Egg-White Lysozyme in Pichia Pastoris and Its Enzymatic Activity

Shunsuke Kawamura+,1, Tamo Fukamizo2, Tomohiro Araki1 and Takao Torikata1

1 Department of Bioscience, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto 869-1404; and 2 Laboratory of Enzyme System Science, Department of Food and Nutrition, Kinki University, Nakamachi, Nara 631-8505

To investigate the structure-function relationships of goose-type lysozyme, a gene coding for ostrich egg-white lysozyme (OEL) was designed based on the published amino acid sequence and constructed by assembling 32 chemically synthesized oligonucleotides. To obtain the recombinant OEL (rOEL), the synthetic gene was fused to the {alpha}-factor signal peptide in the expression vector pPIC9K and expressed in the methylotrophic yeast Pichia pastoris. The secreted protein from the transformed yeast was found to be processed at three different sites, including the correct site. The correctly processed rOEL was purified to homogeneity and shown to be indistinguishable from the authentic form in terms of circular dichroism (CD) spectrum and enzyme activity. Furthermore, the time-course of the reaction catalyzed by OEL was studied using (GlcNAc)n (n = 5 and 6) as the substrate and compared to that of goose egg-white lysozyme (GEL) [Honda and Fukamizo (1998) Biochim. Biophys. Acta 1388, 53–65]. OEL hydrolyzed (GlcNAc)6 in an endo-splitting manner producing mainly (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4, and cleavage to (GlcNAc)3 + (GlcNAc)3 predominated over that to (GlcNAc)2 + (GlcNAc)4. This indicates that OEL hydrolyzes preferentially the third glycosidic linkage from the nonreducing end of (GlcNAc)6 as in the case of GEL. The cleavage pattern seen for (GlcNAc)5 was similar to that seen for (GlcNAc)6. Theoretical analysis of the reaction time-course for OEL revealed that the binding free energy values for subsites B, E, and G were different between OEL and GEL, although these lysozymes were estimated to have the same type of subsite structure.

+ To whom correspondence should be addressed. Tel: +81-9676-7-3918, Fax: +81-9676-7-3960, E-mail: skawa{at}ktmail.ktokai-u.ac.jp


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S. Kawamura, K. Ohno, M. Ohkuma, Y. Chijiiwa, and T. Torikata
Experimental verification of the crucial roles of glu73 in the catalytic activity and structural stability of goose type lysozyme.
J. Biochem., July 1, 2006; 140(1): 75 - 85.
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