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Sorbitol Oxidase from Microorganisms

KOEHI ODA AND KAZUMI HIRAGA

Department of Applied BiologyFaculty of Textile Science

Kyoto Institute of TechnologySakyo-ku, Kyoto 606-8585, Japan

INTRODUCTION

The sorbitol concentration in blood is related to diabetic complications such ascataracts and neuropathies, retinopathies, and nephropathies. Perturbations of thepolyol pathway (FIGURE 1) will increase cofactors such as NADP or NAD in the cell.This makes it more difficult to measure the correct sorbitol concentration by sorbitoldehydrogenase in the clinical field.

In the course of screening for glycerol oxidase–producing microorganisms, wefound an enzyme that oxidized D-sorbitol in the cell-free extract of a strain isolatedfrom soil. From a preliminary study using the purified enzyme, we detected an oxi-dase activity that catalyzed the oxidation of D-sorbitol to form glucose and hydrogenperoxide without any requirement of exogenous cofactors. Thus, the reaction mecha-nism of the sorbitol-oxidizing enzyme differs from that of sorbitol dehydrogenase(EC 1.1.1.14). Hence, we tentatively named the enzyme “sorbitol oxidase (SOX)”.

In this paper, we describe the isolation, some properties, and gene cloning of sor-bitol oxidase from Streptomyces sp. H-7775.1,2

RESULTS AND DISCUSSION

Sorbitol Oxidase from Streptomyces sp. H-7775

Strong SOX activity was detected in the cell-free extract of the isolated strain H-7775. The strain had wall chemotype I and showed typical morphological and physi-ological properties of the genus Streptomyces. Accordingly, this strain was designat-ed Streptomyces sp. H-7775.1

From the mycelia of 20 liters of culture broth, 1.5 mg of SOX was obtained with a

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FIGURE 1. The polyol pathway.

yield of 12.1%. The purified enzyme had a single band in SDS- and native-PAGE.The molecular weight of the purified SOX was estimated to be 45,000 by both SDS-PAGE and Sephadex G-75 column chromatography, indicating that the enzyme was amonomer.

The absorption spectrum of the purified SOX had a typical flavoprotein spectrumwith absorption maxima at 276, 358, and 455 nm and a shoulder around 480 nm(FIGURE 2). A hypsochromic shift of the second absorption band to 358 nm relative tothat of riboflavin at 372 nm was also observed. The spectrum was similar to those offlavoproteins with covalently bound flavin. By the addition of D-sorbitol, the peaks at358 and 455 nm were decreased due to the reduction of flavin. These results indicat-ed that the flavin component was functionally involved in the oxidation of D-sorbitol.The correlation between pH and the fluorescence of the purified SOX was comparedwith that of FAD, FMN, and riboflavin. The fluorescence profile was pH-dependentand was similar to that of FAD, but different from that of FMN or riboflavin.

The flavin prosthetic group could not be liberated from the purified SOX proteinby (1) acidification with 5% trichloroacetic acid, (2) boiling for 5 min, (3) treatmentwith 1% SDS, and (4) dialysis against 3 M KBr/1 mM EDTA for 2 days at 4 °C. Thissuggests that FAD is covalently bound to the SOX. It was calculated that 0.9 moles ofFAD is bound to one mole of SOX.

The enzymatic properties of the SOX are summarized in TABLE 1. The expressionof SOX was not induced by the various sugar alcohols and sugars. The optimum pHand temperature of purified SOX were between 6.5 and 7.5 and 50 °C, respectively.The SOX was stable between pH 7.5 and 10.0 after 24-h incubation at 30 °C and wasstable below 55 °C after 15-min incubation at pH 7.5. D-Sorbitol and D-xylitol wereoxidized most rapidly. The Km values for these substrates were 0.26 mM and 0.38mM, respectively. Glycerol and D-mannose were oxidized at low rates, while D-glu-

ODA & HIRAGA: SOX 455

FIGURE 2. Absorption spectra of the purified SOX. Absorption spectrum of the purifiedSOX (A) in 20 mM potassium phosphate buffer, pH 6.0, at the final concentration of 0.64mg/mL. The insert shows the enlarged spectrum before (B) and after (C) addition of 50 mM D-sorbitol.

cose and D-fructose were not attacked at all. Moreover, the SOX activity was not af-fected by exogenous cofactors such as FAD, NAD, and NADP. One mM monoiodo-acetate (IAA) and N-ethylmaleimide (NEM) inhibited SOX activity. Thiol groupsmight also be involved in the SOX activity.

The reaction product from D-sorbitol was identified by thin-layer chromatographyand 1H-NMR spectroscopy. By comparing with the authentic sample of D-glucose,the product of the enzymatic oxidation was confirmed as glucose.

Thus, the SOX catalyzed the oxidation of D-sorbitol to form glucose and hydrogenperoxide without any requirement of exogenous cofactors. Moreover, it was con-firmed that the SOX did not react with glucose, a reaction product of D-sorbitol.

Construction of a High Expression System in E. coli Cells

Since the expression level of the SOX in Streptomyces sp. H-7775 was very low,we made an attempt to clone and express the SOX gene in E. coli cells.2 Approxi-mately 2000 colonies of the pUC18 genomic library were screened by using anoligonucleotide probe corresponding to the N-terminal amino acid sequence of theSOX, and 1 positive clone was isolated. The gene encoding the SOX was sequenced.The gene consisted of an open reading frame of 1260 bp encoding a protein of 420amino acids with a molecular weight of 45,148. The deduced amino acid sequence ofthe gene had 25.3% identity and 68.1% similarity to that of rat L-gulonolactone oxi-dase3 for the overall amino acids. Nucleotide-binding motifs were not found in thededuced amino acid sequence of the SOX protein. We succeeded in constructing a

ANNALS NEW YORK ACADEMY OF SCIENCES456

TABLE 1. Characteristics of Sorbitol Oxidase from Streptomyces sp. H-7775

Localization intracellular

Inducibility no

Molecular massSDS-PAGE 45 kDagel filtration 45 kDa

Optimum pH 6.5~7.5

pH stability 7.5~10.0(for 24 h at 30 °C)

Optimum temperature 50 °C(pH 7.5, 10 min)

Heat stability below 55 °C(pH 7.5, 15-min incubation)

Substrate specificitya

D-sorbitol 100 (Km = 0.26 mM)D-xylitol 93.5 (Km = 0.38 mM)D-mannitol 55.0 (Km = 7.38 mM)D-arabitol 39.0 (Km = 7.73 mM)glycerol 3.70D-glucose 0D-fructose 0D-mannose 1.00

Inhibitorb

Zn2+ 96.8Hg2+ 90.7IAA 53.8NEM 54.8

Cofactor FAD

Reaction products glucose + H2O2c

aRelative activity, %.bInhibition, %.cSubstrate: D-sorbitol.

high expression system of the SOX gene with a 4000-fold higher activity (40,000units/20 L culture) than that of the Streptomyces sp. H-7775. The enzymatic proper-ties of the recombinant SOX were identified as being similar to those of the enzymefrom Streptomyces sp. H-7775.

CONCLUSIONS

This is the first report of sorbitol oxidase from Streptomyces sp. Based on the fa-vorable properties of SOX and on the successful construction of an overexpressionsystem in E. coli as described above, the novel SOX from Streptomyces sp. H-7775might be useful for enzymatic analysis of sugar alcohols such as D-sorbitol.

REFERENCES

1. HIRAGA, K., M. KITAZAWA, N. KANEKO & K. ODA. 1997. Isolation and some properties ofsorbitol oxidase from Streptomyces sp. H-7775. Biosci. Biotech. Biochem. 61: 1699–1704.

2. HIRAGA, K., T. ETO, I. YOSHIOKA & K. ODA. 1998. Molecular cloning and expression of agene encoding a novel sorbitol oxidase from Streptomyces sp. H-7775. Biosci. Biotech.Biochem. 62: 347–353.

3. KOSHIZAKA, T., M. NISHIKIMI, T. OZAWA & K. YAGI. 1988. J. Biol. Chem. 263: 1619–1621.

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