A composition comprising a coupled enzyme system
A technology of composition and substrate, applied in the direction of digestive system, biocide-containing paint, cardiovascular system diseases, etc., can solve problems such as loss of activity
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[0348] 1. A composition containing sorbitol oxidase, a first substrate and an oxidoreductase, wherein the first substrate can be oxidized to form hydrogen peroxide and a second substrate by sorbitol oxidase, and said A second substrate can be oxidized by an oxidoreductase to form hydrogen peroxide and a product.
[0349] 2. According to the composition of the present invention such as embodiment 1, wherein said first substrate is selected from D-sorbitol, D-xylitol, D-mannitol, D-arabinitol, glycerol, One or more of inositol, 1,3-propanediol, 1,3-butanediol and 1,4-butanediol.
[0350] 3. The composition according to the present invention such as embodiment 2, wherein the first substrate is one or more selected from D-sorbitol or D-xylitol.
[0351] 4. A composition according to the invention such as embodiment 3, wherein the first substrate is D-sorbitol.
[0352] 5. The composition according to embodiment 1 of the present invention, wherein the oxidoreductase is one or mor...
Embodiment 1
[0371] Embodiment 1: Construction of the bacterial strain expressing the sorbitol oxidase of Streptomyces h-7775 in Streptomyces lividans
[0372] The protein sequence of sorbitol oxidase (SEQ ID NO: 1 ) was obtained from published amino acid sequences (see eg Hiraga et al., Biosci. Biotechnol. Biochem., 62:4347-353 [1998]). The signal sequence (SEQ ID NO: 3) of the double arginine pathway of the S. coelicolor SCO6772 gene was obtained from the complete genome sequence of S. coelicolor.
[0373] Sorbitol oxidase was expressed in Streptomyces as a fusion protein of the signal sequence (SEQ ID NO: 3) and sorbitol oxidase (SEQ ID NO: 1) of the SCO6772 protein. For cloning, a restriction site NcoI was introduced at the 5' end of the DNA, which resulted in the addition of the amino acid glycine residue at position 2 (see, SEQ ID NO: 4).
[0374] For cloning, the restriction site BamHI was also introduced at the 3' end of the DNA. The codons of this fusion gene were optimized for ...
Embodiment 2
[0381] Example 2: Expression of sorbitol oxidase from Streptomyces H-7775 in E. coli strain BL21(DE3)pLysS
[0382] The amino acid sequence of the sorbitol oxidase gene from Streptomyces H-7775 SOX gene (published by Hiraga et al. 1998. Bioscience Biotech Biochem. 61: 1699-1704, 1998) was obtained from Sequence Database.
[0383] A synthetic gene (with neutral codons) encoding the H-7775SOX gene was used to express the sorbitol oxidase gene in E. coli strain BL21(DE3)pLysS. The expression vector pET 24a with SOX gene was cloned as Ndel+Bam H1 fragment. The resulting plasmid ( Figure 5a ) were transformed into and propagated in E. coli Top10 cells (Invitrogen, USA). A kanamycin resistant transformant containing the 1.2 kb SOX gene was identified by the direct colony PCR method. SOX positive transformants were grown and plasmid DNA was isolated. Plasmid DNA containing the cloned SOX gene was then used to transform host strain BL21(DE3)pLysS. The entire transformation react...
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