Preparation of recombinant polysaccharide-degrading enzymes from the marine bacterium, Pseudomonas sp. ND137 for the production of protoplasts of Porphyra yezoensis

Abstract

We previously reported that a marine bacterium, strain ND137, produced the enzymes to make protoplasts from the thalli of a marine red alga Porphyra yezoensis. In this study, the bacterium was classified as Pseudomonas sp. based on the sequence of the gene encoding 16S rRNA and the G + C content of the DNA. The β-1,4-xylanase-, cellulase-, β-1,3-xylanase-, porphyranase-, and β-1,4-mannanase-encoding genes were cloned from the genomic library of Pseudomonas sp. ND137 and expressed in Escherichia coli. The protoplasts from Porphyra yezoensis were produced by treating the thalli with several combinations of these recombinant crude enzymes. Treatment of the Porphyra thalli with any of these enzymes on their own failed to produce protoplasts. However, a mixture of β-1,3-xylanase, porphyranase, and β-1,4-mannanase produced protoplasts, although β-1,4-xylanase and cellulase were not observed to have any effect on protoplast production. These results indicate that among the five recombinant crude enzymes, β-1,3-xylanase, porphyranase, and β-1,4-mannanase are essential for producing protoplasts from the thalli of P. yezoensis. The yield of the protoplasts was 1.4 × 104 cells mg−1 wet weight of the thalli, indicating that these recombinant enzymes can be used efficiently for protoplast production from P. yezoensis.