92

NISR RESEARCH GRANT
2002 Young Investigator Research Grant

Construction of the Cellulosome for Targeting the Cell Wall
Degradation in Red Algae and Its Application to MarineBiomass
Yutaka TAMARU
Department of Life Sciences, Faculty of Bioresources, Mie University
Research aims
Marine biomass is comprised of a variety of polysaccharides as structural or storage compounds. The biologicaldegradation to marine biomass occurs through polysaccharolytic enzymes that are produced by organisms ranging
from eukaryotes to microorganisms. Thus, biodegradation
is important for natural and industrial cycling of environmental compounds. Furthermore, its biological degradation
plays a major role in the turnover of CO2. The componentsof cell walls in marine seaweed, especially for red algae include quite unique sugar polymers such as b -1,4-mannan,b -1,3-xylan, and agar, while those in terrestrial plants consist of cellulose, hemicellulose, and lignin. In particular,Porphyra is an important red seaweed which has familiarly
been edible in Japan.
Glycosidases, which generally play a major role in theturnover of polysaccharides in plant biomass, have beenfound either as free enzymes that work synergistically or as
an enzyme complex called cellulosomes. In this project, wetried to construct a cellulosome for targeting the cell wallsof marine seaweed, especially for red algae, Porphyra.
Methods
To construct the cellulosome for degrading the cell wallsof red algae, we used mini-cbpA and engE genes from
Clostridium cellulovorans1) and AgaB from Vibrio sp.

Fig. 1.

strain PO-303, ManA from Vibrio sp. strain MA-138, andTxyA from Alcaligenes sp. strain XY-234. Instead of an
original cellulose-binding domain (CBD) in mini-CbpA, wechanged a carbohydrate-binding domain (CBM) of TxyA
from Alcaligenes sp. strain XY-234. Dokerin domain ofEngE from C. cellulovorans was amplified by PCR to create expression plasmids pET-AgaB, pET-ManA, and pETTxyA, respectively (Fig. 1). The amplified fragments containing each gene and dokerin domain were inserted between NcoI and XhoI sites of pET22b. Four recombinant
proteins were purified from each of the E coli BL21 strains
harboring expression plasmids. To determine the protoplastformation ability of enzymes from three kinds of marine
bacteria, we used a red algae, Porphyra yezoensis.
Results
E. coli BL21 harboring each pET expression plasmid
was grown in 300 ml of LB medium at 37°C to optical density at 600 nm of 0.9. Isopropyl-b -D-thiogalactoside (IPTG)
was then added to a final concentration of 1 mM, and theculture was grown at 30°C for 3 h. After collection of E.
coli cells by centrifugation, the periplasmic proteins wereextracted into 50 ml of extraction buffer (20 mM Tris-HCl[pH 8.0], 10 mM EDTA) by the osmotic method. A 50-ml
aliquot of the supernatant was applied to an Ni2-nirilotriacetic acid agarose column. Each protein was eluted with
200 mM imidazole in 50 mM sodium phosphate buffer

Construction of expression plasmids for cellulosome.

93

(A)

Fig. 2.

(B)

Protoplast formation from Porphyra yezoensis using the recombinant enzymes from marine bacteria.

(pH7.0), and fractions containing each protein were collected. After purification and SDS-PAGE, the final preparation yielded a single band. By using these purified enzymes, we examined the interaction between mini-CbpA
and three kinds of enzymatic subunits. As the result, no interaction bands were detected by native PAGE, indicatingthat dokerin domain of each subunits might have been
lacked by proteolysis in E. coli cells. Next, in order to confirm that three kinds of recombinant enzymes are able to release the protoplasts from Porphyra yezoensis, the three enzymes were tested for protoplast formation. As the result,approximately 4.0104 protoplasts were released in 20 mM
MES buffer (pH 7.5) containing 0.5 M mannitol and oneunit each of three kinds of recombinant enzymes (AgaB,ManA, and TxyA) and under incubation at 22°C for 60 min
with gentle agitation.

Conclusion
We have succeeded in protoplast formation from Porphyra yezoensis by using three kinds of recombinant enzymes from marine bacteria. However, the cellulosome
complex between mini-CbpA and these enzymes was notfound. Since this problem is probably caused by proteolysis
in E. coli, it therefore seems that it is necessary to choose
the host strain as protease-deficient one.
Reference1)

Kosugi, A., Murashima, K., Tamaru, Y., and Doi, R. H. (2002) Cellsurface-anchoring role of N-terminal surface layer homology domains of Clostridium cellulovorans EngE. J. Bacteriol., 184:
884–888.