96

NISR RESEARCH GRANT
2004 Young Investigator Research Grant

Enzymatic Syntheses of Novel Oligosaccharides Using
Haloarchaeal Glycosidases
Rie YATSUNAMI
Department of Bioengineering, Tokyo Institute of Technology
Introduction
Chitin is a b -1,4-linked polymer of N-acethyl-D-glucosamine (GlcNAc) and is the second most abundantbiopolymer in nature after cellulose. Chitinases (EC 3.2.14)
are enzymes, which hydrolyze b -1,4-glycosidic bonds ofchitin. In the more than 80 families of glycosyl hydrolasesthat have been defined on the basis of amino acid sequencehomology, chitinases are classified into two glycosidase
families, 18 and 19.1 In contrast to bacterial, fungal, plantand insect chitinases, studies on chitinase from the third
kingdom, archaea, are very few with the following exceptions: Chitinase genes from hyperthermophile Thermococcus kodakaraensis KOD12 and Pyrococcus furiosus3 wereisolated, cloned and expressed in Escherichia coli, andmembrane-associated chitinase from Thermococcus
chitonophagus was purified and characterized as a nativeenzyme.
Halobacterium species are extremely halophilic archaeathat thrive in environments nearly saturated with salt (4–5M NaCl). So far, characterization of chitinases from
halophilic archaea has not been reported. Recently, thecomplete genome sequence of Halobacterium sp. NRC-1
was reported,4 and this research revealed an open reading
frame which is predicted to encode a chitinase homolog,
which we termed ChiN1. ChiN1 contained three putative
domains: a chitin-binding domain (ChBD), a polycystic
kidney disease I domain (PKD), and a catalytic domain(CatD).
Haloarcula japonica strain TR-1 is an extremely
halophilic archaeon and has large amount of glycoprotein
on its cell surface.5) Previously, the gene encoding the cellsurface glycoprotein (CSG) was cloned and sequenced.6)
Because of the large amount of CSG in Har. japonica, thepromoter of CSG gene is expected to be powerful. Thus, it
may be possible to construct high expression systems offoreign genes in Har. japonica by employing the CSG promoter.
In this study, we report an expression of ChiN1 genefrom Halobacterium sp. NRC-1 including its signal sequence by Har. japonica using the promoter sequence ofCSG gene, and characterization of ChiN1 produced by Har.
japonica. Furthermore, to reveal the function of ChBD andPKD, a truncated ChiN1 (ChiN1CatD), which lacked thepolypeptide region between ChBD and PKD, has been constructed. Characterization of ChiN1CatD revealed that one ofthe physiological function of ChBD and/or PKD might beto enhance the hydrolysis of insoluble chitin.
Results and Discussion
Sequence analysis and protein domains
Genome sequence of Halobacterium sp. NRC-1 revealed
an open reading frame of 1,638 nucleotides which is predicted to encode a chitinase homolog which we termed
ChiN1. The ChiN1 gene encoded 546 amino acid (aa).
However, the predicted signal sequence was found in the Nterminal 28 aa portion, the removal of which would resultin a mature protein of 57 kDa. By comparing of deduced
amino acid sequence among ChiN1 and other chitinases,
ChiN1 seemed to contain three putative domains, a chitinbinding domain (ChBD), a polycystic kidney disease I domain (PKD), and a catalytic domain presumably classified
in family 18 of glycosyl hydrolases (CatD) (Fig. 1).
Recently, a novel translocation pathway for protein secretion was discovered. Because signal peptides in proteinsusing this pathway contain a typical twin-arginine motif, it
was denoted the twin-arginine translocation (Tat) pathway.
In total, 103 proteins with putative signal peptides wereidentified in Halobacterium sp. NRC-1, and more than 60%of these contain a twin-arginine motif. There was a twinarginine motif in the signal peptide of ChiN1, so ChiN1 is
expected to be secreted by Tat pathway.
According to classification of carbohydrate-binding modules (CBMs), the putative ChBD of ChiN1 belongs to family V CBM. Enzymes that have family V CBM are almostchitinases, but a few of chitosanases, xylanases and cellulases also contain CBM of this family.
PKD was first identified in human polycystin-1, the primary protein responsible for polycystic kidney disease. ThePKD was an immunoglobulin (Ig)-like fold mainly composed of ß-sheets. This structure can be found in fibronectin type III domain which is prevalent in chitinases
and cellulases, and presumably function as enzyme-carbohydrate interactions. Due to the similarity of their structure,Fig. 1.

Shematic structure of ChiN1.

97

PKD in chitinases are expected to act like fibronectin typeIII domain.
The deduced amino acid sequence of CatD of ChiN1
showed moderate homology to that of chitianse A of Thermococcus kodakaraensis and chitinase A1 of Bacillus circulans, both of which belong to family 18 chitinases. Thissuggests that ChiN1 belongs to family 18 chitinases.
Expression of chiN1 gene in Har. japonica
The chiN1 gene was subjected to be expressed in Har.
japonica by using its CSG promoter. Har. japonica wastransformed with expression plasmid pChiN9, encoding achiN1 gene downstream of the CSG promoter of Har.
japonica. SDS-PAGE analysis of Har. japonica transformants revealed the presence of a large amount of protein
corresponding to about 70 kDa only in the culture supernatant. SDS-PAGE usually yielded high molecular mass
values for halophilic proteins than calculated molecularmass, due to the reduced ability of them to bind SDS because of its high acidic residues content. The predicted mature ChiN1 contains larger proportion of acidic amino acids(17%) compared to reported chitinases from mesophilic organisms (310%). Chitinase activity of this protein wasdetected by activity staining followed by SDS-PADE, indicating that ChiN1 gene was expressed in Har. japonica. Nterminal amino acid sequence of the purified ChiN1 revealed the first 12 amino acids as ADTPPEWDPDTV. Thissequence was corresponding to the predicted first aminoacid of the mature form of ChiN1 after cleavage of the signal peptide.

Fig. 2.

Effect of reaction pH on the activity of ChiN1 and ChiN1CatD.

Fig. 3. Effect of reaction temperature on the activity of ChiN1 andChiN1CatD.

Characterization of the purified ChiN1
Effects of pH and temperature
To evaluate the roles of additional domains ChBD andPKD, domain-truncated derivative of ChiN1 (ChiN1CatD)
was prepared and characterized. The pH- and temperatureactivity profiles of ChiN1 and ChiN1CatD were almost similar; optimum pH (Fig. 2) and temperature (Fig. 3) of enzyme were around pH 4.5 and 50°C, respectively.
Effects of NaCl
Maximal chitinase activity of ChiN1 was detected at 1 M
NaCl (pH 6.0, 37°C) (Fig. 4). ChiN1 retained only 50% ofoptimal activity at 60 mM NaCl, indicating a salt requirement for its activity. Chitinase activity was very stable overa wide NaCl range from 1–4.5 M (24 h, pH 6.0, 4°C) (Datanot shown). In general, halophilic enzymes require high salt
concentration for retaining their structure and function. Because of its unusual content of acidic residues, ChiN1 waspredicted to be a halophilic enzyme, and these profiles ofChiN1 and ChiN1CatD were almost similar.
Chitin binding activityBinding activity toward insoluble chitin was comparedfor ChiN1 and ChiN1CatD. ChiN1 bound to insoluble chitin.
However, the deletion of ChBD and PKD abolished thebinding activity to insoluble chitin (Date not shown). This
result suggested that these regions were responsible forthe chitin binding. The chitinase activity of ChiN1 andChiN1CatD was compared when soluble or insoluble chitin

Fig. 4.

Effect on NaCl concentration on activity.

Fig. 5. Time course of insoluble chitin degradation by ChiN1 andChiN1CatD.

98

was used as substrate. Both enzymes showed almost equal
activity toward soluble chitin, however, ChiN1 hydrolyzed
insoluble chitin more efficiently than ChiN1CatD. did (Fig.
5). These results indicated that one of the physiological
function of ChBD and/or PKD might be to enhance the hydrolysis of insoluble chitin.
References
1) B. Henrissat, Biochem. J., 280, 309–316 (1991).
2) T. Tanaka, T. Fukui and T. Imanaka, J. Biol. Chem., 276,35625–35629 (2001).
3) J. Gao, M. W. Bauer, K. R. Shockley, M. A. Pysz and R. M. Kelly,Appl. Envion. Microbiol., 69, 3119–3128 (2003).
4) W. V. Ng, S. P. Kennedy, G. G. Mahairas, B. Berquist, M. Pan, H. D.
Shukla, S. R. Lasky, N. S. Baliga, V. Thorsson, J. Sbrogna, S.
Swartzell, D. Weir, J. Hall, T. A. Dahl, R. Welti, Y. A. Goo, B. Leithauser, K. Keller, R. Cruz, M. J. Danson, D. W. Hough, D. G. Maddocks, P. E. Jablonski, M. P. Krebs, C. M. Angevine, H. Dale, T. A.
Isenbarger, R. F. Peck, M. Pohischroder, J. L. Spudich, K.-H. Jung,M. Alam, T. Fretas, S. Hou, C. J. Daniels, P. P. Dennis, A. D. Omer,
H. Ebhardt, T. M. Lowe, P. Liang, M. Riley, L. Hood and S. DasSarma, Proc. Natl. Acad. Sci. U.S.A., 97, 12176–12181 (2000).
5) K. Horikoshi, R. Aono and S. Nakamura, Experientia, 49, 467–502(1993).
6) H. Wakai, S. Nakamura, H. Kawasaki, K. Takada, S. Mizutani, R.
Aono and K. Horikoshi, Extremophiles, 1, 29–35 (1997).