Biosci. Biotechnol. Biochem., 66 (10), 2168–2175, 2002

Cloning and Characterization of the nagA that Encodes b-N-Acetylglucosaminidase from Aspergillus nidulans and Its Expression in Aspergillus oryzae

Sunhwa KIM,1 Ichiro MATSUO,2,* Katsumi AJISAKA,2 Harushi NAKAJIMA,1 and Katsuhiko KITAMOTO1,†

1Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2Meiji Institute of Health Science, Naruda 540, Odawara 250-0862, Japan

Received April 2, 2002; Accepted June 14, 2002

We isolated a b-N-acetylglucosaminidase encoding cell wall digestion, including germination, hyphal gene and its cDNA from the ˆlamentous fungus Asper- growth, branching of hyphae, and hyphal autoly- gillus nidulans, and designated it nagA.ThenagA gene sis.3,4) contained no intron and encoded a polypeptide of 603 b-N-acetylglucosaminidases are also widely dis- amino acids with a putative 19-amino acid signal se- tributed throughout nature. In higher eukaryotes, b- quence. The deduced amino acid sequence was very N-acetylglucosaminidases are lysosomally localized similar to the sequence of Candida albicans Hex1 and and the are generally dimeric. b-N-acetyl- Trichoderma harzianum Nag1. Yeast cells containing glucosaminidase has also shown to be involved in the the nagA cDNA under the control of the GAL1 formation of septa in molds, germ tubes in yeasts, promoter expressed b-N-acetylglucosaminidase activity. and fruit-body formation in a basidiomycete.5) A b- The chromosomal nagA gene of A. nidulans was dis- N-acetylglucosaminidase was puriˆed from an auto- rupted by replacement with the argB marker gene. The lyzed culture of A. nidulans and was found to hydro- disruptant strains expressed low levels of b-N-acetyl- lyze oligomers of chitin produced in the degradation glucosaminidase activity and showed poor growth on a of chitin cell wall by endochitinases.6) medium containing chitobiose as a carbon source. Recently, it has been reported that molecular clon- Aspergillus oryzae strain carrying the nagA gene under ing of b-N-acetylglucosaminidase encoding has the control of the improved glaA promoter produced been done from several bacteria (Alteromonas sp. large amounts of b-N-acetylglucosaminidase in a wheat strain O-7, Serratia marcescens, Vibrio harveyi, V. bran solid culture. furnissii,andEnterobacter sp. strain G-1) and fungi such as T. harzianum7) and C. albicans.8) The gene Key words: b-N-acetylglucosaminidase; nagA; Asper- encoding b-N-acetylglucosaminidase, however, has gillus nidulans not yet been cloned from Aspergillus fungi. In this report, we describe the cloning and characterization Chitin, a b-1,4-linked biopolymer of N-acetyl- of the nagA gene encoding b-N-acetylglucosamini- glucosamine (GlcNAc) is an abundant biopolymer dase from A. nidulans. We also report the successful and one of the major components of fungal cell overexpression of A. nidulans b-N-acetylglucosa- walls. Degradation of chitin aŠects natural eco- minidase in A. oryzae using a wheat bran culture. systems. This abundance has stimulated research for the isolation and characterization of chitinolytic en- Materials and Methods zymes. Many microorganisms enzymatically degrade chitin with two successive steps; degradation of chitin Strains and media. Escherichia coli HB101 (F„ into (GlcNAc)n by exo-(EC 3.2.1.29) and endo- supE hsdS recA ara proA lacY galK rpsL xyl mtl leuB (EC 3.2.1.14) and hydrolysation of the thi )andDH5a (F„ q80dlacZDM15, D(lacZYA- resulting (GlcNAc)n, mainly chitobiose, to GlcNAc argF ) supE recA endA hsdR phoA thi gyrA relA ) by b-N-acetylglucosaminidase (EC 3.2.1.30).1) were used as hosts for DNA manipulations.9) Sac- Chitinase is widely distributed in bacteria, plants, charomyces cerevisiae YPH500 (MATa ura3 lys2 insects, mammals, and fungi.2) In ˆlamentous fungi, ade2 trp1 his3 leu2 )10) was used as a host strain for chitinase seems to have roles in processes involving expression of b-N-acetylglucosaminidase. YNBGal

† To whom correspondence should be addressed. Fax: +81-3-5841-8033; E-mail: akitamo＠mail.ecc.u-tokyo.ac.jp * Present address: Riken (Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Abbreviations: X-GlcNAc, 5-bromo-4-chloro-3-indoyl-b-D-N-glucosaminide; PCR, polymerase chain reaction Cloning of b-N-Acetylglucosaminidase Gene nagA from A. nidulans 2169 (X-GlcNAc) medium (0.17z yeast nitrogen base pression of the nagA gene in S. cerevisiae, the 1.8-kb without amino acids and ammonium sulfate, 0.5z fragment containing the nagA cDNA was inserted ammonium sulfate, 2z galactose, 2z agar, and 0.2 between the HindIII and BamHIsitesoftheyeast mgWml X-GlcNAc [5-bromo-4-chloro-3-indolyl-b-D- expression vector pYES2, downstream of the GAL1 N-acetyl-glucosaminide; purchased from Sigma-Al- promoter. The resultant plasmid, pYcN, was in- drich Fine Chemicals, St. Louis, MO]) was used for troduced into S. cerevisiae YPH500 and cultured on detection of b-N-acetylglucosaminidase activity ex- a YNBGal (X-GlcNAc) plate at 309Cfor3days. pressed by S. cerevisiae strains. Aspergillus nidulans A26 (biA1 veA1 ) was used as the DNA donor. A. Construction of nagA-disruptant strain. For the nidulans A89 (biA1 veA1 argB ) was used as a host disruption of the nagA gene in A. nidulans,the for transformation. YG medium (0.5z yeast extract 0.7-kb EcoRI-EcoRV region in the nagA ORF was and 1.0z glucose) and MM-glucose medium (10 g of replaced with the 2.2-kb fragment carrying the argB 14) glucose, 6.0 g of NaNO3,0.52gofKCl,0.52gof marker gene. The 5.2-kb HindIII fragment car-

MgSO・7H2O, 1.52 g of KH2PO4 and Hutner trace rying the DnagA::argB was introduced into A. nidu- elements per liter (pH 6.5)) were used for culture of lans A89. Chromosomal DNA of Arg+ transfor- A. nidulans. MM-chitobiose medium (MM medium mants that were not stained in blue on a YG plate containing 1z chitobiose instead of glucose, containing 0.2 mgWml X-GlcNAc was prepared and 200 mgWml arginine, 5 mgWml biotin, and 5 mgWml analyzed by PCR with the primers An-GluF and pyridoxin) was used for examination of the ArgB-C (5?-GGCTCGAGATCGACCTACAGCCA- chitobiose use of the nagA disruptant strains. Stan- TTGC-3?) and the primers An-GluF and An-GluR. dard DNA transformation procedures for A. nidu- lans were used.11) Aspergillus oryzae niaD300, which Overexpression of the nagA in A. oryzae. For ex- is a nitrate-reductase gene (niaD ) deˆcient mutant pression of the nagA gene in A. oryzae, the 1.8-kb derived from the wild-type strain RIB4012) was used nagA cDNA fragment was inserted between the NdeI for overexpression of the nagA gene. The transfor- and PmaCI sites of the fungal high-level expression mation of A. oryzae was done basically according to vector pNGA142, downstream from the improved the method of Gomi et al.13) glaA promoter.15) The resultant plasmid pNcN was introduced into A. oryzae niaD300. Some 106 conidia Isolation of the nagA gene from A. nidulans. The of one of the transformants (ONAG1) and the sequence (o9a11a1) was found from the A. nidulans niaD300 strain were inoculated in 20 ml of seeding EST database (University of Oklahoma) by the medium (0.25z yeast extract, 5.6z corn starch, search for T. harzianum nag1 homologue. On the 1.8z peptone, 0.125z KCl, 0.15z MgSO4・7H2O, basis of the nucleotide sequence of the o9a11a1, we and 2z wheatbran)andculturedat309Cfor2days designed the primers NAGp1 (5-ATAAAGATAC- with shaking. A 5-fold dilution of the culture (8 ml) GAGAGACTACACTAGT-3?) and NAGp2 (5?- was inoculated into 10 g of sterilized wheat bran in a AAGCAAAGCACGTCATCGGCGC-3?)andused 300-ml ‰ask and incubated at 309Cfor5days.The PCR on A. nidulans A26 chromosomal DNA. With crude was extracted with 50 ml of water at the 0.5-kb PCR product as a probe, colony hybridi- 309C. After centrifugation, the supernatant of the zation was done on an A. nidulans cosmid library extract solution was used as a crude enzyme solution. (purchased from Fungal Genetics Stock Center). We also examined liquid culture (DPY medium; 2z From one positive clone, a 3.6-kb HindIII fragment dextrin, 1z polypeptone, 0.5z yeast extract, 0.5z was isolated and the nucleotide sequence was KH2PO4, and 0.05z MgSO4・7H2O) for overexpres- analyzed with Model DSQ-1000 DNA sequencer sion of b-N-acetylglucosaminidase at 309Cfor2days (Shimadzu, Kyoto, Japan). To construct a cDNA with shaking. library of A. nidulans, mRNA was prepared from A. SDS-PAGE with the concentration of 10z poly- nidulans A26 grown in YG medium and converted acrylamide was done by the standard method.16) into cDNA using cDNA synthesis Kit (Amersham- b-N-acetylglucosaminidase activity was assayed by Pharmacia). The cDNA fragments were ligated with the method of Sullivan et al.17) One unit of enzyme the yeast expression vector pYES2 (Invitrogen, activity catalyzed the formation of 1 mmol of p- Groningen, Netherlands). For cDNA cloning, nitrophenol from the substrate, p-nitrophenyl-N- primers were designed as follows: An-GluF (sense acetyl-glucosamine (pNP-b-GlcNAc, Sigma), per primer, containing the putative initiation codon), 5?- minute at 379C. b-N-Acetylgalactosaminidase activi- AGAACACTATGGCCTACTTCCG-3? and An- ty was assayed using p-nitrophenyl-N-acetyl-galac- GluR (antisense primer, containing a putative termi- tosamine (pNP-b-GalNAc, Sigma) as the substrate. nation codon) 5?-ATAAAGATACGAGAGAC- TACACTA-3?. Results

Expression of the nagA in S. cerevisiae. For ex- Isolation of nagA gene encoding b-N-acetyl- 2170 S. KIM et al. glucosaminidase from A. nidulans human b-N- b chain (M19735), re- The A. nidulans Expressed Sequence Tag Database spectively. (Advanced Center for Genome Technology, Univer- sity of Oklahoma) was searched for sequences show- Expression of b-N-acetylglucosaminidase activity ing similarity to the T. harzianum nag1 (encoding in S. cerevisiae b-N-acetylglucosaminidase) gene using the BLAST To conˆrm b-N-acetylglucosaminidase activity of algorithm, and a candidate sequence (o9a11a1) was the nagA gene product, the 1.8-kb nagA cDNA frag- found. Two primers, NAGp1 and NAGp2, were con- ment was inserted into a yeast expression vector, structed based on this EST sequence and PCR was pYES2 downstream from the GAL1 promoter, and done with A. nidulans A26 chromosomal DNA. The designated pYcN (Fig. 2-a). pYcN was introduced ampliˆed 0.35-kb DNA fragment was used as a into S. cerevisiae YPH500andculturedonaYNBGal probe, and a cosmid library of A. nidulans genomic plate containing X-GlcNAc for 3 days. The colonies DNA (Fungal Genetics Stock Center) was screened of transformants were strongly stained in blue by b- by colony hybridization. From a positive cosmid, the N-acetylglucosaminidase activity but the control cells 3.6-kb HindIII fragment (Fig. 1a) that hybridized were remained white (Fig. 2-b). It indicates that the with the 0.35-kb probe in the Southern blot analysis nagA gene encodes enzymatically active b-N-acetyl- was isolated. The nucleotide sequence of the frag- glucosaminidase, and the gene is also functional in S. ment had one long open reading frame (ORF) with cerevisiae cells. high similarity to the nag1 gene of T. harzianum,and we designated the gene nagA. To isolate the nagA Characterization of the nagA defective strain cDNA, oligonucleotide primers, An-GluF and An- To examine the phenotypic consequences of a null GluR, were designed based on the genomic sequence allele of the nagA, we did a gene disruption of this around the N-terminus and C-terminus of nagA.The locus. The ORF of nagA in the genome was disrupted cDNA fragment of nagA was ampliˆed from the by the argB gene by in vivo recombination between cDNA library by PCR using the primers An-GluF the introduced 5.2-kb HindIII fragment containing and An-GluR. The ampliˆed 1.8-kb fragment was DnagA::argB and the genomic nagA locus (Fig. 3-a). cloned into pBluescript II and conˆrmed to be nagA The Arg+ transformants were transferred to a YG cDNA by sequencing analysis. Figure 1-b shows the plate containing X-GlcNAc and cultured for 3 days. nucleotide and deduced amino acid sequences of the Although the wild-type strain formed blue colonies, nagA gene.Analysisofthe5? noncoding sequence of seven of the 28 transformants formed white colonies the A. nidulans nagA gene found several sequence on the plate. Chromosomal DNA of two of the can- elements that could be involved in transcription initi- didates, DNAG1 and DNAG2, were prepared and ation.18) One TATA box is present, 140 bp upstream analyzed by PCR. The 0.3-kb fragments were ampli- of the ATG codon. In addition, consensus binding ˆed from DNAG1 and DNAG2 using An-GluF and target sequences for the A. nidulans domain regula- ArgB-C as primers but not from host A89 (Fig. 3-b). tors CreA and PacC19) are present. Comparison of Although the putative 1.8-kb fragment was ampliˆed the complementary and genomic DNA sequences from wild-type A89, the expected 3.3-kb fragments showed that the nagA gene has no intron. The nagA were ampliˆed from the DNAG1 and DNAG2 with cDNA contains a single, large ORF of 1,809 nucleo- An-GluF and An-GluR primers (Fig. 3-c). When the tides, encoding a protein of 603 amino acids. The A89 (wild-type) and DNAG1 (DnagA::argB )were calculated molecular mass of NagA is 68.0 kDa and grown on YG (complex) and MM-glucose (complete) the isoelectric point (pI) is 4.6. The N-terminal media, we observed no diŠerence of the growth rate, hydrophobic sequence is considered to be a secretion cellular morphology, or e‹ciency of conidial forma- signal sequence. Three potential N-glycosylation sites tion between the wild-type strain and the nagA dis- are found in the NagA sequence (Fig. 1b). ruptant (Fig. 3-e). Figure 3-d shows b-N-acetyl- The deduced NagA amino acid sequence was glucosaminidase activity expressed by the A89 and compared to all sequences in the joint GenBank and DNAG1 colonies grown on a YG plate containing X- EMBL databases with the BLAST program. NagA GlcNAc. The nagA disruptant grew normally but showed similarity to and glucosa- was not stained by X-GlcNAc degradation activity minidases from a variety of organisms. There is 69z, (Fig. 3-d). This result indicates that the nagA gene 48z,37z,34z,31z,31z,and28z similarity of encodes a major secretory b-N-acetylglucosamini- amino acids with Penicillium chrysogenum b-N- dase and is not essential for hyphal growth and acetylhexosaminidase (AF056977), C. albicans conidial formation. To examine the role of NagA in b-N-acetylglucosaminidase (L26488), Arabidopsis assimilation of GlcNAc oligomer in A. nidulans,A89 thaliana F3F20.4 gene product (AC007153), and DNAG1 were grown on a MM-chitobiose plate Drosophila melanogaster Hexo1 (AE003480), T. containing chitobiose as a sole carbon source harzianum b-N-acetylglucosaminidase (S83231), D. (Fig. 3-e). A89 grew on the MM-chitobiose medium discoideum b-N-hexosaminidase (J04065), and but DNAG1 showed no growth. Cloning of b-N-Acetylglucosaminidase Gene nagA from A. nidulans 2171

Fig. 1. Nucleotide and Deduced Amino Acid Sequences of the A. nidulans nagA Gene. (a) Restriction map of the A. nidulans nagA gene. (b) Nucleotide and deduced amino acids sequences of the nagA. Potential TATA sequence is underlined. Putative CreA and PacC binding sites are in underlined bold. Potential N-glycosylation sites are indicated by un- derlines. The sequence data reported in this paper have been deposited in the DDBJ, EMBL, and NCBI nucleotide sequence databases under the accession number AB039846.

Overproduction of NagA by solid-state culture in proved glaA promoter, and designated pNcN A. oryzae (Fig. 4a). pNcN was introduced into A. oryzae For high-level secretion of A. nidulans b-N-acetyl- niaD300 and the nitrate-using transformants were glucosaminidase in A. oryzae, the 1.8-kb nagA streaked on fresh medium to obtain homokaryotic cDNA fragment was inserted into the fungal expres- strains. In the selected ˆve strains (ONAG1-5), in- sion vector pNGA142 downstream from the im- troduction of the glaA promoter-nagA DNA frag- 2172 S. KIM et al.

Fig. 2. Expression of nagA cDNA in S. cerevisiae. (a) Structure of the plasmid (pYcN) for expression of the nagA cDNA in a yeast. The GAL1 promoter and the CYC1 terminator regions were used for expression of the nagA cDNA. (b) Yeast strains YPH500 carrying the pYcN or pYES2 vector were grown on a YNBGal (X-GlcNAc) plate at 309C for 3 days. ment was conˆrmed by PCR with their genomic Discussion DNA. We observed that colonies of all of the ˆve strains were more strongly stained on a DPY plate In this study, we isolated a gene (nagA) encoding containing X-GlcNAc than that of wild-type and host the b-N-acetylglucosaminidase from A. nidulans. strains (data not shown). The deduced amino acid sequence of the nagA ORF In the liquid culture of ONAG1, we detected very showed great similarity to b-N-acetylglucosamini- little b-N-acetylglucosaminidase activity in the cul- dases and b-N-hexosaminidases from various eukary- ture medium (data not shown). In commercial en- otes. We conˆrmed the enzymatic activity of the zyme production, Aspergillus strains are often grown nagA gene product by expression of the nagA cDNA in solid-state culture, resulting in high production in S. cerevisiae, which has no background b-N- levels. Therefore, we grew wheat bran solid-state cul- acetylglucosaminidase activity. The S. cerevisiae cells tures of ONAG1 at 309C for 5 days. Crude enzyme expressing the nagA had b-N-acetylglucosaminidase solutions were prepared by extraction from the wheat activity on a YNBGal plate. This result indicates that bran cultures with water. We measured b-N-acetyl- the nagA gene encodes b-N-acetylglucosaminidase glucosaminidase activity of the solutions using pNP- and the product was functional in S. cerevisiae cells. b-GlcNAc as substrate. The ONAG1 solution had a In A. nidulans, the puriˆcation and characterization 60-fold increase in b-N-acetylglucosaminidase activi- of b-N-acetylglucosaminidase was previously re- ty (34.4 unitsWml) compared with the host strain ported from autolyzed cultures.6) Theenzymede- niaD300 (0.58 unitsWml). The ONAG1 solution also grades chitin oligomers and has a molecular mass of had 19.6 unitsWml of b-N-acetylgalactosaminidase 190-kDa, which is much larger than NagA. It activity with pNP-b-GalNAc as substrate. This result remains possible that NagA forms an oligomer with shows the possibility that A. nidulans NagA has both molecular mass of 190 kDa, but it is more possible b-N-acetylglucosaminidase and b-N-acetylgalac- that A. nidulans produces more than two types of b- tosaminidase activities. The enzyme solution showed N-acetylglucosaminidase. Puriˆcation and charac- pH optima of 4–5 and the activity was maximum at terization of NagA will clarify the variation of b-N- 529C under the standard assay conditions (data not acetylglucosaminidases produced by A. nidulans. shown). We found CreA and PacC binding consensus The crude enzyme solution was analyzed by SDS- sequences in the 5? non-coding region of the nagA PAGE (Fig. 4b). The 50 kDa band seen in all lanes gene. In the molecular basis of glucose repression, corresponds to the endogenous A. oryzae Taka- the protein product of the regulatory gene creA is a .20) The enzyme solutions of ONAG1 and negatively acting transcription factor that binds to ONAG2 showed another band with an apparent DNA sequence motifs with the consensus sequence molecularmassof65kDa.Themolecularmasscoin- 5?-(GWC)(CWT)GG(AWG)G-3?.21) At alkaline ambient cides with that of the calculated NagA protein. pH, activated PacC protein binds to DNA with the Cloning of b-N-Acetylglucosaminidase Gene nagA from A. nidulans 2173

Fig. 3. Disruption of the nagA Gene. (a) Strategy of the nagA gene disruption. The 0.7-kb EcoRI-EcoRV fragment in the nagA ORF was replaced with the 2.2-kb fragment containing the argB marker gene. Arrowheads indicate the position and direction of the primers. (b) PCR analysis of the nagA gene dis- ruptant strains with primers An-GluF and ArgB-C. Lane 1, wild-type A89; lane 2 and 3, DnagA::argB transformant; lane 4 marker. (c) PCR analysis of the nagA gene disruptant strains with primers An-GluF and An-GluR. Lane 1, marker; lane 2, wild-type A89; lane 3 and 4, DnagA::argB transformants. (d) Conidia of wild-type (nagA+)andDnagA::argB strain were spotted on a YG plate containing X-GlcNAc and incubated at 309C for 3 days. Arrowhead indicates a colony of the DnagA::argB strain. (e) Wild-type and DnagA::argB strains were streaked on MM-glucose and MM-chitobiose media and incubated at 309C for 5 days.

core consensus 5?-GCCA(AWG)G-3?, activates tran- according to the ``weight-matrices methods''.23) It in- scription of alkaline-expressed genes and represses dicates that the nagA encodes a protein that is trans- transcription of acid-expressed genes.19) The nagA ex- located into the endoplasmic reticulum. When the pression is considered to be repressed by glucose and NagA propeptide is cleaved at the Ala19,thisyieldsa regulated by ambient pH. Chitinase expression in mature protein of 585 amino acids with a calculated fungi is thought to respond to degradation products molecularmassof66.1kDa.Thiscoincidedwiththe that serve as inducers, and to easily metabolizable 65 kDa band observed on a SDS gel arising by the carbon sources that serve as repressors. In the nagA-overexpressing strain of A. oryzae. mycoparasitic fungus T. harzianum, the expression Chitinase and b-N-acetylglucosaminidase of fungi of b-N-acetylglucosaminidase encoding gene nag1 may be involved in processes that need cell wall diges- was induced by the presence of GlcNAc.22) tion, including germination of conidia, tip growth of At the N-terminus of the NagA polypeptide, a pos- hyphae, and hyphal autolysis. In A. nidulans, disrup- sible signal cleavage site following Ala19 was found tion of the chitinase-encoding gene chiA caused the 2174 S. KIM et al.

Fig. 4. Overexpression of the nagA Gene in A. oryzae. (a) Structure of the plasmid pNcN for overexpression of the nagA gene in A. oryzae. The improved glaA promoter and terminator were used for overexpression of the nagA gene. (b) Recombinant b-N-acetylglucosaminidase production by A. oryzae transformants. Crude enzyme solution (12.5 ml, each lane) of A. oryzae transformants was analyzed by SDS-PAGE. Arrowheads indicate the position of NagA (upper) and Taka-amylase (lower). Lanes 1 and 2, overproducing transformant ONAG1; lanes 3 and 4, ONAG2; lanes 5 and 6, host strain A. oryzae niaD300. decrease of germination frequency and hyphal dase in liquid culture.26) It is possible that most of growth.24) We observed that the DnagA strain NagA was bound to the cell wall of A. oryzae in the showed no staining of b-N-acetylglucosaminidase ac- liquid culture. Solid cultivation was more suitable for tivity grown on a plate containing X-GlcNAc. The the NagA production than liquid cultivation. nagA gene is considered to encode a major secretory Some b-N-acetylglucosaminidases hydrolyze both b-N-acetylglucosaminidase in A. nidulans. However, b-GlcNAc and b-GalNac.27) We observed that an en- we observed no phenotypic diŠerences between wild- zyme solution of A. nidulans NagA produced by A. type and the DnagA strains grown on YG and MM- oryzae actedonbothpNP-b-GlcNAc and pNP-b- glucose media. This observation indicates that NagA GalNAc. This result suggests that NagA has broad is not essential for the growth of the ˆlamentous substrate speciˆcity for using several compounds fungi on the medium containing easily metabolizable containing N-acetyl hexosamines. carbon sources. The DNAG1 (DnagA) showed no The reverse hydrolysis activity of b-N-acetyl- growth on a medium containing chitobiose as a sole glucosaminidases derived from jack bean and Bacil- carbon source. This result suggests that the main role lus circulans were used for synthesis of GlcNAcb 1- of NagA in A. nidulans is assimilation of chitin 2Man and Galb 1-4GlcNAcb 1-2Man.28) We expect degradation products. that NagA is readily produced on a large scale by A. When we grew a liquid culture of the nagA ex- oryzae and is applicable to enzymatic syntheses of pressing yeast, no b-N-acetylglucosaminidase activity complex type sugar chains containing GlcNAc or was detected in the medium (data not shown). b-N- GalNAc as components. Acetylglucosaminidase activity of S. cerevisiae ex- pressing the C. albicans HEX1 gene is mostly in the References periplasm.25) The nagA gene product was probably in the periplasm of yeast cells. 1) Cabib, E., The synthesis and degradation of chitin. Although the nagA-overexpressing strain ONAG1 Adv. Enzymol., 59, 59–101 (1987). produced a very low level of b-N-acetylglucosamini- 2) Flach, J., Pilet, P. E., and Jolles, P., What new in chitinase research? 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