Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: purification and characterization

Protein Expression and PuriWcation 38 (2004) 196–204 www.elsevier.com/locate/yprep

Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: puriWcation and characterization Binesh Shresthaa,b, Karine Blondeaub,¤, Willem F. Stevensa, Françoise L. Hegaratb a

Food Engineering and Bioprocess Technology Program, Asian Institute of Technology, Klong Luang, Pathumthani 12120, Thailand b Institut de Génétique et Microbiologie, Université Paris-Sud, Bât 360, Orsay 91405, France Received 28 May 2004, and in revised form 1 August 2004 Available online 5 October 2004

Abstract The chitin deacetylase gene from Colletotrichum lindemuthianum UPS9 was isolated and cloned in Pichia pastoris as a tagged protein with six added terminal histidine residues. The expressed enzyme was recovered from the culture supernatant and further characterized. A single-step puriWcation based on speciWc binding of the histidine residues was achieved. The puriWed enzyme has a molecular mass of 25 kDa and is not glycosylated as determined by mass spectrometry. The activity of the recombinant chitin deacetylase on chitinous substrates was investigated. With chitotetraose as substrate, the optimum temperature and pH for enzyme activity are 60 °C and 8.0, respectively. The speciWc activity of the pure protein is 72 U/mg. One unit of enzyme activity is deWned as the amount of enzyme that produces 1
mol of acetate per minute under the assay conditions employed. The enzyme activity is enhanced in the presence of Co2+ ions. A possible use of the recombinant chitin deacetylase for large-scale biocatalytic conversion of chitin to chitosan is discussed.  2004 Elsevier Inc. All rights reserved. Keywords: Chitin; Chitin deacetylase; Chitosan; Colletotrichum lindemuthianum; Pichia pastoris

The marine waste product chitin, a principal constituent of crustacean carapace, is a polymer of N-acetyl glucosamine. It can be converted into chitosan, the deacetylated form that has many applications in the medical and industrial sectors. Due to the high crystallinity and insolubility of chitin in common solvents, this deacetylation can only be accomplished using harsh chemical conditions. Alternatively, deacetylation can be achieved using the enzyme chitin deacetylase (CDA, EC 3.5.1.41), but for the same reasons, only to a very limited extent. CDAs have been isolated from the fungi Mucor rouxii [1,2], Colletotrichum lindemuthianum (two strains) [3,4], Absidia coerulea [5], Saccharomyces cerevisiae [6], and Aspergillus nidulans [7]. All the enzymes are glyco-

*

Corresponding author. Fax: +33 69 15 63 34. E-mail address: [email protected] (K. Blondeau).

1046-5928/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.08.012

proteins and are secreted either into the periplasmic space or into the culture medium. The genes encoding CDA from M. rouxii [8,9], S. cerevisiae [6,10–12], and C. lindemuthianum ATCC56676 [13,14] have been cloned and the corresponding enzymes have been puriWed. The enzyme of C. lindemuthianum can act on natural chitin [3] whereas other deacetylases can act only on soluble derivatives of chitin or partially deacetylated chitin (PDC). Production and puriWcation of suYcient quantities of native CDA from C. lindemuthianum remains diYcult. The purpose of this work is to apply recombinant DNA techniques to obtain suYcient enzyme for its characterization. In this paper we report the isolation of the cda open reading frame (ORF) from C. lindemuthianum UPS9 and its cloning in Pichia pastoris. An expression system was established for production of a large amount of active CDA. To facilitate puriWcation of the recombinant

B. Shrestha et al. / Protein Expression and PuriWcation 38 (2004) 196–204

protein, a 6£ His tag was added to the C-terminus. The excretion of recombinant CDA in the culture medium of P. pastoris was achieved using the natural secretion signal from C. lindemuthianum and the -secretion signal sequence from S. cerevisiae. A pure non-glycosylated enzyme, active on chitinous substrates, was obtained by a very simple puriWcation method. Its activity has been compared with that of CDAs from other sources.

Materials and methods Materials A multi-copy Pichia expression kit was purchased from Invitrogen, and restriction enzymes, DNA-modifying enzymes, T4-DNA ligase, and Taq DNA polymerase were from Promega. The DNA gel-extraction kit, miniand midi plasmid kit were from Qiagen. Genomic DNA and cDNA libraries of C. lindemuthianum UPS9 were kindly provided by Dr. Richard Laugé, Institut de Biotechnologie des Plantes, Université Paris-XI, France. N-Acetyl chito oligosaccharides (trimer, tetramer, pentamer, and hexamer) were purchased from Sigma. Enzymes and reagents for acetate determination were obtained from Boehringer–Mannheim. A HiTrap chelating column was purchased from Amersham Biosciences, whereas ultraWltration membranes were from Amicon and Millipore. Dialysis cassettes (Slide-A-Lyzer) were from Pierce. All reagents were of analytical grade unless otherwise stated. Strains, plasmids, and media The host P. pastoris strain GS115 (his4) from the multi-copy Pichia expression kit was taken for expression of CDA and grown, transformed, and analyzed according to the manufacturer’s instructions [15]. A shuttle vector pPIC9K carrying the secretion signal of the -mating factor sequence from S. cerevisiae was used. LB medium (10 g bacto-tryptone, 5 g bacto-yeast extract, and 10 g sodium chloride in 1 L) and YEPD medium (10 g yeast extract, 20 g peptone, and 20 g dextrose in 1 L) were used for routine growth and maintenance of Escherichia coli and P. pastoris cells, respectively. LB medium supplemented with ampicillin (100
g/mL) was used to screen the E. coli transformant clones. The buVered glycerol-complex (BMGY) medium pH 6.0 contained per liter 10 g yeast extract, 20 g peptone, 100 mM potassium phosphate, 13.4 g yeast nitrogen base without amino acids (YNB w/o AA), 400
g biotin, and 10 mL glycerol. The buVered methanol-complex (BMMY) medium contained all the components as of BMGY except glycerol is replaced by 5 mL of methanol per liter. The regeneration dextrose base (RD) plates

197

contained 1 M sorbitol, 20 g dextrose, 13.4 g YNB w/o AA, 400
g biotin, L-glutamic acid, L-methionine, Llysine, L-leucine, and L-isoleucine each at 0.05 g and 15 g agar in 1 L. The minimal dextrose (MD) medium contained 13.4 g YNB w/o AA, 400
g biotin, and 20 g dextrose per liter. The minimal methanol (MM) medium contained 13.4 g YNB w/o AA, 400
g biotin, and 5 mL methanol per liter. Synthetic medium (SM) for the growth of Pichia in a bioreactor and preculture preparation comprised the following per liter: glycerol 40 g, FM21 [H3PO4, 5.09 g; K2SO4, 2.38 g; CaSO4 · 2H2O, 0.15 g; MgSO4 · H2O, 1.95 g; and KOH, 0.65 g]; trace elements PTM1 [H3BO3, 0.02 mg; CuSO4 · 5H2O, 6 mg; KI, 0.1 mg; MnSO4 · H2O, 3 mg; Na2MoO4, 0.2 mg; ZnCl2, 20 mg; FeSO4 · 7H2O, 65 mg; and H2SO4, 0.035 g]; and biotin 80
g. The preculture SM medium comprised SM medium (as above) supplemented with nitrogen sources of 4 g (NH4)2SO4 and 12 g H2PO4NH4 in a tartrate buVer (0.1 M, pH 6) in an Erlenmeyer Xask during preculture preparation. Ammonia (15% v/v) was used in the bioreactor culture both to regulate the pH and as a source of nitrogen. The synthetic medium for the fed-batch culture contained 780 g methanol, 5£ PTM1, and biotin 1000
g/L. Casamino acids (1.3 g/L) were directly added in the bioreactor at the beginning of the fed batch to minimize extracellular protease activity. Recombinant DNA techniques and DNA sequencing Standard recombinant DNA methods were carried out according to the methods described in Sambrook et al. [16]. The sequence was determined by dideoxy DNA cycle sequencing using a BigDye terminator Version 3 Cycle Sequencing Kit (Applied Biosystems). Chitin deacetylase gene ampliWcation and cDNA isolation The ORF of CDA was ampliWed from the genomic DNA of C. lindemuthianum UPS9 by using Primer I (5⬘GGAATTCCATATGCACTTCTCGACCCTTCTT-3) and Primer II (5⬘-ACAGCACAGCGGCCGCTTACG CCTTGTACCA-3⬘), synthesized on the basis of the published DNA sequence of the cda gene [13]. Primer III (5⬘-TGGTTTTGCGGCCGCTTAATGGTGATGGTGA TGGTGCGCCTTGTACCAGTTCTC-3⬘) was synthesized with additional introduction of nucleotides encoding the 6£ His tag (italics). ArtiWcial restriction sites for EcoRI (forward) and NotI (backward) were introduced (underlined characters in Primers I, II, and III) in order to achieve directional cloning in phase with the -factor prepro-signal sequence from S. cerevisiae to target the protein to the secretory pathway [17]. AmpliWcation was performed with 20 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min 30 s using a GeneAmp PCR System 2700 from Applied Biosystems. Primers I and II were

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used to amplify the DNA fragment from the genomic DNA of C. lindemuthianum UPS9 that was used as a 32P labeled probe to screen the -phage cDNA library. Phage DNA from a positive clone was extracted and used as a template for polymerase chain reaction using Primers I and III in order to amplify the cDNA encoding the CDA protein. Construction of expression vector and transformation The cDNA fragment isolated was digested by EcoRI and NotI, and puriWed using the QIAquick gel puriWcation kit [18]. The fragment was ligated into the multiple cloning site of the shuttle vector pPIC9K containing the ampicillin resistant marker (Ampr), previously digested with the same set of restriction endonucleases and puriWed. The cda ORF was integrated under the control of the methanol inducible alcohol oxidase promoter (AOX1) of P. pastoris. After transformation of E. coli strain TOP10F⬘ with the pPIC9Kcda construct by electroporation, transformants were selected on LB medium containing ampicillin, 100
g/mL. Plasmid DNA was extracted using a Qiagen plasmid midi kit and the part containing cda ORF was analyzed by sequencing to conWrm the correct integration of cda ORF in phase with the gene encoding -factor secretion signal sequence of S. cerevisiae. Approximately 10
g each of pPIC9Kcda and pPIC9K (as negative control) plasmids were linearized with SalI and transformed to competent P. pastoris cells (GS115 his4) by electroporation (1500 V, 25
FD, and 200 ). This resulted in the integration of the construct in the genome of P. pastoris by homologous recombination. Transformants were selected on RD plates, screened for methanol utilization by patching on MD and MM plates, and scored as Muts (methanol slow utilizing strain) and Mut+ (fast methanol utilizing strain). Cultivation in bioreactor A Mut+ transformant of P. pastoris was used for production of CDA in a bioreactor. A freshly grown single colony was inoculated in 10 mL of YEPD medium (preculture I) and grown overnight at 30 °C with 250 rpm. Preculture I was transferred into 100 mL of preculture SM medium and incubated overnight to obtain preculture II which was subsequently transferred into 1 L of preculture SM medium and incubated overnight to obtain preculture III. A part of it was used to inoculate a bioreactor containing a batch of 1.2 L SM medium keeping the initial OD600 nm around 2. The pH was adjusted at 6, aeration was one volume of sterile air per volume of medium per minute and the oxygen saturation was maintained over 20% by vigorous stirring (1000– 1800 rpm). Upon depletion of glycerol, »24 h after inoculation, a second batch culture was started by adding

new SM medium components directly in the bioreactor. When the entire carbon source was consumed, induction by methanol was initiated with a gradual supply of the feed-medium, the feeding proWle being monitored using a computer-controlled pump to maintain the biomass yield YX/S of 0.3 g biomass per gram of methanol and a theoretical speciWc growth rate of 0.015 per hour. Induction was continued for 72 h after the Wrst addition of methanol. Cell growth was monitored by measuring OD600 using a Hitachi U1100 spectrophotometer. One unit corresponds to 0.15 g of dry cell weight per liter. The residual methanol was analyzed by a FFJA-HPLC column (Waters) using an isocratic mode of 0.8 mL/min of orthophosphoric acid (5 mM). Detection of methanol was performed by refractometry. PuriWcation of recombinant CDA Cells were removed from the culture medium by centrifugation (6500g, 15 min at 4 °C). The clear supernatant (1 L) containing recombinant CDA-6£ His was adjusted to 10 mM imidazole, 50 mM NaH2PO4 and 300 mM NaCl (pH 8.0), Wltered through a 0.2
m membrane Wlter and directly loaded on a HiTrap chelating HP column (5 mL, Pharmacia Biotech) with a Xow rate of 4.5 mL/ min. After sample loading the remaining steps were carried out with Xow rate of 2.5 mL/min. Unbound proteins were eluted with 5 column volumes of 50 mM NaH2PO4 (pH 8.0) buVer containing 300 mM NaCl and 20 mM imidazole. The recombinant CDA-6£ His was eluted with a linear gradient of imidazole (20 ! 300 mM, achieved in 9 column volumes) in the same buVer. The maximum amount of protein was eluted in the third and fourth column volumes. Fractions were collected on the basis of protein concentration as detected by UV280 and assayed for the CDA activity. The fractions with CDA activity were pooled and concentrated using an ultraWltration membrane (Millipore, cut-oV value 10 kDa) and dialyzed against Tris–HCl (30 mM, pH 8.0). Enzyme activity assay CDA activity was determined by an enzymatic assay described by Bergmeyer [19] with modiWcation. Acetate released by the action of CDA on various chitinous substrates was determined via three coupled enzyme reactions. NADH produced was excited at 350 nm and the Xuorescence was measured at 460 nm in a spectroXuorimeter (SFM25 Bio-Tek) to estimate the amount of NADH generated. Enzymatic analysis was carried out by using 50
g of tetra-N-acetyl chitotetraose as substrate in 50 mM phosphate buVer, pH 6.0, in a total volume of 100
L. The incubation time was 15 min at 50 °C. The reaction was terminated by heating at 100 °C for 10 min in a sealed tube prior to acetate determination. One unit of CDA activity is deWned as

B. Shrestha et al. / Protein Expression and PuriWcation 38 (2004) 196–204

1
mol of acetate released per minute under the conditions used above. Polyacrylamide gel electrophoresis and protein concentration determination Polyacrylamide gels (12%) under denaturing and reducing conditions were run according to Laemmli [20]. Protein bands were visualized by staining with Coomassie brilliant blue R and SilverXpress silver staining (Invitrogen). Protein content was determined using a BCA protein assay kit (Pierce). Bovine serum albumin was used as a standard. Mass spectrometry All spectra were acquired in positive-ion mode on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems) equipped with a 337 nm nitrogen laser. Determination of the molecular mass of CDA was performed in linear mode (accelerating voltage 25 kV, grid voltage 93%, guide wire 0.3%, delay 600 ns) with external calibration. Protein solutions were diluted to 40
M with 30% acetonitrile, 0.3% triXuoroacetic acid. Of this solution, 4
L was mixed with 6
L of saturated solution of sinapinic acid in the same diluent and 1.5
L of this premix was then deposited onto the sample plate, allowed to dry at room temperature, and analyzed.

Results Isolation and sequencing of the CDA gene The PCR product obtained from genomic DNA of C. lindemuthianum UPS9 and cda ORF ampliWed from a positive cDNA -phage clone were sequenced and compared. The genomic DNA was found to contain an ORF of 806 bp encoding a preprotein (248 amino acids) with a signal peptide (27 amino acids) and an intron of 62 bp. The ampliWed DNA fragment from genomic DNA of C. lindemuthianum UPS9 showed 98% identity with that of C. lindemuthianum ATCC56676 [13]. Nevertheless, 15 diVerences were observed in nucleotides which were not due to PCR errors since the sequence has been conserved in another strain of C. lindemuthianum (data not shown). Among the 15 diVerences observed, seven indicated diVerences in codon usage for the same amino acid and eight confer changes in amino acids (Table 1). The Wrst change, Leu7 to Phe7, introduces a substitution in the

199

signal peptide which might modify the secretion eYciency of the enzyme by the fungus. It is not known if these changes confer structural or activity variations between the enzymes produced. A comparison has been made between the CDA activity of culture supernatant of C. lindemuthianum UPS9 and C. lindemuthianum ATCC56676 [4] with chitin-50 as substrate (chitin with 50% deacetylated glucosamine units). The speciWc activities are comparable, 0.02 and 0.0195 U/mg, respectively. However, considering that several changes in the amino acid sequence introduce some changes in the catalytic properties of CDA, further study on CDA from C.lindemuthianum UPS9 was carried out. To study pure CDA, cloning and expression of the cda ORF in P. pastoris and characterization of the expressed enzyme were performed in order to explore the properties of recombinant CDA. Cloning of cda ORF and expression of CDA in P. pastoris Upon transformation of P. pastoris with SalI linearized pPIC9Kcda construct, over 2000 clones per microgram of DNA were generated on RD plates (medium without histidine). The clones were screened for methanol utilization following the standard protocol as described in the multi-copy Pichia expression kit manual. All clones were fast methanol utilizing strains (Mut+). Ten Mut+ clones randomly selected were grown overnight in BMGY medium containing glycerol, transferred, and subsequently induced for CDA expression in 100 mL medium containing 0.5% methanol (BMMY) for 96 h. Production and secretion of CDA was analyzed by SDS–PAGE of samples from the culture supernatant. After concentration of the supernatant 10 times, all clones showed a major polypeptide band of »30 kDa which was not detected from the culture supernatant of Pichia transformed with pPIC9K vector without insert. Simultaneously, several other unknown proteins of high molecular weight were detected as well. One clone out of 10 was selected for expression in a bioreactor. The clone was grown in a batch culture till the biomass reached about 20 g/L. The CDA production was induced by a methanol-containing feed during fed-batch culture. The slow methanol addition allows suYcient time for the Pichia clone to utilize all the methanol supplied and avoid accumulation of this toxic carbon source. Samples from the bioreactor were analyzed every 6 h for residual methanol and the latter was not detected throughout the induction period. The total biomass increased exponentially with an observed speciWc growth rate of 0.02 per

Table 1 Comparison of amino acids of CDA from C. lindemuthianum ATCC and UPS9 strains C. lindemuthianum UPS9 C. lindemuthianum ATCC

Phe7 Leu7

Pro57 Ala57

Arg69 Lys69

His125 Gln125

Val126 Leu126

Asn189 Asp189

Note. Complete sequence of CDA C. lindemuthianum UPS9 is deposited in GenBank (Accession No. AY633657).

Ala194 Gly194

Lys219 Arg219

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hour against a theoretical value of 0.015 per hour and 90 g biomass was produced per liter at the end of the cultivation (Fig. 1). The yield was calculated to be »0.26 g dry weight per gram methanol which is close to the expected theoretical value employed in modeling the bioreactor cultivation. Samples of culture supernatant were collected at diVerent times of growth and analyzed by SDS–PAGE. CDA in the crude supernatant could not be detected as a separate protein band on SDS– PAGE (Fig. 2) due to the presence of other proteins. Therefore an enzymatic assay was performed to demonstrate the presence of CDA. The production of CDA started 24 h after methanol induction and continued to increase till the end of fermentation period, i.e., 72 h after

Fig. 1. Production of biomass and CDA in a fed-batch culture. Induction of enzyme synthesis started at time D 0 h. Enzyme activity is measured as acetate released from the substrate tetra-N-acetyl chitotetraose as described in Materials and methods.

induction with an activity of 76 mU/mL (Fig. 1). The speciWc activity of the crude supernatant from P. pastoris was found to be 0.01 U/mg when chitin-50 was used as substrate. This result is in the same range as the speciWc activity of the crude supernatant from C. lindemuthianum UPS9 obtained previously under the same assay conditions. The speciWc activity of the recombinant enzyme from crude culture supernatant using tetra-Nacetyl chitotetraose as a substrate showed a higher value of 0.197 U/mg of protein. PuriWcation of recombinant CDA The puriWcation of was performed in only one step using a HiTrap Chelating HP column and details are provided in Table 2. After elution with imidazole/NaCl the active fractions were pooled. In a typical experiment, a puriWcation factor of 364 could be achieved with an overall yield of 90% of the total activity of the supernatant. SDS–PAGE analysis showed only one band at a position corresponding to a molecular mass of »30 kDa (Fig. 2) using Coomassie blue staining and the silver staining method. The pure protein was submitted to mass spectrometry analysis. The molecular mass was determined to be 25.19 kDa (Fig. 3) as predicted from the amino acid sequence of the cda ORF plus 6 £ His tag. Two major peaks that correspond to the mass of CDA were observed (Fig. 3) with a diVerence of 135 g/ mol corresponding to the mass of Gln28. It might be due to the proteolytic cleavage of the CDA or to a diVerence

Fig. 2. Electrophoretic analysis of recombinant CDA. Samples were run in a 12% polyacrylamide gel under denaturing and reducing conditions. Protein was visualized by staining with Coomassie blue R (1–6) and silver staining (7, 8) to conWrm homogeneity. Lanes 1, 2, and 3: crude bioreactor culture supernatant from 43, 48, and 67 h, respectively (»10
g); lane 4, 6, and 7: molecular weight markers; lane 5 : puriWed CDA (5
g); and lane 8: puriWed CDA (0.5
g).

Table 2 Single-step puriWcation of C. lindemuthianum CDA from P. pastoris Step

Total activity (U)

Total protein (mg)

SpeciWc activity (U/mg)

PuriWcation factor

PuriWcation yield (%)

Supernatanta Ni-agarose

570 516

2892 7.2

0.197 71.67

1 364

100 90

One unit of enzyme activity is deWned as the amount of the enzyme required to produce 1
mol of acetic acid per minute under standard conditions as described in Materials and methods. The CDA assay was carried out at 50 °C. a The volume of supernatant was 1 L.

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201

Fig. 3. MALDI-TOF mass spectra analysis of pure CDA obtained from recombinant P. pastoris. CDA was analyzed as described in Materials and methods. The highest peak corresponds to the mass of amino acid sequence Q28¡A248 + 6£ His and the second peak corresponds to V29¡A248 + 6£ His of recombinant CDA.

in signal peptide cleavage. The molecular mass represents the total mass of amino acids excluding the predicted signal peptide. This analysis also indicates that the protein is not glycosylated, unlike native CDAs from C. lindemuthianum [3,4], M. rouxii [2], and S. cerevisiae [11]. Enzyme activity characterization The eVect of temperature on the activity and the stability of the enzyme is shown in Fig. 4. The optimum temperature for enzyme activity was about 60 °C (Fig. 4A) when tetra-N-acetyl chitotetraose was used as a substrate. The enzyme was stable at high temperatures. More than 50% of the activity was retained after heating of the enzyme at 80 °C for 1 h or at 90 °C for 30 min (Fig. 4B). The optimum pH for activity was 8.0 (Fig. 5A). The eVect of pH on enzyme stability was studied by incubating the enzyme in buVers (50 mM) of diVerent pH at ambient temperature (about 16 °C) for 18 h. It has been observed that incubating the enzyme at pH 8.0 increases the activity by more than 50% (Fig. 5B). The enzyme lost its activity drastically when incubated at a pH less than 5. However, it is stable at higher pH ranges. More than 70% of the total activity was retained after treatment of the enzyme in the pH range 6–12. The enzyme activity in the presence of metal ions was also analyzed and the results are presented in Table 3. The activity was enhanced 26% by adding Co2+ ions to the reaction mixture. Li+ and Mn2+ ions had no eVect on the enzyme activity. Mg2+ and Ca2+ ions had no eVect at a lower concentration of metal but were inhibitory at a higher concentration. The lower concentration of Fe2+ or Cu2+ has no or little eVect but the higher concentration drastically decreases the activity. Ni2+ and Zn2+ clearly inhibited the CDA activity.

Fig. 4. EVects of temperature on the activity (A) and stability (B) of CDA. Enzyme (3 mU) was assayed at various temperatures (A). The same amount of enzyme was subjected to incubation at various temperatures in phosphate buVer (50 mM), pH 8.0, for diVerent periods of time; the remaining activity was measured (B).

Deacetylation of chitinous substrates Recombinant CDA was analyzed for its activity on various chito oligosaccharide substrates. Four diVerent N-acetyl chitooligomers were incubated with enzyme and the amount of acetate released was measured (Table 4). The puriWed enzyme could deacetylate tetra-N-acetyl chitotetraose, penta-N-acetyl chitopentaose, and hexaN-acetyl chitohexaose. The enzyme activity was increased with the number of N-acetyl chitooligomers in N-acetyl-D-glucosamine residues (Table 4). No activity was detected on chitotriose. Preliminary analysis also showed that the crude enzyme from the culture supernatant was active on partially deacetylated chitin (chitin with 50, 65, 70, and 82% degree of deacetylation). No enzyme activity was detected on chitin with 92 and 96% degree of deacetylation (Fig. 6).

Discussion The diVerences in amino acid sequences of CDA of C.lindemuthianum UPS9 observed in the present study did not alter the speciWc activity in crude culture supernatant, when compared with that of C. lindemu-

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Fig. 6. CDA activity on partially deacetylated chitin (CT) of crude culture supernatant. CT-50, CT-65, CT-70, CT-82, CT-90, and CT-96 represent chitin with 50, 65, 70, 82, 90, and 96% degree of deacetylation. One unit of enzyme activity is deWned as 1
mol of acetate released per minute. The enzyme assay was carried out by incubating 1 mL of crude culture supernatant with 5 mg of each substrate at 50 °C.

Fig. 5. EVect of pH on activity (A) and stability (B) of CDA. Pure CDA (3 mU) was incubated with diVerent buVers for 15 min at 60 °C (A). The same amount of enzyme was subjected to diVerent pH at ambient temperature for 18 h and the remaining activity was measured (B). The diVerent buVers used are: citric acid–Na2HPO4, 50 mM (pH 3.0–6.0), KH2PO4–K2HPO4, 50 mM (pH 7.0–8.0), and glycine–NaCl– Na2HPO4, 50 mM (pH 9.0–12.0).

thianum ATCC56676 [4]. The substitution of Ala57 by Pro57 may introduce an extra bend in a chain and a change in amino acid (Gln125 to His125) introduces a cyclic amino acid which may cause a prominent change in a polarity and charge distribution in the polypeptide chain. However, no signiWcant inXuence of these changes

was detected on the CDA activity under the present conditions of enzymatic activity study. In this report we describe the cloning of the CDA gene from the fungus C. lindemuthianum UPS9 and its expression in the yeast P. pastoris using a high cell density cultivation method. The CDA has been expressed as a secreted protein using two diVerent secretion signals, namely, the -secretion signal factor and the native secretion signal from C. lindemuthianum together. In another series of experiments it has been shown that the native secretion signal from C. lindemuthianum by itself is suYcient to express the CDA in culture supernatant (data not shown). Under the same conditions of enzymatic assay using crude culture supernatant on chitin-50 as substrate, the recombinant enzyme and native enzyme seem to have comparable speciWc activity, 0.01 and 0.02 U/mg, respectively. Since culture supernatants are complex media the activity measurement might not be representative.

Table 3 EVects of metal ions on CDA activity Concentration of metal (mM)

Co2+

Li+

Mn2+

Mg2+

Ca2+

Fe2+

Cu2+

Ni2+

Zn2+

Control

0.1 1.0 10.0

126 118 94

105 106 108

103 107 98

105 103 96

106 108 72

101 89 37

85 88 50

43 49 51

45 44 44

100 100 100

Standard deviation among three replicates was in the range between 0.6 and 2.2% activity. Table 4 Deacetylation of N-acetyl chitooligosaccharides by recombinant CDA from P. pastoris Substrate

Acetate released (n mol/mM substrate)a

SpeciWc CDA activity (U/mg)a

Km (mM)

Vmax (nmol of acetate produced per minute)

(GlcNAc)3 (GlcNAc)4 (GlcNAc)5 (GlcNAc)6

No activity 166.3 195.2 332.0

— 138.5 135.6 184.4

— 8.9 5.7 1.5

— 100 81 42

a PuriWed enzyme (48 ng) was used in each of the assays, which were performed in a total volume of 0.1 mL of 50 mM phosphate buVer (pH 8), along with 50
g of each substrate. Incubation was 15 min at 60 °C and the reaction was terminated by heating at 100 °C for 10 min in a sealed tube prior to acetate determination. Acetate released by the action of CDA was determined via three coupled enzyme reactions [19] with modiWcation by microXuorimetry (article in preparation).

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203

optimum temperature and pH for enzyme activity were determined to be 60 °C and 8.0, respectively. Under this optimal conditions, the speciWc activity of recombinant CDA was increased by two times and became 138.5 U/mg when tetra-N-acetyl chitotetraose was used as a substrate. As the enzyme was more active on larger oligomers, the highest speciWc activity of 185 U/mg was obtained on N-acetyl chitohexaose among the three substrates. The enzyme is more active on larger oligomers of acetyl glucosamine. We have compared the activity of puriWed CDA from recombinant P. pastoris with other CDAs from various sources. It was found that the CDA preparation obtained with P. pastoris is more active in the deacetylation of these substrates than the other CDA preparations. Due to scarcity of data for comparison and diVerences in the conditions of enzyme activity measurements it is not easy to make a straightforward comparison. It is noteworthy that all the enzymatic assay methods were based on the quantitation of acetic acid released per minute under the standard conditions employed but the sensitivity of the methods is diVerent. However, it seems justiWed to conclude that the present enzyme preparation is at least one order of magnitude more suitable as a source of CDA (Table 5). Preliminary experiments showed that the activity of crude supernatant on partially deacetylated chitin is promising. The higher the percentage of acetylation of the substrates, the more enzyme activity is detected. Further study is in progress to explore the eVectiveness of the puriWed enzyme on native chitin. The stability of the enzyme at high temperatures and pH makes the enzyme attractive for technical biocatalytic deacetylation of chitin. The high-temperature stability is beneWcial, because it will limit the growth of most microorganisms under processing conditions. The pH stability of the enzyme in

We have developed a rapid and single-step puriWcation of the 6£ His tagged CDA from culture supernatant that yielded a protein with electrophoretic homogeneity. The puriWcation method employed in this study gives an advantage over conventional multi-step puriWcation methods [3,4]. Concentrating proteins, multi-step puriWcation, dialysis, and other intermediate steps cause signiWcant loss of activity and yield of the protein. In the present study, 7.2 mg of pure protein could be obtained in one day from culture supernatant without prior concentration. The yield of the puriWcation has been shown to be reproducible in four diVerent puriWcations performed. The puriWed protein is highly active and has a speciWc activity of 72 U/mg despite the presence of the 6 £ His-tag at the C-terminus of the recombinant protein. The molecular mass as determined by mass spectrometry, 25.2 kDa, of the puriWed protein was in accordance with the amino acid sequence of the mature protein as predicted from the nucleotide sequence excluding the signal peptide. The enzyme is non-glycosylated. This result is supported by the observation that the deduced amino acid sequence of CDA lacks a potential glycosylation site (Asn-X-Ser/Thr) that is recognized by P. pastoris. Despite the absence of glycosylation, the enzyme is active unlike native CDAs from various fungi [2–4,11]. Jaspar-Versali and Clerisse [9] have reported the expression of active glycosylated recombinant CDA of M. rouxii. In another study, total loss of enzyme activity was observed after deglycosylation of the recombinant Cda2p from S. cerevisiae [11]. The activity of the deglycosylated enzyme was restored by adding 1 mM CoCl2. In our case, the recombinant CDA is active without glycosylation and in the absence of Co2+ but is signiWcantly activated by Co2+ ions. Furthermore, the enzyme exhibits very good thermal stability and stability towards a pH range of 6–12. The Table 5 Production, yield and activity of CDA activity from diVerent sources Source

Enzyme type Culture volume Yield (pure enzyme) Amount used/CDA assay Acetate released (nmol/mM of) (GlcNAc)1 (GlcNAc)2 (GlcNAc)3 (GlcNAc)4 (GlcNAc)5 (GlcNAc)6 Reference

P. pastoris

C. lindemuthianum (DSM63144)

C. lindemuthianum (ATCC56676)

S. cerevisiae Cda2p

Heterologous expression from C. lindemuthianum UPS9 1L 7.2 mg 48 ng

Native

Native

2L 930
g 4
g

10 L 656
g —

Overexpression from S. cerevisiae itself 30 g (»2.5 L) 60
g 2.6
g

NA NA 0 166.3b 195.2b 332.0b Present report

0 0 0 29.2c 36.3c 45.42c [3]

NA NA NA NA NA NA [4]

0 35.2a 87.5a 88.9a NA 241.7a [11]

NA, not available. All activities are measured in terms of acetate liberated. a Reaction was carried out for 48 h at 50 °C. b Reaction was carried out for 15 min at 60 °C. c Reaction was carried out for 15 min at 50 °C.

204

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diluted alkaline solutions will allow us to carry out enzymatic deacetylation at a pH at which chitosan does not swell and does not generate a high viscosity that would limit the mobility of the enzyme. The present method of puriWcation described in this work gives access to a large source of pure CDA and oVers the opportunity to further study the action mechanism of CDA on solid and semi-solid substrates. In the near future, the production and puriWcation of large quantities of active CDA from recombinant Pichia will provide the opportunity to elucidate its structure in relation to its mechanism of action and oVer an enzyme source for biocatalytic conversion of chitin into chitosan. Acknowledgments We thank Dr. Richard Laugé for providing cDNA library of C. lindemuthianum. Prof. Armel Guyonvarch is acknowledged for his excellent idea in developing the CDA assay and for providing the bench to work on. We acknowledge the help of Dr. Paulette Decottignies and Dr. Christophe Marchand for mass spectrometry analysis. We thank Dr. Rachel M. Exley, Imperial College, London for her critical review and for correcting the English. Mr. Binesh Shrestha would like to express his sincere gratitude to French Co-operation, the Austrian government, and Asian Institute of Technology, Thailand for support provided. References [1] Y. Araki, E. Ito, A pathway of chitosan formation in Mucor rouxii. Enzymatic deacetylation of chitin, Eur. J. Biochem. 55 (1975) 71–78. [2] D. Kafetzopoulos, A. Martinou, V. Bouriotis, Bioconversion of chitin to chitosan: puriWcation and characterization of chitin deacetylase from Mucor rouxii, Proc. Natl. Acad. Sci. USA 90 (1993) 2564–2568. [3] I. Tsigos, V. Bouriotis, PuriWcation and characterization of chitin deacetylase from Colletotrichum lindemuthianum, J. Biol. Chem. 270 (1995) 26286–26291. [4] K. Tokuyasu, M. Ohinishi-Kameyama, K. Hayashi, PuriWcation and characterization of extracellular chitin deacetylase from Colletotrichum lindemuthianum, Biosci. Biotechnol. Biochem. 60 (1996) 1598–1603.

[5] X.D. Gao, T. Katsumoto, K. Onodera, PuriWcation and characterization of chitin deacetylase from Absidia coerulea, J. Biochem. 117 (1995) 257–263. [6] A. Christodoulidou, V. Bouriotis, G. Thireos, Two sporulationspeciWc chitin deacetylase-encoding genes are required for the ascospore wall rigidity of Saccharomyces cerevisiae, J. Biol. Chem. 271 (1996) 31420–31425. [7] C. Alfonso, O. Nuero, F. Santamaria, F. Reyes, PuriWcation of a heat stable chitin deacetylase from Aspergillus nidulans and its role in cell wall degradation, Curr. Microbiol. 30 (1995) 49–54. [8] D. Kafetzopoulos, G. Thireos, J. Vournakis, V. Bouriotis, The primary structure of a fungal chitin deacetylase reveals the function for two bacterial gene products, Proc. Natl. Acad. Sci. USA 90 (1993) 8005–8008. [9] M.-F. Jaspar-Versali, F. Clerisse, Expression and characterization of recombinant chitin deacetylase, in: A. Domard (Ed.), Advances in Chitin Science, 7th ICCC, vol. 2, Jaques Andre Publisher, 1998, 273–278. [10] C. Mishra, C.E. Semino, K.J. McCreath, H. de la Vega, B.J. Jones, C.A. Specht, P.W. Robbins, Cloning and expression of two chitin deacetylase genes of Saccharomyces cerevisiae, Yeast 13 (1997) 327–336. [11] A. Martinou, D. Koutsioulis, V. Bouriotis, Expression, puriWcation and characterization of a cobalt-activated chitin deacetylase (Cda2p) from Saccharomyces cerevisiae, Protein Expr. Purif. 24 (2002) 111–116. [12] A. Martinou, D. Koutsioulis, V. Bouriotis, Cloning and expression of a chitin deacetylase gene (CDA2) from Saccharomyces cerevisiae in Escherichia coli. PuriWcation and characterization of the cobalt-dependent recombinant enzyme, Enzyme Microb. Tech. 32 (2003) 757–763. [13] K. Tokuyasu, M. Ohnishi-Kameyama, K. Hayashi, Y. Mori, Cloning and expression of chitin deacetylase gene from a deuteromycete, Colletotrichum lindemuthianum, J. Biosci. Bioeng. 87 (1999) 418–423. [14] K. Tokuyasu, S. Kaneko, K. Hayashi, Y. Mori, Production of a recombinant chitin deacetylase in the culture medium of Escherichia coli cells, FEBS Lett. 458 (1999) 23–26. [15] Invitrogen. Multi-copy Pichia Expression Kit Version E: A Manual of Methods for the Isolation and Expression of Recombinant Proteins from Pichia pastoris Strains Containing Multiple Copies of a Particular Gene, 1999. [16] J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989. [17] J.M. Cregg, T.S. Vedvick, W.C. Raschke, Recent advances in the expression of foreign genes in Pichia pastoris, Biotechnology 11 (1993) 905–910. [18] Qiagen. QIAquick Spin Handbook, 2001. [19] H. Bergmeyer, Methods of Enzymatic Analysis, vol. 1, second ed., Verlag Chemie, Weinheim, 1974, pp. 112–117. [20] U. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685.