Molecular Cloning and Purification of an Endochitinase From Serratia

RESEARCH

Molecular Cloning and Purification of an Endochitinase From Serratia marcescens (Nima) AIejandro Ruiz-Sanchez, Ramon Cruz-Camaril10,~Ruben Salcedo-Hernandez, and Jose Eleazar Barboza-Corona*l Jorge E.

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Abstract An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DHSOLF',and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5'-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nirna showed high enzymatic activity within a broad pH range (pH 4.0-10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55°C. Index Entries: Serratia marcescens; Bacillus thuringiensis; chitinases; Cry proteins; fluorogenic substrates.

1. lntroduction Chitin is a homopolymer of l,4-B-linked Nacetyl-B-~glucosamine(GlcNAc) resictues, which is the main component of the exoskeleton of many invertebrate such as crustaceans, insects and spiders, and a stmctural component of the cell walls of most fungi and algae. Because of its composition, this organic compound also serves as a carbon and nitrogen source for many microorganisms. Chitin hydrolytic enzymes (chitinases) are generally classified as endochitinases (EC. 3.2.1.14), which hydrolyze chitin randomly at interna1 sites; and exochitinases, such as chitobiosidases, which cleave GlcNAc from nonreducing ends and release diacetilchitobiose and N-acetyl-B- 1,4-D-glucosaminidases(EC 3.2.1.30). In addition, chitobiases are active on chitobiose,

releasing- GlcNAc monomers (1).From a practica1 point of view, chitinases show potential biotechnological applications, as they may be used as control agents against phytopathogenic fungi as well as on important agricultura1 pest and vectors of diseases ( I d ) .Chitinases have been used in suppressing fungal phytopathogens such as Rhizoctonia solani ( 1 3 ,or in combination with the insecticida1 Cry proteins of Bacillus thuringiensis to enhance their activity against important crop pests such as Eldana saccharina (4) and Spodoptera littoralis (5). Moreover, chitinases may be used in severa1 biotechnological processes, such as the efficient use of shrimp wastes (7). Severa1 genera of bacteria, including Serratia (8,9),Enterohacter (1),and Aeromonas (lo),produce high levels of chitinolytic enzymes. Others,

*Author to whom al1 correspondence and reprint requests should be addressed. 'Instituto de Ciencias, Agrícolas, Universidad de Guanajuato, Guanajuato, Mexico. E-mail:[email protected].'Departamento de Microbiologia,Escuela Nacional de Ciencias Biologica, Instituto Politecnico Nacional, Mexico City, Me~ico.~Departamento de Biotecnología y Bioquímica, CINVESTAV, I.P.N. Irapuato, Gto., Mexico. Mdecular Bioteehndogy O 2005 Humana Press Inc. All ripMs d any nature whatsoevei reserved. 10750085/2005/31:2/103-11 W30.00

7 04 such as B. thuringiensis, produce chitinases in small amounts, although they can be genetically manipulated to improve their chitinase production ( 3 , l l ) .Serratia marcescens is one of the most efficient chitin-degrading agents, and different types of chitinases have been reported from several strains of íhis species, such as ChiA, ChiB, and ChiC1 ;additional to a proteolytic derivative of ChiC 1 (ChiC2), a chitobiase, and a putative chitinbinding protein (CBP;?l) (8).Several genes encoding for chitinases (chi) from S. marcescens strains 2170 (8,9), QMB1466 (12), KCTC2172 (13), BJL200 (14), 27 117 (GenBank accession number L01455), and AV305 (AF085718) have been reported. Although the S. marcescens (Nima) strain produces high levels of chitinases (14a), chi gene homologs have not been reported from this strain. In this article, we describe the cloning and sequence analysis of a chiA allele from S. marcescens (Nima), and the purification and partia1 characterization of the recombinant ChiA Nima protein.

2. Materials and Methods 2.1. Bacteria1 Strains and Plasmids Serratia marcescens (Nima) was kindly provided by R. P. Williams (Baylor College of Medicine) and is part of the ENCB-IPN (Mexico) bacteria1 collection. Al1 genetic constructs were amplified in Escherichia coli DH5aF' [supE44, D lacU169 (F80lacZ D M15) hsdR17 recAl end Al gyrA96 thi-1 relAl]. Plasmid pHT3101 was used as a cloning vector, which contains two replication origins, one functional in E. coli and the other in B. thuringiensis (15). 2.2. Cloning and Nucleotide Seguencing of chiA Gene Total genomic DNA from S. marcescens (Nirna) was extracted and purified as described elsewhere (3). This DNA was used as a template to amplify by polymerase chain reaction (PCR) the chiA gene. Primers were designed according to the initia1 and terminal conserved regions of severa1 reported chiA genes from S. marcescens. Oligonucleotides Sm-3 (5'-CTAGTCTAGAGTTGT

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CAATAATGACAACACCCTGGCTG-3') and Sm-4 (5'-ACATGCATGCCATTTTTAGCGC ATTGGTCTGACCCT-3') were used as forward and reverse primers, respectively, which included additional XhaI and SphI sites (underlined) to allow for direct cloning of amplicons into the pHT3101 vector. The PCR amplifications were performed with PCR SuperMix high fidelity enzymes (Invitrogen) in an iCycler thermocycler (Bio-Rad) for 30 cycles of 94°C for 1 min, 55°C for 1 min, 68°C for 4 min each, followed by a 7-min termination step at 68°C. The PCR product, which tentatively included the putative promoter and ribosome-binding site, was purified using the QIAquick gel extraction kit (Qiagen). The amplicon was then digested with XhaI and SphI, ligated into the same sites in the pHT3101 vector (15),and transfedinto E. coli by electroporation. Transformants were selected in Luria-Bertani (LB) agar supplemented with ampicillin (100 pg/mL) and screened by their activity on the fluorogenic chitin derivative 4-methylumbelliferyl-B-DN,N,N1-triacetylchitotriose[4-MU-(G1cNAc)J (tetrameric fluorescent derivative) and 4-methylumbelliferyl-P-D-NJV'-diacetylchitobioside[4MU-(G~CNAC)~] (trimeric fluorescent derivative) (Sigma) as described previously (3),in a reaction buffer containing 0.05 M citric acid and 0.1 M KH2P04,final concentrations (pH 6.0). Chitinase activity was measured in supernatants and cell extracts (3.11). One colony with high chitinase activity (E. coli DH5aFf/pHT3101-chiA Nirna) was selected and the recombinant plasmid (pHT3101chiA Nima) was recovered and purified with the plasmid midi kit (Quiagen). This plasmid was analyzed by restriction enzyme analysis and the chiA nucleotide sequence was determined by di&oxynucleoti& sequencing (16) using the Sm-3 and Sm-4 primers, as well as interna1 primers to obtain overlapping sequences. These were resolved by automated fluorescent sequencing with an ABI PRISM 377 DNA sequencer. Sequence was compared with other chitinase genes from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov), using the Blast and DNAStar packages.

Endochitinase From Serratia marcescens

2.3. Southern Blof Analysis Chromosomal DNA from S. marcescem (Nima), undigested and digested with SphI andXbaI and the construct pHT3101-chiA Nima were electrophoresed in agarose gels and blotted onto nylon membranes. Blots were hybridized ovemight at 65"C, using the chiA gene amplicon as a probe, previously labeled with digoxigenin, according to the manufacturer's protocol (DIG DNA labeling kit, Roche). 2.4. Purification of ChiA Nima

Escherichia coli DH5a F'lpHT3101-chiA Nima was grown overnight at 37°C in 250 mL of LB broth. After centrifugation (16,00Og, 10 min), the pellet was discarded and the supematant was concentrated 10 times using arnmonium sulfate to 80% saturation. Concentrated proteins were dialyzed overnight against double-distilled water, centrifuged (16,00Og, 10 min), and diluted 10 times in 1X ASSP (0.5 M ammonium sulfate and 0.025 M sodium phosphate at pH 7.0). This sample was loaded onto a HighTrap Phenyl FF column (Amersham Pharmacia Biotech) previously equilibrated with 1X ASSP. Fractions were eluted in a step-decreasing salt gradient of the ASSP buffer (from 1X ASSP to 0.25X ASSP) and chitinase activity of each fraction was determined in a Tumer fluorometer (model450; 340-nm interference filter and 415-nm cut filter) using 4-MU( G ~ C N A Cand ) ~ 4-MU-(GlcNAc)* as substrates. Fractions showing the highest activity were concentrated with 30-kDa cenhifugal filters (Macrosep). To quantifj the enzyme, one unit was defined as the amount of enzyme required to release 1 pM of 4-methylumbelliferone in 1 h ( 3 , l l ) . Concentrated chitinase was treated with Laemmli's disniption buffer (1 7) without B-mercaptoethanol. Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and reactivated by removing SDS with casein-EDTA wash buffer (1% casein, 2 mM EDTA, 40 MTris-HCl, at pH 9.0). Detection of chitinase activity after gel electrophoresis was measured as previously described (11).

2.5. Effect of pH and Temperature The enzymatic activity of concentrated ChiA Nima at a pH range of 4.0 to 10.0 was evaluated with the tetrarnenc and trimericfluorogenic derivatives using a reaction buffer containing acetic acid, MES [2(N-morpholino) ethane sulfonic acid], H3PO4,Tnzma base [Tns(hydroxymethyl) aminomethane] and glycine, with a final concentration of 15 tnM for each component. In addition, chitinase activiíy within a range of temperatures (270°C) was measured at pH 6.0, using 100 pL of diluted (100-1000 times, depending on the activity) purified chitinase preparation and 300 pL of a buffer containing O.1 M citric acid, 0.2 M KH2P04, and 20 FL of ~-MU-(GLCNAC)~ (0.1 mgImL) in a 600-pL final volume.

3. Results 3.1. Cloning and Seguence Analysis of S. marcescens chiA Nima Previous DNA extraction tests indicated that S. marcescens (Nima) harbors no plasmids; therefore, cloning of the chitinase gene was focused on the chromosomal DNA. Once DNA extracts were used as template in the PCR reaction mixture, a distinctive 2.3-Kb amplicon was obtained (data not shown). This amplicon was digested at the linked XbaI and SphI sites and ligated to the pHT3101 vector. The presence of the amplicon in the shuttle vector, as well as in the S. marcescens (Nima) chromosome (digested and undigested) was corroborated by Southem blot analysis (data not shown), using the amplicon as a probe. A distinctive band of approx 5 Kb was detected in the digested chromosome, indicating that this fragment contained the complete chiA Nima gene, as no XbaI and SphI sites were found later during the sequence analysis of the gene (discussed later). Once the amplicon was sequenced, it was registered at the GenBank under the accession number AY566865. The sequence showed an ORF with the potential to encode for a 564amino-acid peptide with a deduced molecular mass of 61 kDa and an isolectric point of 6.84. Whenthe deduced Chii Nima amino acid sequence was compared with other reported ChiA alleles pro-

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duced by other strains of S. marcescens, identities ranged from 92 to 98%. Analyses of the deduced amino acid sequence by the SignalP (http://www.cbs.dtu.dk/services/ SignalPn and the Signal Peptide Prediction (http:/ /bioinfo~matics.leeds.ac.uk/cgi-bidprot-designl Signalxgi) programs, indicated the presence of a putative signal peptide, recognized by both Gramnegative and Gram-positive bacteria, predicting a cleavage site located between Ala-23 and Ala-24. 3.2. Comparision of ChiA Nima With Other Chitinases Further comparison of the ChiA Nima amino acid sequence with other chitinases (Fig. 1) revealed a modular structure composed of a catalytic domain (Gly-251 through Asn-323) and a putative chitinbinding domain (ChBD) (Pro-410 through Val451) (Fig. lA,B), typical of this enzyme family. Also, similar to other type A chitinases encoded by different S. marcescens strains, no fibronectinlike domain (Fn type 111-like domain) was found in the primary stmcture of ChiA Nima, opposite to those reported in B. circulans (18,19) and B . rhuringiensis (3). However, a comparison of the three-dimensional structure of other alleles may indicate the presence of a putative Fn type 111-like domain (see Subheading 4.) (Fig. 1C). The catalytic domain of the ChiA Nima contains the highly conserved amino acids found in the glycosyl hydrolase family (SXGG and DXXDXDXE, Fig. 1A). in addition, toward the C-terminal end, a region showed some identity (18-21%) with chitin-binding domains (ChiBD) and cellulosebinding domains (CBD) (10,19-21). In spite of this low homology, the putative ChiBD of ChiA Nima shows three aromatic residues (Trp-412, Tyr418, and Trp-448),highly conserved in ChiBDs and CBDs from other bacteria (Fig. 1B). Further comparison of the putative chitin-binding domain of ChiA Nima with other S. marcescens alleles shows identities fiom 48% to 100%,although the main difference is the absence of two aromatic residues (Trp, Tyr) at the beginning of the motif (Fig. 2).

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3.3. PurXcation of ChiA Nima and Zymogram Analysis Purification of ChiA Nima fiom culture supernatants was based on previous quantifications of its activity both in cell extracts and supernatants using the tetrameric fluorogenic derivative. Although both extracts showed activity, a large part of the chitinolytic activity (94%) was observed in the supematant of the recombinant E. coli, as compared with the low activity (6%) found in the cell extracts. Therefore, secreted proteins synthesized by the recombinant E. coli DH5a F/pHT3101chiA Nima were concentrated and separated by hydrophobic interaction column chromatography. A series of fractions were collected and their activity on ~ - M U - ( G ~ C N Aand C )~~- M U - ( G ~ C N A C ) ~ was measured. A coincident peak of activity was detected for both substrates, although with the tetrameric derivative it was much higher (696 f 10 U/mg protein) than that observed with the trimeric substrate (13.7 f0.4 U/mg protein) (Fig. 3A). No activity was detected with the dimeric substrate (data not shown). A crude extract and the chromatographic peak fraction were subjected to SDS-PAGE analysis (Fig. 3B) and proteins were renatured in siru and incubated with the fluorescent substrates mentioned previously,including 4-MU-GlcN Ac (dimeric fluorescent derivative), and visualized under ultraviolet light. Only one band of approx 60 kDa was observed when both the purified sample and a crude extract reacted on ~-MU-(G~CNAC)~ (Fig. 3C) and ~-MU-(G~CNAC)~, but no signal was detected when 4-MU-GlcNAc was used as a substrate. These results also showed that one-step hydrophobic chromatography purification of chitinases is an efficient technique, similar to the results reported earlier (14). 3.4. Effect of pH and Temperature on ChiA Nima Activity ChiA Nima showed activity between pH 4.0 and 10.0, although its peak was observed between pH 5.0 and 6.0, representing almost 26% of the total activity (Fig. 4A). In spite of a gradual de-


1Signal peptide Putative FN type Ill-like dornain

Catalytic domain Putative Chitin-binding domain

Fig. 1. (A) Alignment of the catalytic domain of ChiA Nima and other chitinases. Arrows indicate the typical residues found in the family 18 of glycosyl hydrolases [SXGG (k) and DXXDXDXE (?)l. Sequences are from Cellulomonas uda (Cu ChiA, GenBank accession number AY008839), Bacillus cereus (Bce ChiB, GenBank accession number AB041932), Clostridiumparaputrijicum (Cp ChiA, GenBank accession number AB012764), B. thuringiensis kenyae (Btk ChiA74, GenBank accession number AF424979), Enterohacter agglomerans (Ea ChiA, GenBank accession number U59304), B. circulans WL-12 (Bci ChiA, GenBank accession number P20533), Serratia marcescens 2170 (Sm ChiA, GenBank accession number AB015996), S. marcescens (Nima) (Sm NIMA ChiA, GenBank accession number AY566865). (B) Alignment of the chitinbindig domain of ChiA Nima and other bacteria1 chitinases and cellulases. Arrows (fl)indicate aromatic residues, essential in chitin binding (see Subheading 4.). Chitinases and cellulases sequences are from S. marcescens (Nima) (Sm NIMA ChiA, GenBank accession number AY566865), B. circulans WL-12 (Bci ChiA, GenBank accession number P20533), B. circulans WL-12 (Bci ChiD, GenBank accession number D90534),Altermonas sp. strain 0-75 (Al Chi85, GenBank accession number D13762), B. thuringiensis kenyae (Btk ChiA74, GenBank accession number AF424979), B. thuringiensis pakistani (Btp ChiA71, GenBank accession number AAB58579), Bacillus sp. strain N-4 (Bsp CelA, GenBank accession number P06566), Bacillus sp. strain N-4 (Bsp CelB, GenBank accession number P06565), S. marcescens (Sm ChiB, GenBank accession number AB015997), Bacillus sp. strain N186-1 (Bsp-N186 Cel, GenBank accession number CAA83942),Bacillus sp. strainKSM-N252 (Bsp-N252,GenBank accession number BAB62295),Aeromonas hydrophila JP101 (AehChiA92, GenBank accessionnumber AF181852). (C) Schematic representation of the ChiA Nima modular structure.

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Fig. 2. Putative chitin-binding domain of ChiA alleles from different strains of S. marcescens. Aromatic residues involved in chitin binding are indicated by arrows Chitinases are produced by the S. marcescens (Nima) (SmNIMA, GenBank accession number AY566865), S. marcescens 2170 (Sm2170, GenBank accessionnumber AB015996),S. marcescensBJL200 (SmBTL200, GenBank accessionnumberZ36294),S. marcescens QMB 1466 (SmQMB 1466, GenBank accession number X03657), S. marcescens 271 17 (Sr1127 117, GenBank accession number L01455), and S. marcescens AV305 (SmAV305, GenBank accession number AF085718).

(u).

Fig. 3. (A) Hydrolytic activity of a series of fractions from the purification of the Serratia marcescens ChiA Nima by hydrophobic interaction chromatography on two different substrates: 4-MU-(GlcNAc), (-O-) and 4-MU-(GlcNAc), (-O-). For comparison purposes, activity with the trimeric fluorescent derivative was amplified 10 times. Standard deviations were equal or smaller than graph symbols. Arrow shows the point where the buffer was changed from 1X ASSP to 0.25X ASSP; (B) SDS-PAGE analysis of E. coli DH5aF'I pHT3 101-chiA NIMA cmde extract (lane l), and purified ChiA Nima (chromatographic peak fraction) (lane 2); (C) Zymogram analysis of the same gel, using 4-MU-(GlcNAc), as a substrate.

cline in activity toward akaline conditions, these results clearly indicate that ChiA Nirna's activity is higher under acidic situations. In addition, ChiA Nirna activit~was determined under a gradient of ternperatures. Its highest activity was &served between 45 and 6O0C, with an o~timurn at 55°C (Fig. 4B).

4. Discussion The production of prodiogiosin by S. rnarcescens (Nima) has been ,studied ,qince the 1980s (22), but the chitinolytic profile of this strain was still unknown. Recently, we found that S. marcescens (Nirna) is an excellent chitinase producer, with an activity higher than S. marcescens WF, 933,


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Fig. 4. Effect of pH (A) and temperature (B) on the enzymatic activity of concentrated ChiA Nima, determined by the fluorescent degradation of 4-MU-(GlcNAc),. QMB1466, and E5812 (14a). In this work, we cloned an endochitinase gene (chiA Nima) that encoded the Chi-óO (ChiA Nima). This enzyme showed activity mainly on the tetrameric substrate and a weak activity against the trimenc substrate. This indicates that ChiA Nima may have a dual activity (i.e., endochitinase/chitobiosidase), although the main activity is as an endochitinase. Likewise, a combined endochitinase/chitobiosidase activity was reported for the ChiA from S. marcescens BJL200 (21). According to the computer analysis, the signal peptide of ChiA Nima may be recognized by both Gram-negative and Grarn-positive bacteria, which strongly suggests that this signal peptide is recog-

nized by the E. coli transport mechanism, as was demonstrated by the high activity found in secreted proteins, in contrast with the low activity of intracellular extracts. Also, because the signal peptide may be cleaved by Gram-positive bacteria, B. thuringiensis transformed with chiA Nima might cleave and transport the recombinant chitinase. Location of the catalytic domain and the chitinbinding motif was established in ChiA Nima by sequence analysis. Previous reports (9,12,21,23) about ChiA alleles from S. marcescens have established the location of the catalytic domain, but not the position of a chitin-binding motif. Those studies only suggest the implication of some

7 70 amino acids along the ChiA structure on chitinbinding. Likewise, sequence analysis of ChiA Nima indicated a modular structure, which is typical of degrading enzymes of biopolymers as chitin and cellulose. If the putative ChiBD of ChiA Nima is compared with the three-dimensional structure of the highly homologous ChiA from S. marcescem QMB1466 (24), Trp-412 and Tyr418 from ChiA Nima may be highly exposed in a loop, whereas Trp-448 may be located in a nearby irregularly exposed zone. Aromatic amino acids have been shown to play an imponant role in carbohydrate-binding domains, by forming hydrophobic interactions with the sugar rings of their substrate (25). The importante of Trp-687 (Trp448 in ChiA Nima) from B. circulam ChiA has been demonstrated earlier (18,25), as it plays a pivotal role in binding chitin. Also, substitution of Trp-773 (corresponding to Trp-412 in ChiA Nima) with Ala in the chitinase Chi92 from Aeromonus hydrophila JPlOl, led to a spectacular loss of binding activity for powdered and colloidal chitin (20). Interestingly, this latter chitinase contains three chitin-binding domains, one toward the N-tenninus and two toward the C-terminus (10). Altematively, it has been shown that aromatic amino acids Trp-33 and Trp-69, both located in the N-terminal domain, and Trp-245 located in the catalytic center of the ChiA fromS. marcescem 2170, play an imporrant role in chitin binding (23). With this information, it appears that S. marcescens ChtA alleles may have one chitin-binding domain, similar to that of ChiA from B. circulans, but also that different aromatic residues located in the exposed region of the N-tenninus may enhance the chitin binding, similar to ChiA92 from A. hydrophila (10). Still, some reported ChiA alleles from S. marcescens show immnant differences at the beginning of their ChBD (Fig. 2), as Trp-412 is absent in some of these alleles. This absence may interfere in the chitin-binding ability of these enzymes. In addition, sequence analysis of ChiA Nima showed no homology with a common Fn type IlI-like domain, however, it may contain a site with an Fn type m-like fold (Fig. lc), as suggested for the S. marcescens QMB1466 ChiA (24).

Ruiz-Sanchez et al.

Chitinolytic analysis over a range of pH showed that ChiA Nima is more active under acidic conditions. Activity between pH 8.0 and 9.5 represents only 10% of the total activity; however, it is 35 times higher than the activity shown by ChiA74 chitinase from B. rhuringiensis (3). This is an important comparison, as one of the applied purposes of this study is the use of bacterial chitinases as synergistic agents of the insecticida1 Cry proteins of B. rhuringiensis. It is well known that damage to the peritrophic membrane caused by extracts containing chitinases increases the insecticidal activity of Cry proteins (5,6). However, most lepidopteran and dipteran insects susceptible to severa1 Cry proteins possess a midgut content with a pH ranging from 8.0 to 9.5 or even higher (3). Because of the high hydrolytic activity of ChiA Nima, it may show potential to be used in combination with Cry proteins to enhance their insecticidal activity under high pH conditions (26). In conclusion, we were able to clone, sequence, and characterize a natural chitinase variant with high activity from the S. marcescens ChiA family, named ChiA Nima. It shows similarity with alleles from other strains of S. marcescens and a significant chitinolytic activity, which may denote its potential as a control agent for phytopathogenic fungi, when expressed in transgenic plants, or as a synergistic agent with B. thuringiensis insecticidal Cry proteins, for the control of insect pests. Currently, a series of bioassays on severa1 lepidopteran pests are in progress, using a combination of ChiA Nima and Cry - proteins. *

Acknowledgments We are grateful to Beatriz Jiménez for her technical suppOrt. We D. Bideshi and H ~ ~ n - W Park o o for d i c a l reading 0f the manuS C ~ P This ~ . research was supported in part by grmts J35306-B and 44990 from CoNACyT, México. Also, A. R. S. is a graduated student supponed by a CoNACyT fellowshi~-

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