A new α-galactosidase from symbiotic Flavobacterium sp. TN17 reveals four residues essential for α-galactosidase activity of gastrointestinal bacteria

Share Embed


Descrição do Produto

Appl Microbiol Biotechnol (2010) 88:1297–1309 DOI 10.1007/s00253-010-2809-7

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

A new α-galactosidase from symbiotic Flavobacterium sp. TN17 reveals four residues essential for α-galactosidase activity of gastrointestinal bacteria Junpei Zhou & Pengjun Shi & Huoqing Huang & Yanan Cao & Kun Meng & Peilong Yang & Rui Zhang & Xiaoyan Chen & Bin Yao

Received: 14 April 2010 / Revised: 6 July 2010 / Accepted: 30 July 2010 / Published online: 17 August 2010 # Springer-Verlag 2010

Abstract The α-galactosidase gene, galA17, was cloned from Flavobacterium sp. TN17, a symbiotic bacterium isolated from the gut of Batocera horsfieldi larvae. The 2,205-bp full-length gene encodes a 734-residue polypeptide (GalA17) containing a putative 28-residue signal peptide and a catalytic domain belonging to glycosyl hydrolase family 36 (GH 36). The deduced amino acid sequence of galA17 was most similar to a putative αgalactosidase from Pedobacter sp. BAL39 (EDM38577; 66.6% identity) and a characterized α-galactosidase from Carnobacterium piscicola BA (AAL27305; 30.1%). Phylogenetic analysis revealed that GalA17 was similar to GH 36 α-galactosidases from symbiotic bacteria sharing two putative catalytic motifs, KWD and SDXXDXXXR, in which D480, S548, D549, and R556 were essential for α-galactosidase activity based on site-directed mutagenesis. Purified recombinant GalA17 showed apparent optimal activity at pH 5.5 and 45°C; exhibited strong resistance to digestion by trypsin, α-chymotrypsin, collagenase, and proteinase K; and efficiently hydrolyzed several synthetic and natural substrates (p-nitrophenyl-α-D-galactopyranoJ. Zhou : P. Shi : H. Huang : Y. Cao : K. Meng : P. Yang : R. Zhang : X. Chen : B. Yao (*) Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, People’s Republic of China e-mail: [email protected] e-mail: [email protected] J. Zhou College of Life Sciences, Yunnan Normal University, No. 298 December 1st Street, Kunming 650092, People’s Republic of China

side, stachyose, melibiose, raffinose, soybean meal, locust bean gum, and guar gum). Keywords Batocera horsfieldi . Flavobacterium sp. TN17 . α-Galactosidase . Symbiotic bacteria . Site-directed mutagenesis

Introduction Polysaccharides of the α-D-mannan type, such as galactomannans in leguminous seeds, are one of the major constituent groups of hemicellulose (Moreira and Filho 2008) and represent 20–30% of renewable organic matter (Ljungdahl and Eriksson 1985). Galactomannans consist of a main chain of 1,4-linked β-D-mannopyranosyl residues, most of which are substituted at O-6 with α-D-galactopyranosyl side chains. α-Galactosidase (1,6-α-D-galactoside galactohydrolase; melibiase; EC 3.2.1.22) is required to remove the sidechain substituents from galactomannans (e.g., guar gum and locust bean gum) and oligosaccharides (e.g., melibiose, raffinose, and stachyose) (Moreira and Filho 2008). α-Galactosidases are widely distributed in microorganisms, plants, and mammals. Based on their amino acid sequences, most α-galactosidases are confined into glycosyl hydrolase (GH) families 27 and 36 (http://pfam.sanger. ac.uk/; version 24.0). α-Galactosidase is extensively used in processing of food products (Cruz and Park 1982), as an animal feed additive (Baucells et al. 2000), in the pulp and paper industry (Clarke et al. 2000), in the sugar-producing industry (Linden 1982), and in medicinal treatment of Fabry disease (Buja 2009). Digestive enzymes that are produced by symbiotic microorganisms harbored in the gut of wood-feeding insects

1298

are thought to be a potential source of diverse and novel glycosyl hydrolases (Warnecke et al. 2007). Members of the Cerambycidae family (commonly known as longhorned beetles) are particularly noteworthy because they are woodfeeding insects with an estimated 35,000 species worldwide (Smith et al. 2004) and because significant differences of commensal bacterial communities are putatively present in the guts of these species (Schloss et al. 2006). However, few enzymes have been isolated from the symbiotic microorganisms in the gut of longhorned beetles such as Batocera horsfieldi. Thus far, only one lipase (Park et al. 2007), three xylanases (Heo et al. 2006; Zhou et al. 2009, 2010a), and one chromosomal segment showing xylanolytic activity (Zhou et al. 2010b) have been described. In this study, we describe the cloning of a novel αgalactosidase gene from Flavobacterium sp. TN17 harbored in the gut of B. horsfieldi. The gene was expressed in Escherichia coli, and the purified recombinant enzyme (rGalA17) was characterized. The key amino acid residues putatively involved in the catalysis of α-galactosidase from symbiotic bacteria were predicted based on sequence alignment and mutagenesis analysis.

Materials and methods Vectors and reagents The pGEM-T Easy vector (Promega, Madison, WI, USA) and E. coli Trans1-T1 (TransGen, Beijing, China) were used for gene cloning. The pET-22b(+) vector (Novagen, San Diego, CA, USA) and E. coli BL21 (TransGen) were used for gene expression. Nickel-NTA agarose (Qiagen, Valencia, CA, USA) was used to purify the His6-tagged protein. Genomic DNA isolation, DNA purification, and plasmid isolation kits were purchased from Tiangen (Beijing, China). Restriction endonucleases, T4 DNA ligase, DNA polymerase (Taq and Pyrobest), and dNTPs were purchased from TaKaRa (Otsu, Japan). Substrates p-nitrophenyl-α-D-galactopyranoside (pNPG), p-nitrophenol, melibiose, stachyose, guar gum and locust bean gum (from Ceratonia siliqua seeds), and proteases including trypsin, α-chymotrypsin, collagenase, and proteinase K were purchased from Sigma (St. Louis, MO, USA). D-Galactose and raffinose were purchased from Amresco (Solon, OH, USA), and isopropyl-β-D-1-thiogalactopyranoside (IPTG) was purchased from Calbiochem (Darmstadt, Germany). All other chemicals were of analytic grade. Microorganism isolation Luminal contents from the gut of 20 B. horsfieldi larvae were collected, suspended in 0.7% (w/v) NaCl, and spread onto screening agar plates as described previously (Zhou et

Appl Microbiol Biotechnol (2010) 88:1297–1309

al. 2009). The medium for screening contained 1% (w/v) sodium carboxymethyl cellulose, 0.5% (w/v) peptone, and 0.5% (w/v) NaCl. Pure cultures were obtained through repeated streaking on Luria–Bertani (LB) plates and grown at 26°C. One strain, designated TN17, showed significant activity of intracellular α-galactosidase when 1% soybean meal was used as a carbon source in the presence of 0.5% NaCl and 0.2% (NH4)2SO4. The taxon of strain TN17 was identified by comparison of the polymerase chain reaction (PCR)-amplified 16S recombinant DNA (rDNA) sequence using primers 27F and 1492R (Lane 1991) with the known sequences archived in GenBank. Gene cloning and mutagenesis Genomic DNA from strain TN17 was extracted using the Tiangen genomic DNA isolation kit following the manufacturer’s instructions. The degenerate primer set (Table 1; GalAgutF and GalAgutR) was designed using a consensusdegenerate hybrid oligonucleotide primers program (CODEHOP; Rose et al. 2003) based on the conserved blocks of GH 36 α-galactosidases from symbiotic gastrointestinal bacteria (Figs. 1 and 2). Touchdown-PCR was performed as follows to amplify a partial α-galactosidase gene: 94°C for 5 min; then 15 touchdown cycles of 94°C for 30 s, 60°C for 30 s (decreasing by 1°C each cycle), and 72°C for 30 s; followed by 30 cycles of 94°C for 30 s, 44°C for 30 s, and 72°C for 30 s; and one final extension at 72°C for 7 min. The PCR product was gel-purified, ligated to pGEM-T Easy vector, and sequenced by Biomed (Beijing, China). To obtain the full-length α-galactosidase gene (galA17), 4 rounds of thermal asymmetric interlaced-PCR (TAILPCR) were performed with 11 arbitrary degenerate primers (Zhou et al. 2010a) and 8 nested insertion-specific primers (Table 1), using a time-saving and reduced-cost strategy described by Zhou et al. (2010a). The annealing temperatures used for high stringency are shown in Table 1. PCR products of the appropriate size were gel-purified and directly sequenced. Resulting sequences were used to design specific primers (SP3 and SP4 primers) for subsequent round of genome walking. To understand the putative catalytic or essential amino acids for the α-galactosidase activity of rGalA17, mutations were introduced using the Fast Mutagenesis System protocol (TransGen), which utilizes a supercoiled double-stranded DNA vector with the insert of interest and two synthetic oligonucleotide primers containing the desired mutations (Table 1). Mutated plasmids containing staggered nicks were generated by incorporation of the oligonucleotide primers during temperature cycling using Pfu DNA polymerase. The PCR product was treated with DpnI endonuclease for digesting the parental DNA template and selecting mutation-containing plasmids for heterologous expression.

Appl Microbiol Biotechnol (2010) 88:1297–1309

1299

Table 1 Primers used in this study

Tm (°C)b

Primer name

Primer sequence (5′ 3′)a

GalAgutF

GACATGTTCGTGATGGACgayggntggtt

GalAgutR

CGGACTCTGGGTTCACCatytcnggytc

GalA17uSP1

TACCAAAGTACCGATTCCGTTT

GalA17uSP2

AGAGCTGCGTCGTCATTGTTA

GalA17uSP3

GTTGTCCATTGCTCTATAATATCAGT

GalA17uSP4

TGTCTTTTAAGGTAATTTCAGTTTG

GalA17dSP1

AATGACGACGCAGCTCTTGG

GalA17dSP2

ACGGAATCGGTACTTTGGTAAA

GalA17dSP3

GCCATGATGGGAAGATTAGGT

GalA17dSP4

AATCTGGCAAGGAGATCAATACC

rGalA17BF

CGGGATCCGCAACAAATTATCTCAGTAAAAACA

rGalA17HR

CCCAAGCTTGGCCTTTGTAATTTTTAAAACA

GalA17D480AF

GCGTATATCAAATGGGCGTGTAATGCAG

44–60

55

52

55

54

49

53 GalA17D480AR CGCCCATTTGATATACGCCAATTCCGGGT GalA17S548AF

ACAGAATTCTGGCCAGCGGATAACACAG

GalA17S548AR

CGCTGGCCAGAATTCTGTAAAGTATTTC

GalA17D549AF

GAATTCTGGCCAAGTGCGAACACAGATC

53

53 GalA17D549AR CGCACTTGGCCAGAATTCTGTAAAGTAT GalA17D552AF

CCAAGTGATAACACAGCGCCTTTAGAAAG 53

GalA17D552AR CGCTGTGTTATCACTTGGCCAGAATTCT GalA17R556AF

ACAGATCCTTTAGAAGCGGTATATATGC 50

GalA17R556AR CGCTTCTAAAGGATCTGTGTTATCACTT a b

IUPAC/IUB symbols are used, restriction sites are underlined, and sites for mutation analysis are in bold and shaded Tm: annealing temperature. The Tm values of specific primers are used for the high stringency step in TAIL-PCR

All primers were designed and analyzed using Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA) and Oligo 6.0 (Molecular Biology Insights, Cascade, CO, USA) software and synthesized by Sangon (Shanghai, China) or Invitrogen (Carlsbad, CA, USA).

Sequence and phylogenetic analyses Sequence assembly was performed using Vector NTI 10.3 software (InforMax, Gaithersburg, MD, USA). The signal peptide in the deduced amino acid sequence was predicted

1300

Appl Microbiol Biotechnol (2010) 88:1297–1309 Source Actinobacteria; Bifidobacterium adolescentis DSM 20083 |AAD30994|

73 100

Actinobacteria; Bifidobacterium bifidum NCIMB 41171 |ABD96085| Actinobacteria; Bifidobacterium breve 203 |ABB76662|

58

Firmicutes; Eubacterium rectale ATCC 33656 |ACR77113| Firmicutes; Eubacterium rectale ATCC 33656 |ACR74280|

99

Motifs

Human colon Human feces Human feces Human gut Human gut

Firmicutes; Carnobacterium piscicola BA |AAL27305|

55

Firmicutes; Ruminococcus gnavus E1 |ACL13770|

99

100 Bacteroidia; Parabacteroides distasonis ATCC 8503 |YP_001301506|

Bacteroidia; Bacteroides sp. 2_1_7 |ZP_05288634|

100

Bacteroidia; Bacteroides ovatus ATCC 8483 |ZP_02064925|

94 97

Human feces Human gut Human colon Human gut

Bacteroidia; Bacteroides caccae ATCC 43185 |ZP_01961833|

Human gut

Bacteroidia; Bacteroides thetaiotaomicron VPI-5482 |NP_811763|

Human gut

Cluster 1

96

&

Sphingobacteria; Pedobacter heparinus DSM 2366 |YP_003091979| 100

Flavobacteria; Flavobacterium sp. TN17 |GU647091|

97

Batocera horsfieldi gut

Sphingobacteria; Pedobacter sp. BAL39 |ZP_01881920| 86

Sphingobacteria; Spirosoma linguale DSM 74 |ZP_04493119| Thermotogae; Thermotoga maritima |1ZY9|

44

Sphingobacteria; Pedobacter sp. BAL39 |ZP_01884672| Flavobacteria; Flavobacteria bacterium BAL38 |ZP_01734709| 56

Fungi; Dikarya; Trichoderma reesei |1SZN|

45

Metazoa; Craniata; Homo sapiens |3GXN| 54

Sphingobacteria; Rhodothermus marinus DSM 4252 |ACY49470|

47

99

Viridiplantae; Tracheophyta; Oryza sativa |1UAS| Flavobacteria; Flavobacterium johnsoniae UW101 |YP_001197265|

40

Sphingobacteria; Sphingobacterium spiritivorum ATCC 33300 |ZP_03966783| 100 32

Sphingobacteria; Sphingobacterium spiritivorum ATCC 33861 |ZP_04780995|

&

Flavobacteria; Chryseobacterium gleum ATCC 35910 |ZP_03852822| Sphingobacteria; Pedobacter heparinus DSM 2366 |YP_003092109|

99

Sphingobacteria; Pedobacter heparinus DSM 2366 |YP_003090999|

85

Sphingobacteria; Pedobacter heparinus DSM 2366 |YP_003093145|

72 99

Sphingobacteria; Pedobacter heparinus DSM 2366 |YP_003093147|

Flavobacteria; Flavobacteriales bacterium HTCC2170 |ZP_01106700| Sphingobacteria; Chitinophaga pinensis DSM 2588 |YP_003124039|

Cluster 3

Sphingobacteria; Dyadobacter fermentans DSM 18053 |YP_003086005|

57

Cluster 2

67

0.2

Fig. 1 Phylogenetic tree constructed using the neighbor-joining method based on the amino acid sequences of GH 36 α-galactosidases from Bacteroidetes and symbiotic bacteria from gastrointestinal habitats and α-galactosidases with known crystallographic structures. Accession numbers are given at the end of each species name. Strains belonging to Flavobacteria are labeled with triangles. Based on annotation of the

sequences in public databases, the sources from which the strains were isolated are underlined. Sites for mutation analyses contain stars in the motif logos. The enzyme identified in this study is shown in bold type. Bootstrap values (n=1,000 replicates) are reported as percentages. The scale bar represents the number of changes per amino acid position

using SignalP (http://www.cbs.dtu.dk/services/SignalP/). The alignment of DNA and protein sequences was constructed with BLASTN and BLASTP (http://www. ncbi.nlm.nih.gov/BLAST/), respectively. The classification of the glycosyl hydrolase family of GalA17 was determined with the InterPro online tool (http://www.ebi.ac.uk/interpro/). The key functional residues were predicted using Pfam

(http://pfam.sanger.ac.uk/search). The tertiary structure of rGalA17 was predicted by homology modeling using SwissModel (http://swissmodel.expasy.org/). Multiple sequence alignments were performed with Clustal X (Thompson et al. 1997) and then manually adjusted with the BioEdit software package 7.0 (Hall 1999). Logos of sequence motifs comprising the catalytic nucle-

Appl Microbiol Biotechnol (2010) 88:1297–1309

1301

Fig. 2 Amino acid sequence seed alignment (corresponding to Fig. 1) of GH 36 α-galactosidases from Bacteroidetes and symbiotic bacteria in the gastrointestinal habitats and α-galactosidases with known crystallographic structures. Sequences are shown with microbial source as follows (including the accession number): TM, Thermotoga maritima (1ZY9); FB, Flavobacterium sp. BAL38 (ZP_01734709); BB, Bifidobacterium bifidum NCIMB 41171 (ABD96085); CP, Carnobacterium piscicola BA (AAL27305); BO, Bacteroides ovatus ATCC 8483 (EDO12397); GalA17, Flavobacterium sp. TN17 (GU647091). Identical residues are shaded in black, and conserved residues are shaded in gray. Motifs involved in catalysis are framed. Amino acids used for designing degenerate primers are underlined with black bars. The internal peptide sequences identified by LC-ESI-MS/MS are arrowed. The asterisks indicate mutation sites

ophile and acid/base were produced using the Weblogo server (Crooks et al. 2004). Distance matrices for nucleotides and amino acids were calculated according to the Kimura two-parameter model (Kimura 1980) and Poisson correction model (Nei 1987), respectively. Phylogenetic trees were constructed using neighbor-joining algorithms (Saitou and Nei 1987) with MEGA 4.0 (Tamura et al. 2007). The reliability of the resulting trees was assessed using 1,000 bootstrap repetitions.

Expression of galA17 and its mutants To express galA17 in E. coli, the coding sequence of the mature protein without the predicted signal peptide was amplified by PCR using primers rGalA17BF and rGalA17HR (Table 1). The PCR product was gel-purified, digested with BamHI and HindIII, and cloned into the corresponding sites of the pET-22b(+) vector. The recombinant plasmid, pETgalA17, was transformed into E. coli BL21 (DE3)-competent

1302

Appl Microbiol Biotechnol (2010) 88:1297–1309

Fig. 2 (continued)

cells. Transformants were identified by PCR analysis and further confirmed by DNA sequencing. A positive transformant harboring pET-galA17 was picked from a single colony and grown overnight at 37°C in LB medium supplemented with 100 μgml−1 ampicillin. The culture was then inoculated at a 1:100 dilution into fresh LB medium containing ampicillin and was grown aerobically at 37°C to an A600 of approximately 0.8. IPTG was then added to a final concentration of 0.7 mM for induction at 20°C for 20 h. Expression of the rGalA17D480A, rGalA17S548A, rGalA17D549A, rGalA17D552A, and rGalA17R556A mutants in E. coli was performed as described above. Purification and identification of recombinant α-galactosidase To purify the recombinant His6-tagged α-galactosidase (rGalA17), supernatant was collected by centrifugation

and concentrated using the Hollow Fiber Membrane Module (6-kDa MWCO; Motian Membrane Engineering and Technology, Tianjin, China) and an ultrafiltration membrane (PES5000; Sartorius Stedim Biotech, Göttingen, Germany). The crude enzyme was applied to a Ni2+-NTA agarose affinity column with a linear imidazole gradient of 20−300 mM in Tris–HCl buffer (20 mM Tris–HCl, 500 mM NaCl, 10% glycerol, pH 7.6). The eluate was loaded onto the ultrafiltration membrane and further concentrated with solid polyethylene glycol (PEG8000) powder and was then dialyzed three times against 3-l deionized double-distilled H2O. The purified enzyme was stored at −20°C before characterization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 12% running gel. The protein concentration was determined by the Bradford method (Bradford 1976) using bovine serum albumin as a standard. The concentration of each single protein band in

Appl Microbiol Biotechnol (2010) 88:1297–1309

the culture supernatant was quantified with Bio-Profil Bio-1D++ version 10.02 software (Vilber Lourmat, Marne-la-Vallée, France). After denaturation, the purified enzyme was digested with trypsin and then identified by the State Key Laboratory of Biology of Biomembrane and Membrane Technology, Institute of Zoology, Chinese Academy of Science (Beijing, China) using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Enzyme assay α-Galactosidase activity was determined by a modified pNPG method (Cao et al. 2007; Denherder et al. 1992). The standard reaction system included 20 μl of appropriately diluted enzyme, 230 μl of 0.1 M McIlvaine buffer (0.2 M Na2HPO4/0.1 M citric acid), and 250 μl of substrate solution (2 mM pNPG in 0.1 M McIlvaine buffer, pH 5.5). The substrate solution was preincubated at 37°C for 5 min and incubated for an additional 5 min after the addition of the enzyme, then 1.5 ml of 1 M Na2CO3 was added to stop the reaction. Liberated p-nitrophenol was determined by measuring the absorption at 405 nm. As a control, 1.5 ml of 1 M Na2CO3 was added before the addition of the enzyme. Glucose liberated in the hydrolysis of melibiose was detected at 505 nm by the glucose oxidase peroxidase method using a glucose kit (BioSino, Beijing, China) with an incubation time of 30 min (Trinder 1969). The release of reducing sugar from raffinose, stachyose, soybean meal, guar gum, and locust bean gum was determined by the 3,5-dinitrosalicylic acid method at 540 nm with appropriate incubation time (Miller 1959). One unit of α-galactosidase was defined as the amount of enzyme that releases 1 μmol of p-nitrophenol, glucose, or reducing sugar per minute under the above assay conditions unless otherwise noted. Biochemical characterization Characterization of the purified rGalA17 was determined using pNPG as substrate. The optimal pH for αgalactosidase activity of purified rGalA17 was determined at 37°C in buffers with pH ranging from 4.0 to 10.0. The enzyme stability at different pH values was estimated by measuring the residual enzyme activity after incubating the enzyme solution in various buffers at 37°C for 1 h. The buffers used were McIlvaine buffer for pH 4.0–8.0, 0.1 M Tris–HCl for pH 8.0–9.0, and 0.1 M glycine–NaOH for pH 9.0–11.0. The optimal temperature for rGalA17 activity was determined over the range of 0–70°C in McIlvaine buffer (pH 5.5). The thermostability of purified rGalA17 was determined after preincubation of the enzyme in McIlvaine

1303

buffer (pH 5.5) at 37°C, 50°C, or 60°C without substrate for various periods. To investigate the effects of different metal ions and chemical reagents on the purified rGalA17 activity measured in McIlvaine buffer (pH 5.5) at 37°C, 10 mM (final concentration) of NaCl, KCl, CaCl2, LiCl, CoCl2, CrCl3, NiSO4, CuSO4, MgSO4, FeSO4, MnSO4, ZnSO4, Pb (CH3COO)2, AgNO3, HgCl2, ethylenediamine tetraacetic acid (EDTA), SDS, or β-mercaptoethanol was individually added to the reaction solution. To examine resistance to different proteases, purified rGalA17 (100 μgml−1) was incubated at 37°C for 1 h with 10 μgml−1 trypsin (pH 7.0), α-chymotrypsin (pH 7.0), collagenase (pH 7.5), or proteinase K (pH 7.5), and the residual enzyme activity was measured in McIlvaine buffer (pH 5.5) at 37°C. Km, Vmax, and kcat values for purified rGalA17 were determined using 0.05–1.0 mM pNPG, 0.5–5.0 mM stachyose, or 0.5–5.0 mM melibiose as the substrate in McIlvaine buffer (pH 5.5) at 45°C. The data were plotted according to the Lineweaver–Burk method (Lineweaver and Burk 1934). Nucleotide sequence accession numbers The nucleotide sequences for the Flavobacterium sp. TN17 16S rDNA and α-galactosidase gene (galA17) were deposited in GenBank under the accession numbers GU647090 and GU647091, respectively.

Results Strain identification The 16S rDNA sequence of strain TN17 (accession no. GU647090) showed 98.7% identity with that of Flavobacterium johnsoniae DSM 425 (AM230488), 98.5% with Flavobacterium columnare (AY747592), and 98.4% with Flavobacterium aquidurense type strain WB 1.1-56 (AM177392). Thus, strain TN17 was classified into the genus Flavobacterium and deposited in the Agricultural Culture Collection of China under registered number ACCC 03886. The distance tree created by the neighborjoining method also revealed the same classification (data not shown). Gene cloning and sequence analyses Designing degenerate primers with CODEHOP software has proven to be a practical and efficient approach to detect genes containing conserved blocks (Acevedo et al. 2008; Provencher et al. 2003; Rose et al. 2003). Two blocks, [F/L/

1304

Appl Microbiol Biotechnol (2010) 88:1297–1309

V]-[L/V]-[L/M/V]-D-D-G-W-F and E-P-E-M-[V/I]-[N/S][P/E], were found to be conserved in GH 36 αgalactosidases from symbiotic gastrointestinal bacteria (Fig. 2). Using the automatically designed CODEHOP primer pair GalAgutF and GalAgutR (corresponding to conserved blocks DMFVMDDGWF and EPEMVNPDSE, respectively), a galA17 fragment (193 bp) was successfully amplified, revealing the feasibility and efficacy of this primer set in isolating α-galactosidase genes from symbiotic bacteria in the gastrointestinal habitats. The flanking fragments amplified by TAIL-PCR were separated on a 2% agarose gel. PCR products showing the expected differential shift were purified, directly sequenced, and assembled with galA17 fragment to give the 2,205-bp full-length αgalactosidase gene. galA17 encodes a 734-residue polypeptide (GalA17) in which the predicted signal peptide is cleaved (between A28 and Q29) to yield a 706-residue mature polypeptide with a calculated mass of 80.0 kDa and a catalytic domain corresponding to the melibiase family (Pfam) from V289 to G683. The deduced amino acid sequence of GalA17 showed its highest identity (66.6%) with a putative αgalactosidase from Pedobacter sp. BAL39 (EDM38577), followed by the conceptually translated α-galactosidases from Spirosoma linguale DSM 74 (EEO96595; 59.0%), Parabacteroides distasonis ATCC 8503 (ABR41884; 49.7%), and Bacteroides sp. 2_1_7 (ZP_05288634; 49.5%), a characterized α-galactosidase from Carnobacterium piscicola BA (AAL27305; 30.1%), and a putative αgalactosidase from F. johnsoniae UW101 (YP_001197265; 6.3%). A phylogenetic tree was constructed based on the known GH 36 α-galactosidase sequences from Bacteroidetes, symbiotic bacteria in the gastrointestinal habitats, and sequences with known crystallographic structures (Fig. 1). The seed alignment is shown in Fig. 2. High bootstrap

a kDa

values separated these α-galactosidases into three distinct clusters: cluster 1 consisted mostly of α-galactosidases from symbiotic bacteria in the gastrointestinal habitats and shared the motifs KWD and SDXXDXXXR, cluster 2 included α-galactosidases of known crystal structures with motifs KXD and RXXXD, and cluster 3 contained two αgalactosidases from Bacteroidetes. GalA17 from Flavobacterium sp. TN17 was grouped in cluster 1, which is more closely related to the α-galactosidases from symbiotic bacteria in the gastrointestinal habitats than to that from Flavobacterium spp. Because rGalA17 shared very low identity (55% of the maximum activity was retained between pH 5.0 and 7.0 (Fig. 4a). The enzyme was stable over a wide pH range, retaining more than 80% of the initial activity after incubation in buffers ranging from pH 5.0 to 9.0 at 37°C for 1 h (Fig. 4b). Purified rGalA17 showed apparent optimal activity at 45°C when assayed at pH 5.5, retaining >55% of the maximum activity when assayed at 30–50°C (Fig. 4c). rGalA17 was stable at 37°C for 1 h; at temperatures above 60°C, the enzyme activity decreased rapidly after 5 min of preincubation (Fig. 4d). The α-galactosidase activity of purified rGalA17 in the presence of different metal ions or chemical reagents at a concentration of 10 mM is shown in Table 2. The activity was completely inhibited by Ag+, Hg2+, and SDS. Partial inhibition was observed in the presence of Fe2+ and Cr3+. Mn2+, β-mercaptoethanol, Zn2+, and EDTA enhanced the activity by ∼0.2-fold. The addition of other reagents had little or no effect on the enzyme activity. rGalA17 was resistant to trypsin, collagenase, and αchymotrypsin, retaining more than 90% of the initial

Site-directed mutagenesis rGalA17 were mutated based on the Fast Mutagenesis System and further confirmed by DNA sequencing. Expressed in E. coli BL21 (DE3) cells under the same condition, rGalA17 and its mutants were secreted into the culture supernatant and quantified by SDS-PAGE (Fig. 3b) with Bio-Profil Bio-1D++ software. As a result, rGalA17 had an α-galactosidase activity of 102.0 Umg−1 at 37°C using pNPG as the substrate; mutants rGalA17D552A and rGalA17R556A were 81.2% and 27.6%, respectively, of rGalA17 activity; but mutants

b 120

Relative activity (%)

Relative activity (%)

a

activity after incubation at 37°C for 1 h. Under the same conditions, rGalA17 lost ∼35% of its activity in the presence of proteinase K. The Km, Vmax, and kcat values of rGalA17 were 2.47 mM, 476.19 μmolmin−1 mg−1, and 654.62 s−1 towards pNPG, respectively; 14.76 mM, 30.58 μmolmin−1 mg−1, and 42.04 s−1 to stachyose, respectively; and 12.58 mM, 1.70 μmolmin−1 mg–1, and 2.33 s−1 against melibiose, respectively. The specific activities of rGalA17 towards pNPG, stachyose, melibiose, raffinose, soybean meal, locust bean gum, and guar gum were 92.95±4.14, 51.22±0.85, 2.52±0.06, 0.84±0.26, 0.87±0.16, 0.49±0.07, and 0.20± 0.04 Umg−1, respectively.

100 80 60 40 20 0

3

4

6

5

7

8

9

10

11

120 100 80 60 40 20 0

3

4

pH

6

7

8

9

10

11

12

pH

c

d 120

Relative activity (%)

Relative activity (%)

5

100 80 60 40 20 0

0

10

20

30

40

50

60

70

80

Temperature (°C)

Fig. 4 Characterization of purified rGalA17. a Effect of pH on rGalA17. The enzyme activity was determined at 37°C from pH 4.0 to 10.0. b pH stability assay. After preincubation at pH 4.0–11.0 at 37°C for 1 h, the enzyme activity was determined in McIlvaine buffer (pH 5.5) at 37°C. c Effect of temperature on rGalA17 activity

120

37°C 50°C

100

60°C

80 60 40 20 0 0

5

10 15 20 25 30 35 40 45 50 55 60 65

Time (min)

measured in McIlvaine buffer (pH 5.5) at 0–70°C. d Thermostability assay. Purified rGalA17 was preincubated in McIlvaine buffer (pH 5.5) at 37°C, 50°C, or 60°C. Aliquots were removed at specific time points to measure residual activity at 37°C. Error bars represent mean±SD (n=3)

1306

Appl Microbiol Biotechnol (2010) 88:1297–1309

Table 2 Effect of metal ions and chemical reagents on the α-galactosidase activity of purified rGalA17 Chemical

Relative activity (%)a

None Mn2+ β-Mercaptoethanol Zn2+ EDTA Pb2+ Ca2+ Li+ Cu2+ Ni2+ Mg2+ Na+ Co2+

100.0±2.5 123.4±2.2 120.1±1.3 119.4±3.0 118.1±1.4 113.6±1.6 109.7±1.8 108.0±0.6 108.0±0.9 107.6±1.1 106.7±0.9 106.4±7.5 106.0±0.9

K2+ Cr2+ Fe2+ SDS Ag+ Hg2+

105.4±6.4 74.1±5.8 28.3±2.1 0.0 0.0 0.0

a

Values represent the mean±SD (n=3) relative to untreated control samples

rGalA17D480A, rGalA17S548A, and rGalA17D549A showed no α-galactosidase activity (Table 3).

Discussion Gut microorganisms, especially those from termite (Breznak and Brune 1994), rumen (Ferrer et al. 2005), and human (Xu et al. 2007), can play important roles in their host’s ability to absorb nutrients. Bacteria from the genus Flavobacterium have been identified in the gut of Oberea linearis (Bahar and Demirbag 2007) and Anoplophora glabripennis (Schloss

Table 3 α-Galactosidase activity of rGalA17 and its mutants Mutant

Motifa

Relative activity (%)b

rGalA17 rGalA17D480A rGalA17S548A rGalA17D549A rGalA17D552A rGalA17R556A

KXD & SDXXDXXXR KXD SDXXDXXXR SDXXDXXXR SDXXDXXXR SDXXDXXXR

100 0 0 0 81.2±3.3 27.6±4.1

a

The mutated amino acid in the corresponding motif is shown in bold

b

Values represent the mean±SD (n=3) relative to rGalA17

et al. 2006). In this study, Flavobacterium sp. TN17 was isolated from the gut of B. horsfieldi and had significant α-galactosidase activity when induced with soybean meal. To date, α-galactosidases from symbiotic Flavobacterium spp. have never been reported. Thus, this is the first report on the cloning, expression, and characterization of an αgalactosidase from a symbiotic Flavobacterium strain in the gut of B. horsfieldi larvae. The deduced amino acid sequence of galA17 shared its highest identity (66.6%) with a putative α-galactosidase from Pedobacter sp. BAL39 (EDM38577), a bacterium belonging to Sphingobacteria rather than Flavobacteria. Furthermore, GalA17 shared little identity (6.3–13.6%) with the putative GH 36 α-galactosidases from Flavobacteria spp. recorded in the public database (ZP_01734709, YP_001197265, ZP_03852822, ZP_01106700; Fig. 1). Based on phylogenetic analysis, the GH 36 α-galactosidases from Bacteroidetes were separated into three clusters. Each of them contained interlaced Flavobacteria and Sphingobacteria α-galactosidase lineage twigs as shown in Fig. 1, suggesting that the evolution of Bacteroidetes α-galactosidases might not be closely coordinated with the classification of these bacteria. The gastrointestinal habitat—a fluid environment with low dissolved oxygen concentration—might be the main force involved in α-galactosidase evolution, as the phylogenetic tree put the α-galactosidases from symbiotic gastrointestinal bacteria in a separate cluster, and these microbial sources contained high G+C Gram-positive bacteria (Actinobacteria), low G+C Gram-positive bacteria (Firmicutes), and Bacteroidetes. Due to the inability to directly predict the putative active sites and homology modeling of rGalA17, the catalytic amino acid residues of rGalA17 remain unknown. In most cases, hydrolysis of the glycosidic bond by glycoside hydrolases is catalyzed by two amino acid residues, a general acid residue (proton donor) and a nucleophile/base residue (Davies and Henrissat 1995). As shown in Figs. 1 and 2, rGalA17 was grouped in a cluster sharing motifs KWD and SDXXDXXXR, not the KXD and RXXXD motifs of known α-galactosidases. Experimental and structural analyses indicate that D in KXD is the nucleophilic residue whereas D in RXXXD is the acid/base residue, and that K and R contribute to ligand binding (Comfort et al. 2007; Fujimoto et al. 2003; Garman and Garboczi 2004; Golubev et al. 2004). Based on the alignment and phylogenetic analyses in Figs. 1 and 2, we identified the presence of three D residues (D480, D549, D552) in motifs KWD and SDXXDXXXR. To identify which D matched those in KXD and RXXXD, site-directed mutagenesis was conducted. Almost no α-galactosidase activity was detected for mutants rGalA17D480A and rGalA17D549A, whereas rGalA17D552A retained ∼80% of the activity of rGalA17. This result suggested that D480

Appl Microbiol Biotechnol (2010) 88:1297–1309

and D549 might be the nucleophilic and acid/base residues of rGalA17, respectively, and might also contribute to the catalysis of other α-galactosidases in cluster 1 (Figs. 1 and 2). Furthermore, K in the KWD motif might be involved in ligand binding. The functions of S and R in SDXXDXXXR are unknown due to the lack of tertiary structure modeling and the substantial sequence differences between RXXXD and SDXXDXXXR. Mutants of these residues (rGalA17S548A and rGalA17R556A) were virtually inactive, revealing their important roles in rGalA17 and other α-galactosidases in cluster 1. The crystal structure of α-galactosidase from Trichoderma reesei (1SZN) indicates that C and W residues participate in substrate binding (Golubev et al. 2004). The drastic inhibition of rGalA17 enzyme activity in the presence of Ag+ and Hg2+ and its substantial increase by βmercaptoethanol suggest that sulfhydryl (or thiol) groups may exist in the active sites of the enzyme and are required for catalysis. Because Hg2+ can oxidize the indole ring, it likely interacts with W residue in the catalytic domain of the enzyme. Characterization of a GH 36 α-galactosidase from Bacteroidetes or from symbiotic bacteria in the gut of B. horsfieldi larvae has not been reported. Members of this group previously described are generally hypothetical based on conceptual translation of genomes (Fig. 1). The partially characterized α-galactosidase AAL27305 from C. piscicola BA (Figs. 1 and 2; Coombs and Brenchley 2001) shared the highest identity with GalA17 and was only 30.1%. Furthermore, α-galactosidases characterized from symbiotic Firmicutes, the most dominant bacterial phylum in human gut (Mahowald et al. 2009), have never been reported. To date, only three GH 36 α-galactosidases (AAD30994, ABD96085, and ABB76662) have been characterized from bacteria in gastrointestinal habitats (Goulas et al. 2009; van den Broek et al. 1999; Zhao et al. 2008). All of these are from Bifidobacteria, an important member of the human gastrointestinal microflora (Schell et al. 2002), and share 21.1–22.1% identity with GalA17 (Fig. 1). Using pNPG as a substrate, the apparent optimal pH (5.5) and temperature (45°C) of rGalA17 were close to those of the three abovementioned α-galactosidases from Bifidobacteria (pH 5.5–6.5; temperature 45–50°C). The reported pH stability (pH 4.5– 9.0), temperature stability (
Lihat lebih banyak...

Comentários

Copyright © 2017 DADOSPDF Inc.