0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.11.023
Short Communication
Cloning, characterization and phylogenetic relationships of stxI,
a endoxylanase-encoding gene from Streptomyces thermonitri
Wcans
NTU-88
Hsueh-Ling Cheng
a, Pei-Min Wang
a, Yu-Chi Chen
b,
Shang-Shyng Yang
a,c, Yo-Chia Chen
a,¤a Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung, Taiwan, ROC b Department of Food Science, National Pingtung University of Science and Technology, Pingtung, Taiwan, ROC
c Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan, ROC Received 28 August 2006; received in revised form 17 November 2006; accepted 20 November 2006
Available online 9 January 2007
Abstract
A thermostable xylanase gene (stxI) obtained from Streptomyces thermonitriWcans NTU-88 on domain analysis revealed an N-termi-nal catalytic domain featuring homology to a known xylanase within the glycoside hydrolase family 11. Recombinant STXI retained more than 60% of its activity following its incubation for at 60 °C for 24 h. These characteristics were close to thermophile and mesophile
Streptomyces strains. The main hydrolysis products of xylan degraded by STXI included large xylooligosaccharide fragments. These
results indicated that STXI was a typical endoxylanase. As regards the phylogenetic relationships of GH11, STXI and the other xylanase deriving from Streptomyces were included in a subgroup of the aerobic bacterial group. This result implied that the evolutionary relation-ships between the various xylanases deriving from Streptomyces strains were convergent.
© 2006 Elsevier Ltd. All rights reserved.
Keywords: Streptomyces thermonitriWcans; Xylanase gene; Phylogenetic analysis; Glycosyl hydrolase family 11
1. Introduction
Xylan, the major hemicellulose component of the plant cell wall, is a polymer with a linear backbone of -1,4-D
-xylopyranoside residues, residues which are commonly substituted by acetyl, arabinosyl, and glucuronosyl groups (Collins et al., 2005). Due to the structural complexity of xylan, the synergistic actions of a series of enzymes are nec-essary to hydrolyze xylan into simple sugars. Amongst the various xylanolytic enzymes, endo--1,4-xylanase (-1,4-D
-xylan -xylanohydrolase; EC 3.2.1.8) has been termed “true xylanase” for its speciWcity to xylan degradation (Collins et al., 2005), the enzyme randomly cleaving the -1,4
glycosidic bonds in the xylan backbone, thus bringing about a major reduction in the degree of polymerization of the substrate (Collins et al., 2005).
Endo--1,4-xylanases of glycoside hydrolase family 11 (GH11) are commonly found in fungi, bacteria and actino-myces (Collins et al., 2005). Microorganisms distributed in various environments typically develop various features as regards xylanase so as to acclimatize their growth to the prevailing conditions (Collins et al., 2005; Kulkarni et al., 1999). Mesophilic Streptomyces is one of the major bacteria types found to be growing within normal agricultural-waste composts, and xylanolytic activities are one of the major contributing factors to hemicellulose degradation. During solid-state fermentation, however, the temperature of the bulk of the compost will typically rise from ambient tem-perature to a temtem-perature range of around 50–70 °C, thus a range of thermostable xylanases are necessary for the activ-ities of mesophilic Streptomyces (Jang and Chen, 2003).
* Corresponding author. Tel.: +886 8 7703202x5181; fax: +886 8 7740550.
This feature is also important to many industrial applica-tions such as hemicellulose bioconversion of lignocellulose. In this study, we investigated the biochemical characteris-tics of the xylanase of Streptomyces thermonitriWcans NTU-88 by cloning a xylanase gene into Escherichia coli. We also discuss the phylogenetic relationships of GH11 as regards S. thermonitriWcans NTU-88 and other xylanolytic micro-organisms, by analysing certain of their amino-acid sequences.
2. Methods
2.1. Microorganism and DNA extraction
S. thermonitriWcans NTU-88 was grown for Wve days at 50 °C in a Mandels–Reese broth (Jang and Chen, 2003). The enriched biomass was collected for the genomic DNA extraction. The protocol for DNA extraction was based upon phenol–chloroform extraction (Chen et al., 2003). 2.2. Cloning procedures and recombinant xylanase expression
The PCR reaction adopted herein was used for the ampliWcation of xylanase genes obtained from the genomic DNA samples extracted from S. thermonitriWcans NTU-88. Two primers, stxF (5⬘CCAGACCGGCACCCACAACG-GC-3⬘) and stxR (5⬘GCTGACCGTGGGCCAGGTCC-3⬘) were used in the PCR reaction. The puriWed PCR product (stxI gene) was cloned to the pGEM-T-Easy vector and pET21a (Novagen Inc., Germany) for DNA-sequence determination and STXI expression, respectively. The stxI gene-transformed clone was grown in Luria–Bertani broth for for the induction of STXI (Huang et al., 2005). The molecular mass of the recombinant STXI was estimated by means of a slab–gel zymogram as previously described by Theather (Theather and Wood, 1982).
2.3. Enzyme assays
The xylanase activity of recombinant STXI was mea-sured by a method as described in 2005 by Huang et al. (2005). For estimation of the reaction temperature and pH optima of STX, the relative activity of xylanase was deter-mined at diVerent temperature levels as also diVerent pH values. The relative temperature stability of the tested xylanase was determined by incubating the enzyme, asepti-cally, in the absence of any substrate, and at various diVer-ent temperatures (10–100 °C) for a period of 4 h (Sengupta et al., 2000). The inXuence of a variety of metal ions upon
the enzyme’s activity was investigated by addition of vari-ous diVerent metal salts (Ca2+, Co2+, Cu2+, Fe2+, Hg2+,
Mg2+, Mn2+ and Ni2+) at a Wnal metal ion concentration of 10 mM in the reaction mixture (Sengupta et al., 2000). The speciWcity of STXI was tested for its ability to hydrolyse a variety of presented substrates. Hydrolysis products of xylan were identiWed by thin-layer chromatography (TLC).
2.4. Phylogenetic analysis
The deduced amino-acid sequence of stxI was aligned by use of CLUSTAL X software (Thompson et al., 1997) with available GH11 xylanase sequences retrieved from the CAZy database (Henrissat and Bairoch, 1996). The phylo-genetic tree based upon the neighbour-joining (NJ) algo-rithm was generated by using MEGA software (Kumar et al., 2004). The resultant unrooted tree topologies were evaluated in light of a bootstrap analysis of the neighbour-joining method based upon 1000 resamplings using the seq-boot and consense programs in the MEGA package.
3. Results and discussion
3.1. The nucleotide and deduced amino-acid sequences of stxI
The total length of the stxI was 839 bp and it contained a putative coding region encoding a polypeptide of amino-acids with a molecular mass of 30.4 kDa. Analysis of the induced crude extracts by zymogram revealed that the activity of xylanase was remained and featured one clear band with an apparent molecular weight of 32 kDa (Fig. 1). An analysis of stxI revealed that the DNA featured an overall G + C content of 64.96% and a G + C content of 92.1% in the third position of the codons. This nucleotide composition featuring a rather high G + C content was typ-ical of many thermophilic microorganisms (Gilkes et al., 1991). According to the sequence-based glycosyl hydrolase classiWcation (Henrissat and Bairoch, 1996), a putative
Fig. 1. Zymogram analysis of E. coli lysate using xylan substrate and Congo-red staining. Lane: 1: Protein standards. Lane 2: Cell lysate of E. coli BL21 (DE3) harboring pET21C-stxI.
Fig. 2. Neighbour-joining tree based upon partial GH11 xylanase sequences of 47 xylanolytic microbes. The numbers at the nodes indicate the level (%) of bootstrap support based upon a neighbour-joining analysis of 1000 resampled data sets. The accession numbers of amino-acid sequences are listed at the end of the scientiWc name of microorganisms.
Streptomyces sp. S38 CAA67143 Streptomyces olivaceoviridis CAC19491 Streptomyces sp. EC3 CAA56935 Streptomyces lividans AAC06114 Streptomyces viridosporus AAF09501
Streptomyces thermonitrificans NTU88 Streptomyces thermoviolaceus BAA19778 Streptomyces thermocyaneoviolaceus AAF04601
Cellulomonas fimi CAA54145 Thermobifida fusca AAV64879
Nonomuraea flexuosa CAD48747 Bacillus subtilis CAB13776
Geobacillus stearothermophilus AAB72117 Bacillus firmus AAQ14588
Paenibacillus sp. W61 BAE93061 Aeromonas punctata BAA06837 Gibberella zeae AAP41129
Fusarium oxysporum f. sp. lycopersici AAK27975 Humicola grisea var. thermoidea AAG16891
Magnaporthe grisea AAC41683 Phanerochaete chrysosporium AAG44995 Emericella nidulans CAA90073
Emericella nidulans CAA90074 Chaetomium thermophilum CAD48750
Scytalidium acidophilum AAQ22691
Aureobasidium pullulans var. melanigenum BAB69655 Penicillium purpurogenum CAA90390
Aspergillus niger CAA01470 Cryptococcus sp. S2 BAA09698 Phaedon cochleariae CAA76932 Trichoderma viride AAP83925
Hypocrea jecorina AAB29346 Claviceps purpurea CAA76570 Thermomyces lanuginosus AAB94633 Plectosphaerella cucumerina ABA08462 Setosphaeria turcica CAB52417
Ascochyta pisi CAA93120 Ruminococcus albus AAA85198
Ruminococcus sp. CAA90271 Pseudobutyrivibrio xylanivorans CAD65888
Clostridium saccharobutylicum AAA23287 Polyplastron multivesiculatum CAD56867
Orpinomyces sp. PC2 AAD04194
Neocallimastix patriciarum AAF14365 Piromyces sp. CAA62969
Neocallimastix frontalis CAA57820 Piromyces communis AAG18439
58 69 100 96 92 68 100 100 99 99 96 66 41 94 93 90 89 87 84 78 56 57 78 65 53 39 38 33 21 15 8 63 83 81 71 82 55 36 51 23 31 71 0.1 aerobic bacteria aerobic fungi anaerobic fungi anaerobic bacteria
conserved domain of GH family 11 was detected at posi-tion 106–516 of STXI. Interestingly, an addiposi-tional cellu-lose-binding domain (CBD) conserved with CBD family two was found at the C-terminal (position 582–834) of STXI. Some types of CBD are found to be able to stabilize the structure of xylanase. Such modules have also been found to be present within mesophilic bacteria such as Cellulomononas Wmi and Bacillus spp. Thus, CBD2 was thought to be capable of thermostabilizing the activity of the xylanase from Streptomyces when in a mesophilic environment (Collins et al., 2005; Gilkes et al., 1991). 3.2. Enzyme characteristics of STXI
STXI remained active over a very broad pH range of 5–9 and xylanase was active between 30 and 60 °C with an opti-mum at around 50 °C. The enzyme showed rather good sta-bility from 10 to 50 °C, whereas above 60 °C the enzyme’s stability decreased rapidly. The thermostability of the enzyme appeared to be quite pronounced within the meso-philic range. The biochemical characteristics of the recom-binant STXI are similar to those reported for many of the enzymes of Streptomyces strains isolated from compost (Ninawe and Kuhad, 2005, 2006; Ruiz-Arribas et al., 1998). Various metal ions examined revealed either an enhancing or inhibiting eVects were insigniWcant except Hg2+. These
results suggested that the xylanases were not a metallo-protein (Heck et al., 2006).
STXI appeared to be speciWc for xylan degradation, the highest activity being found on oat-spelt xylan; however, the enzyme also displayed diminished activity toward carbo-xylmethyl cellulose, avicel and -glucan as compared to xylan substrates. The hydrolysis products released from oat-spelt xylan by STXI consisted of a mixture of large xylooli-gosaccharide fragments, whereas xylotriose, xylobiose and xylose were not visualized in the TLC plate. This indicated that the mode of action of xylanase was of an endo-type hydrolysis and this type of xylanase could be applied in the prebleaching of eucalyptus kraft pulp (Dhillon et al., 2000). 3.3. Phylogenetic relationships
A relatedness tree was constructed for GH11, the tree including the sequence of the catalytic region of STXI in addition to the sequences of the sequences of GH11 deriv-ing from various microorganisms. The analysis of NJ revealed the division of the homology to GH11 xylanase into four groups, including aerobic bacteria, aerobic fungi, anaerobic bacteria and anaerobic fungi (Fig. 2). The GH11 xylanases of Streptomyces were clustered in the subgroup of aerobic bacterium group. The sequence homology of these diVerent xylanases appeared to be consistent with a hypothesis that the xylanases of GH11 evolved together within the Streptomyces that contained them.
In 1991, Glikes et al. reported that the -1,4-glycanases of various microorganisms arose from limited progenitor sequences by mutation, domain fusion and shuZing.
These changes could give critical insights into the ways in which the enzymes interact with certain substrates, or how these enzymes “Wt into” their environments. For example, non-catalytic thermostabilizing domains have been found to be present in xylanases of thermophilic bacteria (Gilkes et al., 1991), whereas the conserved triad (Val-Val-Asn or Val-Val-Asp) has been identiWed as being present only in the xylanases from alkaliphilic organisms (Kulkarni et al., 1999). In this study, the phylogenetic relationships of GH11 (Fig. 2) suggested that some elements of the catalytic region within GH11 xylanases could vary according to the oxygen requirement of xylanolytic microorganisms.
Acknowledgements
The Wnancial support of the National Science Council of Taiwan (Grant No. NSC 94-2313-B-020 -018) for this research is greatly appreciated.
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