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/lp/springer-journals/cloning-of-novel-bacterial-xylanases-from-lignocellulose-enriched-gEYRtgXwJl
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- Springer Journals
- Copyright
- Copyright © 2019 by The Author(s)
- Subject
- Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
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- 2191-0855
- DOI
- 10.1186/s13568-019-0847-9
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- See Article on Publisher Site
Abstract
Xylanases are in important class of industrial enzymes that are essential for the complete hydrolysis of lignocellulosic biomass into fermentable sugars. In the present study, we report the cloning of novel xylanases with interesting prop- erties from compost metagenomics libraries. Controlled composting of lignocellulosic materials was used to enrich the microbial population in lignocellulolytic organisms. DNA extracted from the compost samples was used to con- struct metagenomics libraries, which were screened for xylanase activity. In total, 40 clones exhibiting xylanase activity were identified and the thermostability of the discovered xylanases was assayed directly from the library clones. Five genes, including one belonging to the more rare family GH8, were selected for subcloning and the enzymes were expressed in recombinant form in E. coli. Preliminary characterization of the metagenome-derived xylanases revealed interesting properties of the novel enzymes, such as high thermostability and specific activity, and differences in hydrolysis profiles. One enzyme was found to perform better than a standard Trichoderma reesei xylanase in the hydrolysis of lignocellulose at elevated temperatures. Keywords: Xylanase, Metagenomics, Lignocellulose, Compost, Screening, Cloning Introduction biocatalysts is therefore clearly a priority for future com- Lignocellulosic biomass represents an attractive raw mercial production. In particular, enzymes with good material base for the production of alternative fuels in the thermal stability considered crucial for applications (Vii- future due to its great abundance and renewability (Ged- kari et al. 2007; Kumar et al. 2018, 2019). des et al. 2011). Producing renewable fuels and chemicals Xylanases (endo-β-1,4-xylanases, EC 3.2.1.8) are an from lignocellulosic biomass along the biochemical con- important class of lignocellulose degrading enzymes that version route requires the hydrolysis of the polysaccha- cleave the β-1,4-glycosidic backbone linkages in xylan, ride components of biomass, cellulose and hemicellulose, the most prominent non-cellulosic polysaccharide in the into their constituent sugars. Enzymatic hydrolysis has cell walls of land plants (Girio et al. 2010). Xylan is espe- been identified as the most efficient means to this end cially abundant in the cell walls of hardwoods and grasses (Girio et al. 2010), but current enzyme systems remain where it can represent up to 35% of total dry mass (Saha inefficient and consequently high doses of expensive 2003). Xylanases are grouped into families of glycosyl enzymes are required. Enzymatic hydrolysis is cited as hydrolases (GH) based on sequence homology (Henris- one of the limiting factors in the production of lignocel- sat 1991). The first xylanases to be discovered belonged lulosic bioethanol (Horn et al. 2012; Klein-Marcuscha- to families GH10 and GH11, and these families remain mer et al. 2012), and the development of more efficient clearly the best characterized. Families GH10 and GH11 both contain over 200 characterized members and have over 30 structure entries listed in PDB according to the *Correspondence: simo.ellila@vtt.fi carbohydrate active enzymes (CAZy) database (Cantarel VTT Technical Research Centre of Finland, P.O. Box 1000, 02044, et al. 2009). More recently, family GH30 has been found Vuorimiehentie, Espoo, Finland to include several xylanases (St John et al. 2010), many Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Ellilä et al. AMB Expr (2019) 9:124 Page 2 of 12 of which appear to be active specifically on glucuron - at − 80 °C for DNA extraction. Total DNA was isolated oxylan. A handful of xylanases have also been reported using the PowerSoil DNA isolation kit (MoBio Labora- from family GH8 (Pollet et al. 2010). Xylanases are often tories) and the bacterial and fungal communities were multidomain proteins, containing one or more additional monitored using 16S and 18S rRNA gene-targeting PCR- domains such as carbohydrate-binding modules. DGGE. The other two replicates ran untouched to obtain Xylanases are already used in several industrial appli- information about the biodegradability of substrates. cations ranging from the manufacture of food and feed Samples for constructing the metagenomic libraries were to the bleaching of kraft pulp (Kumar et al. 2016, 2018). selected based on substrate biodegradation, and bacterial Xylanases are also important components of enzyme and fungal community succession. Samples from several preparations currently on the market and utilized in time points and enrichment conditions were pooled for commercial lignocellulosic bioethanol plants. The most library construction. common enzyme mixtures are derived from the fungus Trichoderma reesei, which can secrete large amounts of Construction of metagenomic DNA libraries enzymes (Merino and Cherry 2007). However, as T. reesei For the construction of large-insert metagenomic librar- is a mesophilic fungus, its enzymes can suffer from low ies, high molecular weight DNA was isolated by enzy- stability when confronted with high temperatures and/or matic lysis followed by hot phenol treatment and the inhibitor concentrations (Rahikainen et al. 2013). There is isolated DNA was purified with pulse-field gel electro - therefore a clear need for more thermostable xylanases. phoresis as described in Kielak et al. (2009). Metagen- Such enzymes can be isolated from thermophilic organ- omic libraries were prepared from gel-extracted DNA isms and engineered for improved thermostability, as with the CopyControl Fosmid Library Production kit reviewed by Kumar et al. (2018). (Epicentre) according to manufacturer’s instructions. While we still struggle to deconstruct lignocellulose in Two separate libraries, FOS-37 °C-CEL and FOS- a controlled manner, a wide variety of fungi and bacteria 50 °C-CEL, were prepared from DNA samples from 37 are capable of using it as an energy and carbon source in to 50 °C composts, respectively. Average insert size and nature (Rubin 2008). Metagenomics methodology allows library size was estimated from NotI restriction digests the genetic study of these microbes directly from their of fosmids isolated from 18 to 40 random clones. Before habitats and grants access to the enormous biocatalytic screening, the libraries were amplified to 10 clones. The diversity that they represent (Ferrer et al. 2009). Both amplified libraries were stored in 20% glycerol at − 80 °C. activity- and sequence-based metagenomics methods A separate plasmid library (PLA-50 °C-CEL) was pre- have proven excellent in uncovering novel lignocellulose- pared from samples from the 50 °C composts as fol- degrading enzymes (Montella et al. 2016). In the present lows: DNA was isolated using the PowerSoil kit (MoBio), study, we report the cloning of novel bacterial xylanases blunt-ended using T4 polymerase, A-overhangs added from metagenomic libraries derived from the composting using Taq polymerase, dephosphorylated using antarc- of lignocellulosic materials. Several interesting enzymes tic phosphatase (NEB) and finally TOPO-gated into the were uncovered, including a thermostable GH11 xyla- pCR2.1-TOPO vector (Invitrogen). The TOPO-gations nase performing better than Xyn11A from T. reesei in the were transformed into OmniMAX 2-T1 cells. The library hydrolysis of hydrothermally pre-treated wheat straw at size was estimated in a similar manner to the fosmid elevated temperatures. library, but using the restriction enzymes EcoRI/NcoI. Materials and methods Enrichment of lignocellulosic microbes by controlled Screening of metagenomic libraries for xylanases composting The functional screen for xylanase activity was con - Composting was conducted under controlled conditions ducted on LB-agar plates containing 0.1% (w/v) (The CEN standard 14046) at 37 °C and 50 °C. Steam Azurin-crosslinked xylan from oat spelt (AZCL-xylan, exploded spruce (from the University of Lund), cutter Megazyme). For the screening of the fosmid libraries, chips and Whitman filter paper were used in separate Copycontrol Fosmid Autoinduction Solution (Epicentre, composts to enrich lignocellulolytic species in the micro- USA) was added to screening plates in order to increase bial communities. Triplicate composts were run per assay sensitivity. The screen was conducted at tenfold substrate. coverage of each primary library clone number to pro- The biodegradation of the substrates was followed by vide the maximal amount of unique xylanase-positive quantitative online measurement of CO evolution for clones. E. coli clones surrounded by blue halos were 20 weeks. One of three replicate composting bioreac- picked, repeat plated and unique clones isolated using tors was sampled regularly and the samples were frozen restriction analysis. Ellilä et al. AMB Expr (2019) 9:124 Page 3 of 12 Sequencing and sequence analysis incubating the lysates in water baths at given tempera- The BigDye Terminator v3.1 Cycle Sequencing Kit and tures, taking samples at intervals, quantifying the xyla- an ABI Prism 3100 Genetic Analyzer (Applied Biosys- nase activity using the EnzCheck assay and comparing to tems) were used to sequence all plasmids and fosmid a pre-incubation reference to calculate residual activity. terminal sequences. Fosmid clones identified in the xyla - nase-activity screen were pooled and sequenced using Subcloning a 454 GS FLX Titanium instrument (Roche). The reads Fosmid (or plasmid) DNA was recovered from 5 mL cul- were assembled into contigs using Newbler (v. 2.0.00.20) tures of each clone using the QIAprep Spin Miniprep kit and assigned to specific clones by aligning with the vec - (Qiagen) and used as templates for PCR. Primers were tor end sequences generated using Sanger sequencing. designed for the amplification of the selected genes (with - All assembled contigs above 1 kb are deposited in Gen- out signal sequences) and incorporation of the genes into Bank under accessions KX236210.1–KX236308.1. the pASK-IBA16 expression vector (IBA GmbH). PCR- Analysis and annotation of the available vector amplification of the xylanase genes was done using the sequences was performed using Geneious Pro soft- iProof high-fidelity DNA polymerase (Bio-Rad). Cloning ware (Kearse et al. 2012). A downloadable version of was performed using the recombination-based In-Fusion the carbohydrate active enzymes (CAZy) database was Advantage PCR cloning system (Clontech) and the com- retrieved from the CAZymes Analysis Toolkit web- pleted reactions were used to transform Stellar compe- site (Park et al. 2010). The obtained vector sequences tent E. coli cells (Clontech) according to manufacturer’s were used in standard BlastP queries against the whole instructions. The constructed expression vectors were CAZy database to locate carbohydrate active enzymes recovered using the QIAprep kit and correct sequences (CAZymes). Open reading frames (ORFs) were predicted verified by Sanger sequencing. using Glimmer 3 (Delcher et al. 2007). Putative xylanase- encoding ORFs were used in new BlastP queries against Expression and purification of recombinant xylanases the CAZy database to find closest homologues, and The subcloned plasmids were used to transform XL-1 in many cases, an annotation for associated domains. Blue E. coli cells (Stratagene) using standard procedures. Signal sequence prediction was performed using Sig- The clones were grown in LB medium containing 100 μg/ nalP Server (Petersen et al. 2011). Annotated xylanase mL carbenicillin in 1.5 L volume at 30 °C with 250 rpm sequences were deposited in GenBank under accessions shaking. At OD ≥ 0.7 the expression of the recombi- MF171165.1–MF171184.1. nant genes was induced by the addition of anhydrotetra- cycline (Clontech). After 2 h, the cells were harvested by centrifugation, washed with 90 mL of 50 mM Tris/HCl pH 8.0 and pelleted by centrifugation. Initial thermostability assay from cell lysates To recover the periplasmic protein, the cell pellets Whole cell lysates for all unique xylanase-positive E. were resuspended in 50 mL of 50 mM Tris/HCl pH 8.0. coli clones were prepared from 500 mL cultures using To these suspensions sucrose, EDTA and lysozyme were standard methods. In brief, when OD of the cultures added to final concentrations of 20% (w/v), 1 mM and reached 0.7, enzyme expression was induced using 1 mL 0.5 mg/mL, respectively. The suspensions were kept on of CopyControl Induction Solution (fosmid clones) or slow rotation for 30 min at room temperature to allow IPTG (plasmid clones). The cells were harvested 15–20 h cell wall lysis, followed by addition of 50 mL of ice-cold (fosmid clones) or 3 h (plasmid clone) post-induction by water. The suspension was kept on ice for 15 min with centrifugation and resuspended in 5 mL volume of assay gentle intermittent inversions. The spheroblasts were pel - buffer (50 mM Na-citrate, pH 6.5) and lysed by sonica - leted by centrifugation and the supernatants (the peri- tion. Cellular debris was pelleted by centrifugation, and plasmic extracts) transferred to a fresh tube. the supernatants (cell lysates) collected and stored at The periplasmic extracts were loaded onto 25 mL − 20 °C. StrepTactin Sepharose High Performance columns (Inv- Xylanase activity from the whole cell lysates was meas- itrogen) and purified using an ÄKTAmicro purifica - ured using the fluorescence-based EnzCheck Ultra tion system (GE Healthcare). Fractions were gathered Xylanase Assay Kit (Invitrogen), according to the manu- and samples run on precast SDS-PAGE gels and visual- facturer’s instructions. The substrate used in the kit ized using the Criterion Stain Free Imager (Bio-Rad). is 6,8-difluoro-4-methylumbelliferyl β-d-xylobioside Fractions containing visible bands of correct size were (DiFMUX ). Reactions were carried out on microplates pooled and concentrated to a final volume of 3 mL using in 50 mM Na-citrate pH 5 at 40 °C for 30 min, with flu - Vivaspin 20 (GE Healthcare) sample concentrators, with orescence measured at 40-s intervals. The thermosta - a cut-off of 10 kDa. The buffer was exchanged for 50 mM bility of the xylanases in the cell lysates was assayed by Ellilä et al. AMB Expr (2019) 9:124 Page 4 of 12 Na-citrate pH 5.0 using Econo-Pac 10DG desalting col- the xylanases were used as components of cellulolytic umns (Bio-Rad), and the concentrated purified recom - enzyme mixtures. For hydrolysis at 45 °C, an enzyme binant xylanases stored at − 20 °C for further study. The mixture comprising T. reesei enzymes was created by protein concentrations of the purified proteins were combining CBHI (TrCel7A), EGII (TrCel5A) and XYN2 measured using the Lowry-based Bio-Rad DC II protein (TrXyn11A) at a mass ratio of 70:20:10, and the enzyme assay kit using BSA as standard. mixture used in hydrolysis at an overall dose of 16 mg/g dry matter. β-Glucosidase (Cel3A) from Aspergillus niger was further dosed at an activity based concentration of Preliminary characterization of novel xylanases 30 U/g dry matter. Similar enzyme mixtures were pre- Xylanase activity of the recombinant enzymes was meas- pared with the purified metagenomic xylanases replac - ured on 1% birchwood xylan (Roth) in 50 mM Na-citrate ing the T. reesei xylanase (Xyn11A), while in one mixture pH 5.0. The standard reaction was conducted at 50 °C xylanase was omitted. The purification of the reference for 5 min, and released reducing sugars quantified using enzymes has been described elsewhere (Suurnäkki et al. DNS, using xylose as standard. One unit of catalytic 2000). activity (U) was defined as the amount of enzyme capa - Separate hydrolysis reactions were performed at a ble of releasing 1 µmol of reducing sugar from xylan per higher temperature (55 °C) using the two metagenomic minute under the assay conditions and standard errors xylanases that were found to be thermostable. The ref - calculated from triplicate reactions. This assay was used erence mixture of thermostable enzymes comprised in further applications unless otherwise noted. CBHI from Acremonium thermophilum (AtCel7A), The pH-profiles of the xylanases were determined CBHII from Clostridium thermocellum (CtCel6A) and using three buffer systems: Na HPO /citric acid (McIl- 2 4 EGII from Thermoascus aurantiacus (TaCel5A) (Viikari vane’s buffers—pH 3.0–6.5), Tris/HCl (pH 7.0–8.5), and et al. 2007), kindly provided by ROAL Oy. The enzymes glycine/NaOH (9.0–10.5). Buffers were prepared for 0.5 were used at an overall dose of 10 mg/g dry matter at pH unit increments and 1% (w/v) birchwood xylan (Roth) a ratio 50:20:20:10 (CBHI:CBHII:EGII:Xylanase) and solutions prepared in each buffer. Xylanase activity was β-glucosidase from T. aurantiacus (TaCel3A) added at then determined using the standard method. 30 U/g dry matter. To assay the optimal temperature for each xylanase, The substrate used was wheat straw thermally pre - reactions were conducted at different temperatures over treated at 190 °C for 15 min (BioGold—Estonia), pre- a range 20–90 °C, with buffer pH adjusted to be optimal pared as a homogenous slurry in 50 mM Na-citrate pH for each enzyme based on the previous assay. Thermo - 5.0 using an immersion blender. Triplicate hydrolysis stability was assayed by incubating the enzymes in water reactions were performed in 500 µL volume in micro- baths at 60 °C and 70 °C and sampling at regular interval centrifuge tubes. Reactions were terminated by adding to quantify residual xylanase activity. 10 μL of 10 M NaOH and unhydrolyzed substrate was The activity of xylanases was quantified on three sub - removed by centrifuging the tubes at 4000 rpm for 5 min. strates: birchwood glucuronoxylan (Roth), wheat ara- Reducing sugars from the supernatants were measured binoxylan (Megazyme) and carboxymethyl cellulose using the p-hydroxy benzoic acid hydrazide (PAHBAH) (CMC—Sigma). All substrates were prepared as 1% solu- method (Lever et al. 1973), with glucose as standard. tions in 50 mM Na-citrate buffers of optimal pH for each enzyme. Activity toward each substrate was measured as previously with glucose serving as standard for CMC Results activity. Creation and screening of compost metagenomic libraries To study the products of birchwood xylan hydroly- Controlled composts were created using lignocellulosic sis, the standard xylanase assay was performed for 18 h materials (Whatman filter paper, steam exploded spruce instead of 5 min using 1 U/mL enzyme, and the released and cutter chips) to enrich microbial communities pro- reaction products were analysed using high-perfor- ducing cellulolytic and hemicellulolytic enzymes. The mance liquid chromatography (HPLC). A CarboPac PA garden waste inoculum originated from the Ämmässuo 1 column was used on an ICS-3000 ion chromatogra- composting plant (YTV, Finland). Composting was con- phy system (Dionex). Xylo-oligosaccharides of a DP of ducted at 37 °C and 50 °C to enrich for meso- and ther- 1–6 (xylose to xylohexaose—Megazyme) were used as mophilic organisms, respectively. The biodegradation of standards. lignocellulose containing substrates was clearly more effi - cient at 50 °C. For example, after 20 weeks, 61% of cutter Hydrolysis of hydrothermally pre‑treated wheat straw chips were converted to CO whereas at 37 °C the degra- To assess the enzymes’ efficiency on a technical lig - dation was less than 20%. nocellulosic substrate, steam-exploded wheat straw, Ellilä et al. AMB Expr (2019) 9:124 Page 5 of 12 Compost samples from several time-points and rep- (EnzCheck—Invitrogen), which allowed activity meas- resenting different materials used for enrichment were urement in all 40 lysates. The lysates were incubated at pooled, metagenomic DNA was extracted from the 60 °C for a total duration of 3 days, and residual activ- microbial communities and used to construct DNA ity was quantified from samples (Fig. 1). Most lysates libraries using both fosmid and plasmid vectors. The were found to rapidly lose their xylanase activity by 6 h details of the constructed compost metagenomic libraries of incubation, but a small number displayed considerable can be seen in Table 1. The primary fosmid libraries FOS- stability at this temperature. In particular, lysates repre- 37 °C-CEL and FOS-50 °C-CEL were found to contain senting two clones (XYL38 and XYL40) retained virtually roughly 43.000 and 40.000 clones, respectively. Based all their xylanase activity for the whole duration of incu- on the number of clones containing a unique insert and bation. These clones were therefore of particular interest the average insert size both libraries were estimated have for further study. captured about 1.2 Gb of unique sequence. The plasmid library PLA-50 °C-CEL yielded a far greater number of Sequencing clones: 760,000. On average 95% of the clones contained All unique xylanase-positive fosmid clones were pooled a unique insert with an average size of 5.4 kb, bringing and sequenced using 454-pyrosequencing. This approach the total size of this library to 3.9 Gb, or about three yielded a total of 40.6 Mb of sequence data. 9 entire fos- times the size of the fosmid libraries. mid inserts could be completely sequenced and assigned The libraries were screened to ten times coverage of the to specific clones using the fosmid terminal sequences primary library clone number on agar plates containing the chromogenic substrate AZCL-xylan. FOS-37 °C-CEL and FOS-50 °C-CEL fosmid libraries and the PLA- 50 °C-CEL plasmid library yielded 21, 18 and 1 unique xylanase hit(s), respectively (Table 1). Accordingly, the hit rates for the fosmid libraries were 1/58 and 1/68 Mb, while for the plasmid library the hit rate was significantly lower at 1/3900 Mb. These numbers are in line with fig - ures (1/14–1/1024 Mb) previously reported for endoglu- canase screens (Duan and Feng 2010). However, it is not readily apparent why the hit rate for the plasmid library was so much lower than for the fosmid libraries. Thermostability screen of metagenomics library clones An initial assay was devised to identify thermostable xylanases directly from cell lysates of the metagen- omic library clones. We were unable to measure xyla- Fig. 1 Initial thermostability assay of xylanase-positive metagenomic library clones. Whole cell lysates of the positive metagenomic clones nase activity from all the lysates using birchwood xylan were incubated in a water bath at 60 °C for 3 days. Samples were as substrate (Data not shown). We therefore opted for taken at regular intervals and residual xylanase activity quantified the sensitive fluorescent xylanase substrate DiFMUX using the EnzCheck xylanase assay Table 1 Details regarding the metagenomic fosmid and plasmid libraries constructed and screened for xylanase activity in this study Library FOS‑37 °C‑ CEL FOS‑50 °C‑ CEL PLA‑50 °C‑ CEL Vector Fosmid—pCC1FOS Fosmid—pCC1FOS Plasmid—pCR2.1-TOPO Number of unique clones 43,000 40,000 760,000 Average insert size 28.5 kb 30.8 kb 5.4 kb Total library size 1226 Mb 1232 Mb 3900 Mb No. of xylanase hits 21 18 1 Hit rate 1/58 Mb 1/68 Mb 1/3.9 Gb Positive clone identifiers XYL1–21 XYL22–39 XYL40 Number of clones was estimated using serial dilution and plating of the primary library. Average insert size was estimated based on restriction digests of 30–40 individual library clones Ellilä et al. AMB Expr (2019) 9:124 Page 6 of 12 generated by Sanger sequencing. Additionally, larger and disruption in a fashion similar to CBMs (Kataeva contigs of over 10 kb could be assigned to one of the et al. 2002). Finally, the GH10 xylanase in an unassigned termini of five fosmids. A further round of 454-pyrose - sequence (contig 74) contained an N-terminal sequence quencing was performed on the ten most thermostable of roughly 100 aa, with homology to cadherin-like clones by LGC Genomics GmbH (Berlin). This run gen - (CADG) sequences. Bacterial CADG domains have also erated 35.1 Mb of sequence and 16 assembled contigs of been shown to be active in carbohydrate binding (Frai- over 1 kb. In total, we were able to assign over 20 kb of berg et al. 2011). sequence to 20 of the fosmid clones. The single unique Six enzymes were shortlisted for subcloning and puri- hit from the plasmid library (XYL40) was sequenced fication. Three xylanases were chosen based on the initial using Sanger sequencing. thermostability assay: XYL40 (GH11/CBM60), XYL38 The annotation of the sequence data set revealed (GH10) and XYL35 (GH11). Three others were chosen 20 unique putative xylanases, of which 15 could be due to their unique sequences. Two were the xylanases traced back to a particular metagenomic library clone belonging to the less studied xylanase-containing families (Table 2). Of these the clear majority (13) belonged to of glycosyl hydrolases, family GH8 (XYL32) and GH30 family GH11, while five were members of family GH10. (XYL13), while one was selected due to its complex One putative xylanase representing both families GH8 domain organization. This xylanase, XYL21 (GH10/FN3/ and GH30 were also found. The majority of identi - FN3/CBM2), contained two fibronectin type III domains fied genes encoded multidomain proteins, with puta - (FN3) linking a catalytic GH10 domain to a CBM2-type tive carbohydrate-binding modules from families 2, 6, carbohydrate-binding module. An FN3-domain has pre- 22, 60 and 64 accompanying the catalytic domains. In viously been described in conjunction with a GH10 xyla- addition, the putative xylanase in clone 21 encoded for nase (Kim do et al. 2009). All the six selected xylanases fibronectin type III domains (FN3). FN3-domains have were amplified from fosmid or plasmid DNA using PCR previously been reported in other glycosyl hydrolases, and incorporated into the pASK-IBA16 E. coli periplas- where they have been implicated in substrate binding mic secretion vector for recombinant protein production. Table 2 The novel putative xylanase sequences discovered in the annotation of the metagenomic sequence dataset Library Sequence GenBank ID Domain organization of xylanase FOS-37 °C-CEL XYL1 MF171170 [GH11]-[CBM60] XYL3 MF171171 [GH11]-[CBM60] XYL7 MF171172 [GH11]-[CBM60] XYL11 MF171173 [GH11]-[CBM60] XYL12 MF171174 [GH11]-[CBM60] XYL13 A MF171176 [GH30] B MF171175 [GH11]-[CBM64] XYL18 MF171177 [GH10] XYL19 MF171178 [GH11]-[CBM60] XYL21 MF171179 [GH10]-[FN3]-[FN3]-[CBM2] FOS-50 °C-CEL XYL25 MF171180 [GH11]-[CBM2] XYL32 MF171181 [GH8] XYL35 MF171182 [GH11] XYL38 MF171183 [GH10] PLA-50 °C-CEL XYL40 MF171184 [GH11]-[CBM60] Unassigned sequence Contig. 56 MF171165 [GH11]-[CBM60] Contig. 74 MF171166 [CADG]-[GH10]-[CBM6]-[CBM22]-[CBM22] Contig. 228 MF171167 [GH10]-[CBM2] (partial) Contig. 1219 MF171168 [GH11]-[CBM60] Contig. 1238 MF171169 [GH11] Xylanase domain structure is indicated using the abbreviations of families of glycosyl hydrolases (GH) and carbohydrate-binding modules (CBMs) in brackets. The genes selected for subcloning and further study are indicated in italics [CADG] cadherin-like domain Ellilä et al. AMB Expr (2019) 9:124 Page 7 of 12 Recombinant protein production had very similar pH profiles with peak activity occurring The recombinant xylanases were produced in E. coli at pH 6.5. XYL38 fared slightly better at a more alkaline shake flask cultures and the protein recovered by peri - pH, retaining over 70% activity at pH 9. XYL35 had the plasmic extraction and purified using StrepTag affin - narrowest pH profile with a pH-optimum of 7.0. ity chromatography. The approach yielded sufficient The optimal temperature for all the purified xylanases amounts (> 1 mg) of recombinant protein for preliminary was measured by performing reactions at different tem - characterization of five of the selected xylanases (Fig. 2). peratures over a range of 20 °C to 90 °C (Fig. 3b). The The five recombinant enzymes were found to be active on highest value found was 80 °C for XYL38, followed by both the small-molecule EnzCheck substrate and birch- 70 °C for both XYL40 and XYL35. Interestingly, XYL32 wood xylan (Roth). Only in the case of the family GH30 displayed high activity at 20 °C and could therefore be enzyme (XYL13) the protein yields and quality were not suitable for application where cold-active enzymes are sufficient to allow the study of enzyme properties. required. The activity of the enzymes was additionally quanti - Preliminary characterization of novel xylanases fied on wheat arabinoxylan and carboxymethyl cellu - The purified recombinant xylanases were characterized lose (CMC). CMC was included as a control for possible in terms of temperature and pH optima, thermostabil- endoglucanase activity. T. reesei Xyn11A was included as ity, substrate specificity and hydrolysis profile. The results a reference enzyme in this assay. Higher activities were are summarized in Fig. 3 and in Table 3. measured on wheat straw arabinoxylan than on birch- The pH profiles of the purified xylanases were assayed wood glucuronoxylan for all enzymes. This substrate over a pH range 3.0–10.5 (Fig. 3a). None of the novel preference was more striking in the case of XYL35, which enzymes was active at the acidic pH of 3, but the xyla- had over threefold higher activity on wheat straw xylan. nases XYL32 and XYL21 show considerable activity at XYL21 was the only enzyme for which CMCase activity pH 4 with 70% and 60% activity, respectively. Both of could be detected under these assay conditions. The spe - these enzymes also have relatively low pH optima. The cific activity on CMC (1.8 U/mg) was over tenfold lower pH optimum of XYL32 was 5.0–5.5, while XYL21 had a than that observed for arabinoxylan. very broad pH profile in general, with maximal activity Figure 3c also allows comparison of the specific activi - occurring over a range of pH 5–7 and still retaining over ties of the different enzymes. Interestingly both enzymes 50% activity at pH 10. The most thermostable enzymes belonging to family GH10, XYL21 and XYL38, had signif- based on the initial characterization, XYL38 and XYL40, icantly lower specific activities than the other enzymes, 17.4 and 7.2 U/mg, respectively. All enzymes belonging to family GH11, including the reference enzyme TrX- yn11A, had specific activities on birchwood xylan in the 240–500 U/mg range, while the specific activity of the GH8 xylanase XYL32 was nearly twice that of TrXyn11A at 1050 U/mg. We also quantified the specific activities of the purified xylanases on the DiFMUX model sub- strate (Table 3). A difference in specific activities of over 10 -fold was observed with this substrate, with the GH8 xylanase XYL32 having clearly the lowest level of activity on this substrate. The hydrolysis products generated by the metagenomic xylanases from birchwood xylan were analyzed using high-performance liquid chromatography (HPLC). The GH10 xylanase XYL38 was found to degrade the xylan to the furthest extent, with xylose (X ) being the most abun- dant reaction product in the hydrolysate (Fig. 3d). In our assay, the GH11 enzymes XYL40 and TrXyn11A had very Fig. 2 SDS-PAGE analysis of the five purified recombinant similar hydrolysis profiles, with xylobiose (X ) being the metagenomic xylanases. Two micrograms of each purified enzyme dominant reaction product. The hydrolysis products pro - ™ ™ was loaded on a 4–20% Criterion TGX Stain-Free Precast gradient duced by the three remaining enzymes (XYL21, XYL32 gel (Bio-Rad) and visualized on a Bio-Rad Criterion Strain-free imaging and XYL35) contained primarily xylobiose and -triose. system. Lanes: M—Prestained Precision Plus protein standard The thermostability of the purified recombinant (Bio-Rad), 1—XYL35 (GH11), 2—XYL40 (GH11-CBM60), 3—XYL38 (GH10), 4—XYL32 (GH8), 5—XYL21 (GH10 + FN3 + FN3 + CBM2) enzymes was quantified at 60 °C and 70 °C (Fig. 4). The Ellilä et al. AMB Expr (2019) 9:124 Page 8 of 12 Fig. 3 Preliminary characterization of the purified recombinant xylanases. a The relative activity of the purified recombinant xylanases on birchwood xylan as a function of pH. b Temperature optima were measured by conducting the standard xylanase assay at different temperatures over a range of 20 °C to 90 °C. c Specific activity of the purified xylanases on birchwood glucuronoxylan (GX) and wheat arabinoxylan (AX). d Xylo-oligosaccharides (X –X ) released by recombinant xylanases from birchwood glucuronoxylan as measured by HPLC. Results plotted as relative 1 6 to original substrate mass (w/w) Table 3 Characteristics of the purified recombinant xylanases Xylanase XYL21 XYL32 XYL35 XYL38 XYL40 Domain organization [GH10]-[FN3]-[FN3]-[CBM2] [GH8] [GH11] [GH10] [GH11]-[CBM60] Size (aa) 602 385 196 370 325 Mw (kDa) 63.2 44.3 24 41.9 34.9 pI 7.5 5.4 9.8 6.9 8.1 pH optimum 5.5 5.5 7.0 6.5 6.5 Temp. optimum 60 40 70 80 70 XOS produced from GX X /X X /X X –X X /X X 2 3 2 3 2 4 1 2 2 GX (U/mg) 17.2 1048 293 7.1 281 AX (U/mg) 23.8 1551 1062 13.8 399 DiFMUX (U/mg) 0.110 0.0072 10.0 0.106 1.94 Symbol used in legends ■ ▼ ▲ ● Figures presented for molecular weight and pI were calculated based on the amino acid sequences of the native secreted proteins excluding the purification tag GX birchwood glucuronoxylan, AX wheat straw arabinoxylan Ellilä et al. AMB Expr (2019) 9:124 Page 9 of 12 Fig. 4 Stability of the purified metagenomic xylanases at 60 °C (a) and 70 °C (b). The purified enzymes were incubated in water baths at 60 °C (a) and 70 °C (b) for a total duration of 3 days and residual xylanase activity was quantified using the standard assay Hydrolysis of pre‑treated wheat straw reference TrXyn11A and the multidomain XYL21 were The performance of the purified recombinant xylanases found to lose all activity at 60 °C by the first time-point in the degradation of hydrothermally pretreated wheat (2 h). XYL32 and XYL35 were of intermediate stabil- straw was evaluated using model enzyme cocktails ity, gradually losing most of their activity during the (Fig. 5). Hydrolysis was performed at 45 °C and 55 °C, first 6 h of incubation. XYL38 and XYL40 were found combining the novel xylanases with the core cellulases of to be equally stable in purified recombinant form as T. reesei and thermostable enzymes provided by Roal Oy, in the initial assay conducted on the E. coli whole cell respectively. lysates (Fig. 1). Both enzymes remained virtually stable All xylanases improved the hydrolysis of pretreated at 60 °C, while XYL40 was found to remain remarkably wheat straw at 45 °C compared to the reference cellu- stable even at 70 °C, with a half-life of roughly 6 h. lases alone (dashed line in Fig. 5a). The increase was most notable at the initial 4 h time point. After this time point Fig. 5 Hydrolysis of hydrothermally pre-treated wheat straw using novel xylanases as components of multienzyme mixtures. a Hydrolysis at 45 °C using mesophilic enzymes: T. reesei cellulases ( TrCel7A, TrCel5A) and A. niger glucosidase (AnCel3A), supplemented with xylanase according to figure inset. b Hydrolysis at 55 °C using thermostable enzymes: CBHI from Acremonium thermophilum (AtCel7A), CBHII from Clostridium thermocellum (CtCel6A) in addition to EGII and β-glucosidase from Thermoascus aurantiacus ( TaCel5A and TaCel3A). Xylanase was included according to figure inset Ellilä et al. AMB Expr (2019) 9:124 Page 10 of 12 the xylanases could be divided into three groups based subcloned and studied in more detail due to their rather on performance: The best hydrolysis yields were achieved unique primary sequences. when the cellulases were supplemented with TrXyn11A The recombinant XYL38 (GH10) and XYL40 (GH11) or the most thermostable xylanase XYL40. The lowest proved to be very stable at 60 °C, and the latter even yields were achieved using XYL35 or XYL38, with only a retained activity for long periods at 70 °C. Although sev- minor improvement over the core cellulase mixture. The eral more thermostable enzymes have been reported performance of XYL32 and XYL21 could be described as (Kumar et al. 2018), few have been tested in lignocel- intermediate. It is interesting to note that the enzymes lulose hydrolysis as components of cellulolytic enzyme that performed the best in this assay, TrXyn11A and cocktails. XYL40 was found to perform better that XYL40, share a very similar reaction rate and hydrolysis Xyn11A from T. reesei in hydrolysis experiments carried profile (Fig. 3d). out on an authentic lignocellulosic substrate at 55 °C. The effect of xylanase addition was also clearly observ - This enzyme therefore shows potential toward lignocel - able using the mixture of thermostable enzymes at 55 °C lulose hydrolysis as a component of a cocktail of thermo- (Fig. 5b). XYL40 and the TrXyn11A provided similar stable enzymes. yields up to 24 h, after which XYL40 performed better, The other thermostable enzyme XYL38 performed most probably due to inactivation of TrXyn11A. XYL38 rather poorly in the wheat straw hydrolysis experiments. surprisingly failed to increase yields at all after 4 h. The This enzyme had a very low specific activity toward birch result suggests that XYL40 could be a useful thermosta- wood and wheat straw xylans, from which it released ble xylanase for use in lignocellulose degradation, while xylose in addition to xylobiose, and showed activity on for XYL38 its poor specific activity hinders its use. carboxymethylcellulose. GH10 enzymes generally have broad substrate specificities, and activity on cellulose has Discussion been reported (Chu et al. 2017; Wang et al. 2019). The In the present study, we set out to identify novel xylanases release of xylose as the main product suggests XYL38 desirable characteristics, with particular emphasis on might display exo-activity (Juturu and Wu 2014). The thermostable enzymes that are considered advantageous data suggests that XYL38 is a rather unusual xylanase for many applications (Kumar et al. 2018). Metagenomics and considerably different from its closest characterized offers an attractive toolset for the discovery of enzymes homologue Xyn10A from Acidothermus cellulolyticus with novel backbones and/or improved characteristics. 11B (50.3% identity). This enzyme was inactive on CMC Activity-based plate screening of metagenomic libraries and did not produce detectable amounts of xylose from is an efficient method for identifying desired enzymes birchwood xylan (Barabote et al. 2010). The other recom - that are expressible in the cloning host, but screening binantly produced GH10 and GH11 xylanases, XYL21 large DNA-libraries can quickly become very labor-inten- (GH10-FN3-FN3-CBM2) and XYL35 (GH11) did not sive. We aimed to lower the number of clones needed to display any particularly attractive properties considering be screened (increased hit-rate) by first enriching desired potential industrial applications. microbial species using target carbon sources. Enriching We also expressed a novel xylanase belonging to the has previously been applied using lignocellulosic mate- less-studied family GH8 (XYL32). This enzyme had the rials such as rice straw (Reddy et al. 2013), switchgrass highest specific activity among the tested xylanases on (Allgaier et al. 2010), Napier grass (Kanokratana et al. both types of polymeric xylan. It also had the lowest tem- 2018) and crystalline cellulose (Mori et al. 2014). perature optimum (40 °C) among all the xylanases, and The number of clones to be screened can also be low - retained over 60% of this activity at 20 °C. Its high activity ered by increasing the amount of sequence per clone, at low temperature and acidic pH optimum (5.5) might e.g. by using large-insert fosmid vectors (Colombo et al. make it suitable for e.g. certain food applications (Dornez 2016; Lewin et al. 2017). However, in large-insert vectors et al. 2011). The enzyme also showed low but detectible the enzymes are expressed from their native promoters, activity on the fluorescent EnzCheck substrate. Another which can result in low expression in the cloning host (E. GH8 xylanase was previously found to be completely coli). In our study, reducing sugar-based xylanase activ- inactive on the substrate (Ge et al. 2007). The hydroly - ity assays could not detect activity from several of our sis profile of XYL32 (Fig. 3d) suggests that it is clearly an fosmid clones. A fluorescence-based microplate assay endo-acting xylanase. It shares 42% and 43% identity with proved more sensitive and allowed early insight to the two characterized GH8 endo-xylanases, Xyn8A from thermostability of the uncovered xylanases. Using this Bacteroides intestinalis (Hong et al. 2014) and TtGH8 data we shortlisted two GH11 xylanases (XYL40 and from Teredinibacter turnerae (Fowler et al. 2018), both XYL35) and one GH10 xylanase (XYL38) for further of which also released xylotriose as their main hydrolysis study. Two other xylanases (XYL21 and XYL32) were also product from xylan. Ellilä et al. AMB Expr (2019) 9:124 Page 11 of 12 Author details In addition to xylanases, our dataset comprised a sig- VTT Technical Research Centre of Finland, P.O. Box 1000, 02044, Vuorimie- nificant amount of sequence with numerous putative hentie, Espoo, Finland. Institute of Biotechnology, University of Helsinki, ORFs encoding additional carbohydrate active enzymes P.O.Box 56, 00014 Helsinki, Finland. (CAZymes), several of which are related to the depo- Received: 21 February 2019 Accepted: 25 July 2019 lymerization of xylan. For example, a genomic insert (GenBank KX236224.1) containing the most thermo- stable xylanase XYL40, also encoded a putative GH115 α-d -glucuronidase, GH51 α-l -arabinofuranosidase and a GH9 endoglucanase. This demonstrates one of the References strengths of combining functional screening of large- Allgaier M, Reddy A, Park JI, Ivanova N, D’haeseleer P, Lowry S, Sapra R, Hazen TC, Simmons BA, VanderGheynst JS, Hugenholtz P (2010) Targeted insert vectors with high throughput sequencing: Using discovery of glycoside hydrolases from a switchgrass-adapted com- this approach it is possible to uncover whole operons post community. PLoS ONE 5:e8812. https ://doi.org/10.1371/journ of genes with interrelated functions, and discover novel al.pone.00088 12 Barabote RD, Parales JV, Guo YY, Labavitch JM, Parales RE, Berry AM (2010) enzymes for which plate-screening substrates might Xyn10A, a thermostable endoxylanase from Acidothermus cellulolyticus not be available. 11B. Appl Environ Microbiol 76:7363–7366. https ://doi.org/10.1128/ In summary, we report here the use of environmen- AEM.01326 -10 Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B tal enrichment using controlled composting of target (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert materials, followed by the construction and screening resource for glycogenomics. Nucleic Acids Res 37:D233–D238. https :// of metagenomic DNA libraries for novel xylanases. doi.org/10.1093/nar/gkn66 3 Chu Y, Tu T, Penttinen L, Xue X, Wang X, Yi Z, Gong L, Rouvinen J, Luo H, Five novel xylanases were expressed, purified and pre - Hakulinen N, Yao B, Su X (2017) Insights into the roles of non-catalytic liminarily characterized. All enzymes were found to be residues in the active site of a GH10 xylanase with activity on cellulose. J active not only on screening and small-molecule sub- Biol Chem 292:19315–19327. https ://doi.org/10.1074/jbc.M117.80776 8 Colombo LT, de Oliveira MNV, Carneiro DG, de Souza RA, Alvim MCT, dos San- strates, but also on the technical lignocellulosic feed- tos JC, da Silva CC, Vidigal PMP, da Silveira WB, Passos FML (2016) Apply- stock wheat straw, with one enzyme (XYL40) displaying ing functional metagenomics to search for novel lignocellulosic enzymes superior performance in wheat straw hydrolysis com- in a microbial consortium derived from a thermophilic composting phase of sugarcane bagasse and cow manure. Antonie van Leeuwen- pared to TrXyn11A. hoek, Int J Gen Mol Microbiol 109:1217–1233. https ://doi.org/10.1007/ s1048 2-016-0723-4 Acknowledgements Delcher AL, Bratke KA, Powers EC, Salzberg SL (2007) Identifying bacterial The authors wish to thank Roal Oyj for providing the thermostable reference genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679. enzymes and Biogold for the pretreated wheat straw. Birgit Hillebrandt-Chel- https ://doi.org/10.1093/bioin forma tics/btm00 9 laoui is graciously acknowledged for expert technical assistance. Dornez E, Verjans P, Arnaut F, Delcour JA, Courtin CM (2011) Use of psychro- philic xylanases provides insight into the xylanase functionality in bread Authors’ contributions making. J Agric Food Chem 59:9553–9562. https ://doi.org/10.1021/jf201 SE subcloned genes, purified recombinant proteins, performed enzyme 752g and hydrolysis assays, and wrote the paper, PB constructed and screened Duan CJ, Feng JX (2010) Mining metagenomes for novel cellulase genes. Bio- metagenomics libraries and supervised the work, MN performed microbial technol Lett 32:1765–1775. https ://doi.org/10.1007/s1052 9-010-0356-z enrichment and DNA extraction, LP performed 454-sequencing and assembly, Ferrer M, Beloqui A, Timmis KN, Golyshin PN (2009) Metagenomics for mining MI, AK and KK conceived and supervised the work. All authors discussed the new genetic resources of microbial communities. J Mol Microbiol Bio- results and contributed to the final manuscript. All authors read and approved technol 16:109–123. https ://doi.org/10.1159/00014 2898 the final manuscript. Fowler CA, Hemsworth GR, Cuskin F, Hart S, Turkenburg J, Gilbert HJ, Walton PH, Davies GJ (2018) Structure and function of a glycoside hydrolase fam- Funding ily 8 endoxylanase from Teredinibacter turnerae. Acta Crystallogr D Struct Support for this work was provided by the EU 7th framework-projects NEMO Biol 74:946–955. https ://doi.org/10.1107/s2059 79831 80097 37 (Novel high performance enzymes and micro-organisms for conversion of Fraiberg M, Borovok I, Bayer EA, Weiner RM, Lamed R (2011) Cadherin domains lignocellulosic biomass to bioethanol), Grant no. 222699 and DISCO ( Targeted in the polysaccharide-degrading marine bacterium Saccharophagus DISCOvery of novel cellulases and hemicellulases and their reaction mecha- degradans 2-40 are carbohydrate-binding modules. 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