Molecular Cloning, Expression and Characterization of a Novel Feruloyl Esterase Enzyme from the Symbionts of Termite (Coptotermes Formosanus) Gut

BMB reports

Molecular cloning, expression and characterization of a novel feruloyl esterase enzyme from the symbionts of termite (Coptotermes formosanus) gut

Matam Chandrasekharaiah*, Appoothy Thulasi, M Bagath, Duvvuri Prasanna Kumar, Sunil Singh Santosh, Chenniappan Palanivel, Vazhakkala Lyju Jose & K.T. Sampath Rumen Microbiology Laboratory, Animal Nutrition Division, National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore, India

Termites play an important role in the degradation of dead bran, barley straw, maize and sugar-cane bagasse and to arabi- plant materials and have acquired endogenous and symbiotic nose in the pectins of sugar beet and spinach. FA also medi- cellulose digestion capabilities. The feruloyl esterase enzyme ates the polysaccharide-protein crosslink via tyrosine or cys- (FAE) gene amplified from the metagenomic DNA of Copto- teine residues. Feruloyl esterase (FAE) is an enzyme capable of termes formosanus gut was cloned in the TA cloning vector hydrolyzing sugar-phenolic acid ester linkages. FAE forms a and subcloned into a pET32a expression vector. The Ft3-7 part of the enzyme complex that acts collectively and synerg- gene has 84% sequence identity with Clostridium saccha- istically to completely hydrolyze xylan to its monomers. There rolyticum and shows amino acid sequence identity with pre- has recently been considerable interest in a large number of dicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. potential applications of these enzymes due to their roles in The sequence analysis reveals that probably Ft3-7 could be a many biotechnological processes, in various industries (chemi- new gene and that its molecular mass was 18.5 kDa. The ac- cals, fuel, animal feed, textile and laundry, pulp and paper (2), tivity of the recombinant enzyme (Ft3-7) produced in Escheri- food and agriculture and the pharmaceutical industry. FA is a chia coli (E.coli) was 21.4 U with substrate ethyl ferulate and precursor for vanillin and its access through biotechnological its specific activity was 24.6 U/mg protein. The optimum pH methods was crucial in the quest for natural vanillin (3, 4). As and temperature for enzyme activity were 7.0 and 37oC, res- well as being exploited as a hydrolase, FAE was shown to be a pectively. The substrate utilization preferences and sequence good catalyst in synthesizing sugar-phenolic esters (5, 6). Some similarity of the Ft3-7 place it in the type-D sub-class of FAE. of the ester-linked substituents on plant cell wall poly- [BMB reports 2011; 44(1): 52-57] saccharides retard or inhibit microbial infection (7). Phenolic components of the plant cell wall, especially p-coumaric acid, FA and p-hydroxybenzaldehyde, inhibit the growth of rumen INTRODUCTION microorganisms (8) and phenolic acids derived from plant cell walls have long been used as food preservatives (9) to inhibit Ferulic acid (FA) is the most abundant hydroxycinnamic acid microbial growth. FAEs, also known as cinnamoyl esterases present in the cell walls of several plants. It is to be found co- (E.C. 3.1.1.73), represent a subclass of carboxylic acid este- valently linked to polysaccharides by ester bonds and to com- rase. FAEs have been classified into four subclasses: types A, ponents of lignin through ether linkages (1). It plays an im- B, C and D based on their specificity for aromatic substrates portant role in maintaining the structural integrity of the plant and their varying ability to release diferulic acid (diFA) from es- cell wall matrix, which partially limits the biodegradability of terified substrates. Type A is active on methyl ferulate (MFA), non-lignified plant cell wall polysaccharides and regulates the methyl p-coumarate (MpCA) and methyl sinapate (MSA). growth by the cross-linking of cell wall polymers. These FAEs have sequences related to those of lipases and are FAs are esterified to arabinose in the arabinoxylans of wheat able to hydrolyze synthetic ferulate dehydrodimers. Type B FAEs are specific against MFA, MpCA, methyl caffeate (MCA), *Corresponding author. Tel: +918025711304; Fax: +918025711420; but not MSA. These enzymes do not release diFA and show se- E-mail: [email protected] quence similarities to carboxylic esterase family 1 - acetyl xy- DOI 10.5483/BMBRep.2011.44.1.52 lan esterase. Type C and D act on all four hydroxycinnamic acid methyl esters. Type C enzymes do not release diFAs from Received 23 August 2010, Accepted 3 December 2010 model and complex substrates, whereas type D enzymes are Keywords: Cloning, Expression, Feruloyl esterase, Metagenome, able to hydrolyze dimers. Type D is active on the substrates Termite MFA, MSA, MpCA and MCA. These FAEs have sequence sim-

52 BMB reports http://bmbreports.org Characterization of feruloylesterase from termites Matam Chandrasekharaiah, et al.

ilarity to Xylanase (10, 11). acetylase of Clostridium saccharolyticumi (ADL05911) with Termites are considered to be one of the most important bi- 87% identity. This Ft3-7 sequence shows a high homology to oreactors on the planet, converting cellulose into the ubiq- the Endoxylanase, xylanase/chitin deacetylase, putative carbo- uitous energy source, glucose (12). The main role of these gut hydrate esterase family 4 protein, endo-1,4-beta-xylanase (EC symbionts was thought to relate more to anaerobic respiration 3.2.1.8), indicating an evolutionary relationship among FAEs, and nitrogen fixation than cellulose digestion (13). Now it has acetyl xylan esters and certain lipases. Ft3-7 gave 61 % iden- been observed that endosymbiotic and termite-derived cellu- tity to the predicted xylanase/chitin deacetylase enzyme of the lases are both present in termites (14, 15) supporting the con- butyrate-producing bacterium accession no CBL40699 and nection that the cellulases of both endosymbiont and termite 59% to the polysaccharide deacetylase family of Clostridiales origin are important to termite cellulose digestion. There have bacterium ZP_04667659, 59% to the hypothetical protein of also been reports of FAEs that are part of large multidomain Clostridium bolteae ZP_02084187 and 46% to the Clostri- structures, such as cellulosomes. The metagenomic approach, dium asparagiforme strain no. ZP_03762399 (Fig. 2A). The a culture independent method, has been used to clone a num- other domain of Ft3-7 has a sequence identity of 90% with the ber of genes, including FAE genes. Therefore, the objective of ABC transporter related protein of Clostridium saccha- the present study was to amplify FAE enzyme encoding se- rolyticum WM1(ADL05909), 68% with ATPase component of quences from the microorganisms present in termite gut by butyrate-producing bacterium SSC/2 (CBL37620), 62% with metagenomic approach. The amplified FAE gene was further ABC-type multidrug transport system, ATPase component of expressed and characterized by sequence and activity based Faecalibacterium prausnitzii L2-6 (CBK99948) (Fig. 2B). studied. Hence, we have isolated DNA from symbionts of Ft3-7 has polysaccharide deacetylase/xylanase domains Coptotermes formosanus gut, amplified, cloned and over-ex- from residues 51 to 143 amino acids (AAs), which correspond pressed the recombinant protein in E.coli. The Purified re- to FAE type-D. This FAE module hydrolyzes the ester linkage combinant enzyme was biochemically characterized. between arabinofuranoside side chains and FA. There are reports that FAE can also hydrolyze xylan/ acetyl or 4-O-methyl- RESULTS AND DISCUSSION glucuronyl moieties (16). One of these ORFs present in Ft3-7 encodes a putative protein that features an ABC transporter/ Nucleotide sequence and expression of the FAE gene ATP-binding domain from AA residues of 352-436. ABC trans- The FAE gene was amplified from the symbionts of Copto- porters are a large family of proteins involved in the transport termes formosanus gut metagenomic DNA using enzyme-spe- of a wide variety of different compounds like sugars, ions, pep- cific primers. The obtained PCR product of 1.3 kb was cloned tides and more complex organic molecules. Thus, all available into the pGEMT-Easy TA cloning vector for DNA sequence evidence indicates that Ft3-7 is a component of the FAE analysis and the FAE gene was subcloned into the Eco RI digested pET32a expression vector (Fig. 1). The resulting clone was named pET32a Ft3-7 recombinant plasmid. The positive clone plasmids carrying our gene of interest were sequenced. The 1.3 kb nucleotide sequence of the Ft3-7 positive clone was compared with the genome database, which shows 84% primary sequence identity with Clostridium saccharolyticum and a query coverage of 52%. On translation of the above nucleotide sequence using the EXPAsy proteomics server, the predicted protein sequence matched with polysaccharide de-

Fig. 2. Comparisons of Ft3-7 amino acid sequence. (A) Alignment of Ft3-7 polysaccharide deacetylase domain with Clostridium saccharolyticum WM1, Clostridium bolteae, butyrate-producing bacte- Fig. 1. Restriction enzyme analysis of Ft3-7. Lane 1: 1,000-100 rium SS3/4 and Clostridium saccharolyticum K10; (B) Conserved bp DNA ladder (top to bottom); Lane 2: +ve clone of Ft3-7 in regions of Ft3-7 ATP domain with Clostridium saccharolyticum pGEMT Easy vector; Lane 3: +ve clone of Ft3-7 in pET32a ex- WM1, butyrate-producing bacterium SSC/2, Faecalibacterium praus- pression vector; Lane 4: 10-1 kb DNA ladder (top to bottom). nitzii L2-6 and Clostridium sp. SS2/1. http://bmbreports.org BMB reports 53 Characterization of feruloylesterase from termites Matam Chandrasekharaiah, et al.

complex. of protein. The activity of the Ft3-7 produced in Escherichia coli was found to be 21.4 U and its specific activity was 24.6 FAE activity of the recombinant Ft3-7 protein U/mg protein. To confirm that the transformants were producing FAE, the culture was induced with IPTG and the expressed protein was pu- Temperature and pH effect on the activity of Ft3-7 rified by affinity chromatography. The molecular mass of Ft3-7 This purified enzyme Ft3-7 was active at a pH range of 6-9. was confirmed by SDS-PAGE and MALDI-TOF mass spectrom- The optimal pH of the enzyme was 7 because it achieves its etry as 18.5 kDa (Fig. 3A). Ft3-7 showed the same molecular maximum activity at this condition (Fig. 4A). The enzyme ac- mass under both denaturing and nondenaturing conditions tivity remained at a high level when the pH was between 7 (Fig. 3B). The purified protein was dialyzed and concentrated and 8. However when the pH was out of the 6 to 8 range, before the studies on enzyme activity. The activity assay was Ft3-7 showed very little activity and when the pH was below 5 based on the measurement of FA released from the substrate the activity was completely lost. This enzyme Ft3-7 showed its ethyl ferulate. The substrate and enzyme were prepared as de- main activity in the temperature range of 10-40oC. Its max- scribed in Materials and Methods. After incubating the re- imum activity was observed when Ft3-7 was incubated at a action mixture at 37oC for different time intervals, the released temperature between 35-40oC (Fig. 4B). When the temperature FA was analyzed by HPLC using a Nucleosil C18 column. was higher than 50oC, enzyme activity was quickly lost. One unit (U) of enzyme activity was defined as the amount of Enzyme activity remained stable below 45oC. enzyme that catalyses the reaction of 1 μM of substrate per minute and its specific activity was given in units per milligram

Fig. 3. Analysis of recombinant protein Ft3-7 expression on PAGE. (A) SDS-PAGE of the fractions obtained during purification of recombinant Ft3-7 from E.coli BL21 cell extract. Lane 1, molecular mass markers; lane 2, cell lysate of uninduced E.coli BL21 cells; lane 3, cell lysate of induced E coli BL21 cells; lane 4 flow-through of Ni-NTA Biorad column and lane 5, Ft3-7 (FAE) purified protein; (B) Native-PAGE of purified Ft3-7 protein compared with native PAGE molecular markers as follows. Lane 1, bovine serum albumin (BSA) 67 kDa; lane 2, Ovalbumin 43 kDa; lane 3, Trypsine soyabean inhibitor 20.1 kDa and lane 4, purified Ft3-7 protein.

Fig. 4. Effects of pH and temperature on the activity and stability of the enzyme. (A) Influence of pH on the activity of Ft3-7 from C. formosanus gut. Enzyme activity was measured at pH 4-9. The highest level of activity, 22 U/mg, was obtained at pH 7-7.5 and was set at 100%. (B) Influence of temperature on the activity of Ft3-7 from C. formosanus gut. Enzyme assay was measured according to the slandered assay from 10- 60oC. The highest level of activity, 24.6 U/mg, was obtained at 40°C and was set at 100%.

54 BMB reports http://bmbreports.org Characterization of feruloylesterase from termites Matam Chandrasekharaiah, et al.

Release of phenolics from crude plant cell walls Collection of termites and DNA isolation The ability of the purified Ft3-7 protein to release phenolic Worker termites were collected from small breaches in nests, compounds from crude plant cell wall materials in the form of wood or foraging trails. 50-60 worker termites were randomly crop residues was tested. The enzyme was found to be able to chosen from each colony. Immediately after collection, the ter- release FA and diFA from wheat bran and spent grain in com- mites were transported to the laboratory. The exterior surfaces bination with xylanase (17). When the 20 mU Ft3-7 protein had been washed with distilled water and then the entire gut was incubated with wheat bran in the presence of 2 U xyla- was collected from these individuals by using a pair of sterile nase at 37oC for 6 hr, 72% of FA and 28% of diFA was forceps. The genomic DNA from the symbionts of termite’s gut released. On the other hand, with spent grain 46% of FA and was isolated by the CTAB (Cetyl Trimethyl Ammonium Bro- 23% of diFA was released. However, these measurements mide) method (22) with the some modifications. The Purity of have only been carried out on phenolic acid-substituted xylan the isolated genomic DNA was checked using a spectrophoto- polysaccharide isolated from wheat straw and rarely on un- meter. The plasmid DNA from the E.coli strains was isolated treated crude cell wall materials (18). It was found that phe- and purified using a Gene Jet plasmid Miniprep Kit from nolic acids were released from the same substrates of wheat Fermentas and a Gene Jet gel extraction kit (Fermentas) was bran and spent grain in the presence of enzymes xylanase and used for extracting DNA from agarose gels. FAE (19). Our results are agreeing the Crepin et al. 2004 findings (20). The synergistic nature of the association between xy- PCR amplification and cloning of the FAE gene lanases and FAE activity has now been accepted and reports of PCR was performed with purified genomic DNA as a template, the purification of FAE have either concluded that FAE activity using primers 5'-CTC GCT AGC GAC AAA TTT CCT GTA could only be detected in the presence of xylanases or that in GCA G-3' and 5'-CAC CTC GAG TTC ATG GAA GAA ATA the presence of xylanase activity FAE activity increased. One TGG-3' (16) The amplified gene was sent for the sequencing explanation why FAE activity has been detected in only a few and based on the sequence obtained the following enzyme strains is the lack of a complete set of xylanase enzymes, re- specific primers have been designed: 23F (5'-AAA ATA CGT sulting in low and perhaps undetectable FAE activity (21). The ATG GCA GCT TCC CT) and 31R (5'-ACA GCG TAC TGC results suggest that Ft3-7 is able to release phenolic residues GGT CCG GCA GGT AAG AGA T) to amplify the near from wheat bran and spent grain. full-length FAE gene. A high fidelity Novagen KOD XL DNA The FAE from the symbionts of termites (Coptotermes for- polymerase was used for DNA amplification which was car- mosanus) gut was cloned and an expressed protein of Ft3-7 ried out with 35 cycles of denaturation (45s at 95oC), anneal- was purified and characterized in this work. Ft3-7 is distinct ing (1 min at 63oC) and extension (2 min at 72oC), followed by from the other subclasses of type B and C FAE which does not 10 min of further extension at 72oC. The PCR product was pu- release 5-5' diFA (20). However, Ft3-7 releases 5-5' diFA from rified by the StrataPrep PCR cleanup kit (Stratagene) and li- plant cell wall materials. The enhancement of FAE activity by gated into the pGEMT-Easy TA cloning vector according to the xylanase has also been elucidated. Therefore, based on these manufacturer's instructions. The pGEMT easy vector carrying sequence similarity with xylanase and substrate utilization the FAE gene was digested with the Eco R1 enzyme and the properties of Ft3-7, we propose that it is indeed a novel type-D produced fragment was gel-purified before its subcloning into FAE as predicted from a comparative sequence analysis. These an expression vector. The Gel-purified FAE gene was ligated findings enable us to develop superior fibrolytic enzymes that with the EcoR1 digested pET32a expression vector and trans- are impractical for commercial enzyme production. Thus, formed by electroporation into E.coli BL 21. cloning of the desired genes is essential for the commercial production of plant cell wall polysaccharide-degrading en- High-level expression and purification of Ft3-7 recombinant zymes. protein E.coli cells containing the recombinant plasmid pET32a-Ft3-7 MATERIALS AND METHODS were cultured overnight on a LB agar plate containing ampicillin (100 μg/ml). The culture obtained was then inoculated Bacterial strains, vectors and media into 500 ml of LB broth containing ampicillin (100 μg/ml) and For cloning the FAE gene from the metagenomic DNA isolated allowed to grow at 37oC until it reached an optical density of from endosymbionts of termites, E.coli DH5α was employed 0.6 at 600 nm. At this point, 150 μM of IPTG (isopropyl-α- as a host cell, with the pGEMT-Easy (Promega) TA cloning vec- thio-galactopyranoside) was added and the cells were re-in- tor being used as the host-vector system. The pET32a (Novagen) cubated for 6 hrs at 37ºC. The bacterial culture was centri- vector and an E.coli BL 21strain were used for protein fuged at 6,000 × g for 10 min to separate the cells from the expression. E.coli was grown at 37oC in Luria-Bertani (LB) agar media. The induced bacterial cell pellets were resuspended in plates containing 60 μg ampicillin/ml, X-Gal and IPTG for the 10 ml of buffer A (10 mM TRIS, 100 mM NaCl, pH 8.0) and selection of positive clones. lysed by ultra sonication on ice. After centrifugation at 15,000 × http://bmbreports.org BMB reports 55 Characterization of feruloylesterase from termites Matam Chandrasekharaiah, et al.

g for 10 min at 4oC, a supernatant fraction containing a re- the residual activity was measured by the standard assay. All combinant fusion protein was loaded on pre-equilibrated col- the assays were done in triplicate. The optimum pH was meas- umns containing Ni-NTA agarose affinity resin and incubated ured by running the standard assay at different pH values. The for binding. The Ni-NTA resin was washed with 50 ml of buf- buffers used were a citrate/phosphate buffer (0.2 M) for pH 4-5 fer B (10 mM TRIS-Cl pH 8.0, 100 mM NaCl and 5 mM and a sodium phosphate buffer (0.2 M) for pH 6-9. The pH Imidazole) to remove non-specific proteins from the column. was monitored before and after the reaction. Significant The affinity-bound recombinant protein was eluted in buffer C changes in pH due to the reaction were not observed. consisting of 10 mM TRIS-Cl pH 8.0, 200 mM NaCl and 500 mM Imidazole. The purity of the recombinant fusion protein Sequence alignments was determined by SDS-PAGE and the protein concentration AA sequence alignments were performed by using the BLAST was determined by the dye-binding procedure of Bradford et programs (26) at the server of the National Center for Biotech- al. 1976 (23) using bovine serum albumin as a standard. nology Information (www.ncbi.nlm.nih.gov) and ClustalW (www.ebi.ac.uk). SDS-PAGE analysis The purified recombinant protein was analyzed on SDS-PAGE Acknowledgements according to the method of Laemmli (24). The stacking gel was The authors are gratefully acknowledging the financial support 4% polyacrylamide and the running gel was 12.5% poly- of ICAR/NAIP for conducting this research. acrylamide, used for the separation of polypeptides. Samples containing 1 mg/ml protein were solubilised by boiling for 2 REFERENCES min in a sample buffer containing 5% glycerol 0.1 M Tris-HCl, (pH 6.8), 2% SDS and 100 μM mercaptoethanol along with 1. Jeffries, T. W. (1990) Biodegradation of lignin carbohy- 0.1% of bromophenol blue. The molecular mass of the re- drate complexes. Biodegradation 1, 163-176. solved proteins was calculated by using Novagen markers con- 2. Sigoillot, C., Camarero, S., Vidal, T., Record, E., Asther, taining the 9 precise recombinant proteins of 225, 150, 100, M., Perez-Boada, M. and Martinez, M. J. (2005) Compar- 75, 50, 35, 25, 15 and 10 kDa. ison of different fungal enzymes for bleaching high-quality paper pulps. J. Biotechnol. 115, 333-343. 3. Ou, S. and Kwok, K. C. (2004) Ferulic acid: pharmaceut- Enzyme assay ical functions, preparation and applications in foods. J. Sci. The recombinant protein was subjected to dialysis and con- Food Agric. 84, 1261-1269. centrated by ultrafiltration using Sartorius concentrators with a 4. Nethaji, M. and Pattabhi, V. (1988) Structure of 3-(4-Hydroxy- 10-kDa cutoff. The protein so obtained was used for FAE activ- 3-methoxyphenyl)-2-propenoic acid (ferulic acid). 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