Cloning, Expression and Characterization of Highly Active Recombinant Thermostable Cellulase from Thermotoga naphthophila

Cellulose is the most abundant biopolymer on Earth. Plants produce about 180 billion tons of cellulose annually through photosynthesis process (Festucci et al., 2007). It is a linear polysaccharide with many glucose molecules that are polymerized with β 1,4 glycosidic linkages (Nishida et al., 2007). The D- glucose residues in linear polymer of cellulose are interconnected and stabilized via hydrogen bonding or van der Waals forces and make it overall recalcitrance (Cheng et al., 2011). Cellulosic raw material in the form of agriculture and industrial waste could not be exploited efficiently. Therefore, it is needed to develop considerable economic and cost efficient procedure for utilization of cellulose as carbon source. It is important to convert the inexhaustible organic mass to useful product (Clarke, 1997).

Cellulases (EC 3.2.1.4) are responsible for hydrolysis of β 1,4 linkage of cellulose to fermentable sugars which can be utilized as energy source (Dienes et al., 2004). The cellulases have wide range of application in different field such as in farm animal feed industry for the improvement of feed digestion (Karmakar and Ray, 2011), in juice industry for maceration process (Kuhad et al., 2011), in leather industry for final finishing step of fiber (Anish et al., 2007), in paper industry for de-inking (Bhat, 2000) and in textile industry for softening and finishing of cotton fabrics (Adsul et al., 2007).

Production of cellulases has been reported from bacteria (Park et al., 2011), fungi (Kataoka and Ishikawa, 2014), animals (Smant et al., 1998) and plants (Urbanowicz et al., 2007). Bacteria have potential advantage over the others because of fast growth rate and easy to handle (Liu et al., 2011;Maki et al., 2009; Lynd et al., 2002). Bacteria secrete three different categories of cellulases including endoglucanases (EC 3.2.1.4), β-glucosidases (EC 3.2.1.21) and cellobiohydrolase (EC 3.2.1.91) which act synergically to degrade complex cellulose material into simple sugar components (Matsui et al., 2013; Beeson et al., 2015). Industry requires thermostable enzyme which should be stable for long time and can tolerate the harsh temperature conditions. Thermophiles and hyperthermophiles are the microbes that have ability for the production of thermostable protein as compared to mesophiles.

Thermotoga naphthophila is an anaerobic hyperthermophile. The cells are rod shaped that shows optimal growth at 90°C and are involved in the production of thermostable enzymes (Sabir et al., 2017). The present study demonstrates the production and characterization of recombinant thermostable cellulase from this hyperthermophile and the utilization of this cellulase for paper de-inking process.

The chemicals used in the study were of analytical grade and were purchased from Sigma Aldrich (St. Louis, MO, USA). The PCR cloning kit, DNA extraction kit, ligation kit and restriction endonucleases were purchased from Thermoscientific, LifeSciences, USA.

The cellulase gene from T. naphthophila RUK-10 was amplified using the CEL-TN-F (CATATGTATCTCAAAGAGCTTTC) and CEL-TN-R (TCATGAGACCACCTCCACG) as forward and reverse primers, respectively, using the genomic DNA of T. naphthophila RUK-10 as template. The forward primer contained the unique Nde I restriction site. The genomic DNA of T. naphthophila RUK-10 was purchased from Leibniz Institute DSMZ German Collection of Microorganism and Cell Culture. The amplified PCR product was purified from agarose gel using DNA purification kit and the purified PCR product was ligated in pTZ57R/T using T4 DNA ligase. The ligated material was utilized for the transformation of E. coli DH5α competent cells. The positive clones were selected on the basis of blue white screening (Sambrook and Russel, 2001). Plasmid DNA was isolated and the presence of insert in the recombinant pTZ57R/T was confirmed by restriction digestion using Nde I and Hind III endonucleases. DNA sequencing of the restriction confirmed recombinant pTZ57R/T was performed (Sanger et al., 1977) using M13 forward and reverse primers and the obtained DNA and deduced amino acid sequences were utilized for homology and comparative analysis using the BLAST and Clustal Omega programs (Tayyab et al., 2011).

Regarding the expression studies, the gene was transfered from recombinant pTZ57R/T to pET28a already restricted with same restriction enzymes and was utilized for the transformation of BL21 CodonPlus(DE3) competent cells. The cells were grown in LB medium supplemented with kanamycin (50 µg/mL). The overnight grown culture of BL21 CodonPlus (DE3) cells having recombinant pET28a was diluted to 1% and was incubated at 37°C till the OD reached to 0.4 at 660 nm. The cells were induced with 0.5 mM IPTG and were further incubated for 6 h in shaker at 37°C under shaking conditions (I3000, Labtech, Korea). Centrifugation was performed, supernatant was discarded and the cellular pellet was resuspended in 50 mM sodium acetate buffer (pH 4.8). The cells were lyzed by sonication (Sonics, Newtorn, USA). The soluble and insoluble production of cellulase was examined by activity assay and SDS-PAGE analysis (Laemmli, 1970). The expression was also analyzed at 20 and 25°C. For this purpose, the recombinant cells after attaining the OD to 0.4 were shifted to selected temperature before induction. The induction was done as described above with the post induction time of 22 h at the selected temperature.

The soluble part after sonication was applied to Ni-NTA agarose column (Wang et al., 2015) pre-equilibrated with 50 mM sodium acetate (pH 4.8) with 500 mM NaCl and 50 mM imidazole. The elusion was done with same buffer having 500 mM NaCl and 250 mM imidazole. The fractions were collected and were utilized for activity assay (Pereira et al., 2010). The purity of fractions was analyzed on SDS-PAGE. The protein concentrations of fractions were calculated by Bradford method (Bradford, 1976). The purified recombinant cellulase (CELTN) was utilized for further studies.

CELTN activity was examined using sodium carboxymethyl cellulose as substrate (Xiao et al., 2005). The enzyme activity assay mixture was prepared by mixing 50 µL of recombinant cellulase with 1% sodium carboxymethyl cellulose in 50 mM sodium acetate buffer (pH 4.8) containing 2.5 mM cobalt. The reaction mixture was incubated at 90°C for 30 min in water bath (D-91126, Memmert, Germany). DNS reagent (1mL) was added to the above assay mixture followed by boiling for 10 min. The absorbance was recorded at 540 nm. One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 µmol of glucose per minute under optimal conditions.

CELTN activity was examined at wide range of temperature ranging from 40 to 100°C in 50 mM sodium acetate buffer (pH 4.8) (Bajaj et al., 2009).

The enzyme activity was evaluated by examining the CELTN activity at wide range of pH using 50 mM of each of sodium acetate buffer (3-5), sodium phosphate buffer (5-7) and Tris HCl buffer (7-9) at 90°C using sodium carboxymethyl cellulose as substrate.

The dependency of CELTN on metal ion was explored by examining the CELTN activity in the presence of 1 mM of EDTA or Ca2+, Co2+, Zn,2+ Mg2+ or Mn2+ at 90°C in sodium acetate buffer (pH 4.8) (Pereira et al., 2010). The CELTN activity was also analyzed in the presence of ionic (SDS) and non-ionic (Tween-80 and Triton X-100) detergents at final concentration of 1% (Chakraborty and Mahajan, 2014; Tayyab et al., 2011).

Regarding the thermostability studies, the CELTN was incubated at 90°C in the presence and absence of cobalt. The sample was withdrawn after every 10 min and was utilized for examining the CELTN residual activity.

Effect of substrate concentrations on enzyme activity was analyzed by measuring the CELTN activity using various concentrations of sodium carboxymethyl cellulose ranging from 0.1 to 1% in sodium acetate buffer (pH 4.8) at 90°C. The data obtained was utilized for developing Lineweaver Burk Plot and for the estimation of kinetic parameters (Mansoor et al., 2018).

Paper de-inking ability of CELTN was explored as the process is environment friendly without production of any hazardous pollutants. Two papers (Rizvi Paper Products, Lahore, Punjab, Pakistan) with 15 x 20 cm dimensions were taken. Pen (Dollar, Industries Private Ltd, Karachi, Pakistan) and Ballpoint (Piano, Sayyad Engineers Ltd, Lahore, Pakistan) were utilized for writing on the pages. Each page was divided into two half’s (15 x 10 cm). One was taken as negative control in each case and was not treated with enzyme whereas the second half was treated with enzyme. All the paper pieces were passed through the process of pulping and maceration. The pulping was done by soaking the paper in hot water for 2 h followed by incubation in 0.1% Tween-80 for maceration process and was oven dried at 50°C for 30 min (Mohandass and Raghukumar, 2005). After the completion of maceration process, each of the experimental pages was incubated at 50°C in 50 ml of sodium acetate buffer (pH 4.8) containing 50 µl of enzyme with 2.5 mM concentration of CoCl2 for 30 min. The incubation condition for the negative control was same except the presence of enzyme. After enzymatic treatment the page was dried and picture was taken for record (Pala et al., 2004).

The PCR using the gene specific primers and genomic DNA of T. naphthophila RKU-10 as template resulted in the amplification of 1 kb cellulase gene. The purified PCR was cloned in pTZ57R/T and restriction digestion with Nde I and Hind III confirmed the liberation of 1 kb insert in the recombinant vector. The sequence of confirmed recombinant vector was utilized for homology analysis. The nucleotide sequence comparative analysis of cellulase from T. naphthophila showed 99% identity with Thermotoga sp. Cell2 and Thermotoga sp. RQ2, 97% with Thermotoga maritima and 93% with Thermotoga petrophila strain RUK1. Amino acid based sequence alignment of cellulase from T. naphthophila RKU-10 confirmed this cellulase as a member of M42 Family of aminopeptidases. Comparative analysis of this cellulase with various members of M42 Family of aminopeptidases indicated the presence of conserved amino acids responsible for the incorporation of metal ions and for the enzymatic activity including His, Asp, Glu, Glu, Asp and His. However second last Asp that was conserved in this Family was replaced with Glu219 in case of CELTN (Fig. 1).

The expression studies at 20, 25 and 37°C demonstrated the high level production of CELTN at 25°C as compared to 20°C or 37°C. The maximal soluble production of CELTN was recorded when the BL21 CodonPlus (DE3) cells having cellulase gene in pET28a were induced with 0.5 mM IPTG with the post induction incubation of 22 h at 25°C. The SDS-PAGE analysis of the purified CELTN after Ni-NTA affinity chromatography resulted in the appearance of single band having molecular mass of 39 kDa (Fig. 2).

The increase in temperature from 40 to 90°C showed a linear increase in CELTN activity with a maximal activity at 90°C. Further increase in temperature to 100°C resulted in decreased enzymatic activity (Fig. 3). The CELTN activity was increased with the increase in pH. Optimal activity was recorded between pH 4 and 5 while further increase in pH resulted in decreased activity. Further studies on enzyme activity between pH 4 to 5 demonstrated the maximal enzymatic activity at pH 4.8 (Fig. 4) when 50 mM Sodium acetate buffer was utilized for CELTN activity analysis.

The abolishment of CELTN activity in the presence of 1 mM EDTA indicated the dependency of CELTN on metal ions as cofactor. The presence of Mn2+, Zn2+, Ca2+ and Mg2+ did not put significant effect on the CELTN activity whereas the presence of 1 mM Co2+ resulted in 1.3 fold increases in CELTN activity (Table I). The optimal enzymatic activity was recorded when the cobalt was utilized at a final concentration of 2.5 mM in the activity assay. The presence of detergents showed an inhibitory effect on CELTN activity. Presence of SDS, Tween 80 and Triton X-100 at final concentration of 1% reduced the CELTN activity to 8, 10 and 14%, respectively (Table I).

Thermostability studies demonstrated that recombinant enzyme retained 50% residual activity at 90°C after an incubation of 1.67 h in the absence of metal ion whereas the presence of 2.5 mM Co2+ showed stabilizing effect and CELTN retained more than 70% residual activity after an incubation of 1.67h at 90°C (Fig. 5).

The enzyme activity was increased with the increased in substrate concentration following the Michaelis Menten equation till the achievement of saturation level. Line Weaver Burk plot demonstrated the Km and Vmax values of 0.22 mg/mL and 2500 µmoles/min when sodium carboxymethyl cellulose was utilized as substrate (Fig. 6).

The comparison of control with the experimental paper for the removal of ink clearly demonstrated the ability of CELTN to remove the pen and ball point ink as compared to control. However, the intensity of removal of pen ink was quiet high as compared to ball point ink (Fig. 7).

Thermostable cellulases have wide range of application in many industries. The cellulase produced in current study has strong potential for its utilization in paper and poultry industry. Current study deals with the characterization of recombinant thermostable cellulase from T. naphthophila and explores its utilization for paper de-inking process.

The sequence homology analysis indicated the high level identity of CELTN with M42 Family of aminopeptidases. Homology based on amino acid sequence of cellulase from present study indicated the Thermotoga maritima Tm-1049 as closest homologue with 98.5% identity whereas CELTN shared 36.5% identity with FrvX aminopeptidase from P. horikoshi. Interestingly these three strains are members of M42 Family of aminopeptidase that share the conserved amino acids (His, Asp, Glu, Asp and His) for the binding of metal ion and for the enzymatic activity. However in sequence of CELTN the second last conserved Asp is replaced with Glu219 but this change is simply the replacement of one negatively charged amino acid with the other negatively charged amino acid. According to sequence of CELTN, His60, Asp166, Glu196, Glu197, Glu219 and His307 are the conserved amino acids responsible for the accommodation of two cobalt ions with the help of water molecule. Asp166, His307, Asp197 and water molecule are responsible for the accommodation of first metal ion whereas Asp166, His60, Asp219 and water molecule are responsible for the binding of second metal ion. It follows a similar mechanism of action as described by Russo and Baumann (2004). CELTN active site conserved amino acids are same as that of FrvX aminopeptidase (Fig. 1), further experimentation will be performed in-order to explore the aminopeptidase activity of CELTN.

Expression studies of CELTN, demonstrated the higher level production of recombinant protein at 25°C whereas its closest homologue T. maritima showed the optimal production of cellulase at 20°C (Pereira et al., 2010). Similarly maximal production of endoglucanase from E. cellulosolvens was recorded at 18°C (Yoda et al., 2005) whereas the cellobiohydrolase from C. saccharolyticus (Park et al., 2011) and endocellulase from P. furiosus (Kataoka and Ishikawa, 2014) showed their maximal production at 30°C.

Maximal CELTN production was recorded when the cells were induced with 0.5 mM IPTG that is well aligned to 0.5 mM for E. cellulosolvens (Yoda et al., 2005) and C. saccharolyticus (Park et al., 2011), 0.4 mM with the same enzyme from T. maritima (Pereira et al., 2010), whereas a lower concentration of 0.1 mM was required for the maximal production of endocellulase from P. furiosus (Kataoka and Ishikawa, 2014).

SDS-PAGE analysis of the purified protein showed the molecular mass of 39 kDa which is in agreement with 38.3 kDa for AcCel12B from Acidothermus cellulolyticus (Wang et al., 2015), 37 kDa cellulase from F. nodosum (Wang et al., 2015) and 35 kDa Cel5A from T. maritima (Pereira et al., 2010) while the two cellulases from T. neapolitana showed the molecular size of 29 and 30 kDa (Bok et al., 1998).

The CELTN exhibited optimal activity at 90°C that agrees with cellulase from T. neopolitana and P. furiosus which showed maximal activity between 90 to 100°C whereas this value is quiet high as compared to other cellulase from Cellulomonas sp. (Chakraborty and Mahajan, 2014), C. saccharolyticus (Park et al., 2011), T. maritima (Pereira et al., 2010) and Geobacillus WSUCFI (Rastogi et al., 2010) which revealed highest activity at 60, 80, 80 and 70°C.

The enzyme retained more than 75% residual activity at 90°C with 2.5 mM Co+2 after 1.67h of incubation that is comparable with recombinant RmCel12A from R. marinus which showed more than 50% residual activity at 90°C for 2.5h (Wicher et al., 2001). This cellulase was found more stable as compared to recombinant GH5 from C. saccharolyticus which showed 50% residual activity after 23h of incubation at 80°C (Park et al., 2011) and AcCel12B from A. cellulolyticus 11B which showed 50% residual activity at 70°C after 2h of incubation (Wang et al., 2015). However, Eg1A cellulase from P. furiosus showed higher level stability with more than 50% residual activity at 95°C after 40h of incubation (Bauer et al., 1999).

The pH studies indicated the highest CELTN activity at pH 4.8 that is similar to cellulases from T. maritima (Pereira et al., 2010), C. saccharolyticus (Park et al., 2011), Geobacillus WSUCFI and Bacillus strain M9 (Rastogi et al., 2010) having highest cellulase activities between pH 4.8 to 5 whereas the cellulase from R. marinus (Halldorsdottir et al., 1998), Streptomyces sp. (Solingen et al., 2001), T. neopolitana (Bok et al., 1998), E. cellulosolvens (Yoda et al., 2005), P. furiosus (Kataoka and Ishikawa, 2014) and Cellulomonas sp. (Chakraborty and Mahajan, 2014) showed maximal cellulytic activities at pH between 5.5 to 8.

The abolishment of enzymatic activity in the presence of 1 mM EDTA clearly indicated the metal dependency of enzyme. The presence of Co2+ showed an enhancement of CELTN activity. Previous reports on cellulases also confirm the role of Co2+ in the enhancement of cellulolytic activity of Cellulomonas sp. (Chakraborty and Mahajan, 2014), T. fusca (Ferchak and Pye, 1983), A. cellulyticus (Wang et al., 2015), M. circinelloides (Saha, 2004) and C. paradoxa (Lucas et al., 2001).

The presence of ionic and non-ionic detergents showed inhibitory effect on the CELTN activity. Same pattern of inhibition was recorded for cellulases form Bacillus strain M9 that also showed its highest activity under acidic conditions (Bajaj et al., 2009) whereas the alkaline cellulase from Bacillus sp. SMIA-2 showed stability with 95% residual activity in the presence of 0.25% SDS (Ladeira et al., 2015).

CELTN showed Km value of 0.22 mg/mL that is quiet low as compared to 1.08 mg/mL for WSUCFI from Geobacillus sp. (Rastogi et al., 2010), 3.11 mg/mL for DUSELR13 from Bacillus sp. (Rastogi et al., 2010), 3.6 mg/mL for P. fluorescens (Bakare et al., 2005), 4.97 mg/mL for A. anitratus (Ekperigin, 2007), 7.90 mg/mL for Branhamella sp. (Ekperigin, 2007), 19 mg/mL from T. curvata (Stutzenberger, 1971) and 25 mg/mL for AcCel12B from A. cellulyticus (Wang et al., 2015). The low Km value of CELTN indicted high affinity of enzyme for its substrate.

The CELTN showed higher level cellulytic activity of 12464 Umg-1 as compared to 1536 and 1219 Umg-1 for CelB and CelA from Thermotoga neapolitana (Bok et al., 1998), 735 Umg-1 for GH12 endoglucanase from Thermotoga petrophila (Haq et al., 2015), 66 Umg-1 for thermostable Cellobiohydrolase from Thermotoga petrophila (Haq et al., 2018) and 4.23 Umg-1 for hyper-thermostable endoglucanase from Thermotoga meritima (Zhang et al., 2015). Low km and high affinity of CELTN for its substrate is the basis for its higher level activity. The higher level activity might be correlated with the replacement of conserved Asp in M42 Family with Glu at 219 position in the sequence of CELTN. However, 3D x-ray crystallographic structure determination will be required in order to confirm this.

The CELTN has strong potential for paper de-inking of pen ink as compared to ball point ink. Previously, Patil and Dhake (2014) utilized β-glucosidases from Penicillium purpurogenum for de-inking of newspaper ink.

This study successfully described the characterization of a recombinant thermostable cellulase from Thermotoga naphthophila a new member of M42 family of aminopeptidases. The ability of enzyme to show its activity at a wide range of temperature, its stability to high temperature, pH and de-inking ability are the characteristics that make this cellulase unique. Large scale production of this enzyme will be required in future for its possible use in industry of Pakistan.

This work was supported by funds provided by Higher Education Commission, Pakistan.

The authors have no conflict of interest.