Optimization of Conditions for Maximal Production of Recombinant Thermostable Cellulase from Thermotoga naphthophila using E. coli BL21-CodonPlus (DE3) as Expression Host

Cellulose is the most abundant organic compound on earth and is the principal component of plant cell wall (Wang et al., 2015). It is a homo-polymer of glucose where monomers are linked each other through β-1,4 glycosidic linkage (Kang et al., 2011). A variety of industrial and agricultural wastes have cellulose as a major component which can be transformed into simple sugar via the hydrolysis process. Two methods are available for hydrolysis of cellulose to its monomeric units. The chemical hydrolysis method requires the treatment of cellulose with strong acid under high temperature >300°C at 25 MPa which make this method as cost efficient with poor quality and low yield of final product (Deguchi et al., 2006). Enzymatic hydrolysis method is preferred over chemical hydrolysis due to its environment friendly nature. Cellulases are biocatalyst required for digestion of cellulose under ordinary temperature and pH conditions which is not hazardous to environment (Koomnok, 2005).

On the basis of catalytic action, the cellulases have been divided into the three major classes including endoglucanases (EC 3.2.1.4), β-glucosidase (EC 3.2.1.21) and exoglucanase (EC 3.2.1.91) which are responsible for the de-polymerization of complex cellulose to simple glucose units (Karmakar and Ray, 2011). Cellulases have been immensely used in various industries including textile industry for improvement of fiber softness (Galante et al., 1998; Sreenath et al., 1996), in paper and pulp industry for refinement and strengthing of paper (Singh et al., 2007; Akhtar, 1994; Bhat, 2000), in bioethanol production for saccharification of lignocellulosic materials (Sukumaran et al., 2005; Kuhad et al., 2010; Gupta et al., 2011), in wine and brewery industry for improving the quality and yield of the fermented product (Singh et al., 2007; Galante et al., 1998; Bamforth, 2009), in food processing industry for the improvement of extraction procedure, cloud stability, texture and clarification of fruit and vegetable juices (Minussi et al., 2002; Carvalho et al., 2008), in animal feed industry for the improvement of nutritional value of feed by adding the digestion process and ultimately the performance of animals (Dhiman et al., 2002), in agriculture for enhancement of crop growth by controlling plant diseases (Chet et al., 1998; Harman and Kubicek, 1998) and in detergent industry for improving colour brightness, feel and dirt removal from garments (Sukumaran et al., 2005; Singh et al., 2007; Sharyo et al., 1978).

Recombinant DNA technology played significant role for the fulfilment of industrial requirement of enzymes. Selection of appropriate expression vector and its compatibility with expression system is very important for the successful production of recombinant protein. An efficient expression system which can fulfil the industrial demand of enzyme in a limited time with low cost is required in order to meet the industrial demand (Porro et al., 2005). Optimization of conditions is another strategy being followed by scientists for the higher level production of recombinant proteins (Larentis et al., 2011; Couto et al., 2017; Mohajeri et al., 2016; Morowvat et al., 2015; Muntari et al., 2012).

Present study deals with the utilization of BL21-CodonPlus (DE3) a prokaryotic expression system as expression host and pET28a having cellulase gene from T. naphthophila as expression vector for the production of recombinant cellulase. The conditions including inducer concentration, post induction time, medium composition, temperature and pH were analyzed for the enhanced production of recombinant cellulase.

All the chemicals utilized in this study were of analytical grade and were purchased from Merck, Life Sciences, Darmstadt, Germany.

Recently, we have characterized a recombinant thermostable cellulase from T. naphthophila (Khalid et al., 2019). Cellulase gene from T. naphthophila was cloned in pET28a and was expressed in BL21-CodonPlus (DE3) cells. The recombinant cells harboring the pET28a with cellulase gene from T. naphthophila were utilized for the optimizing of conditions for the maximal production of recombinant thermostable cellulase.

Regarding the production of recombinant cellulase, the overnight grown recombinant BL21-CodonPlus (DE3) cells were diluted to 1% with fresh LB medium and was incubated at 37°C under shaking conditions (I3000, Lab Tech, Korea) till the OD660 reached to 0.4. The cells were induced with 1 mM IPTG followed by further incubation at 37°C. The production of recombinant protein was analyzed after lyzing the cells by sonication (Sonics, Newtorn, USA) (Mansoor et al., 2018).

Cellulase activity was determined in 50 mM sodium acetate buffer (pH 4.8) using carboxymethyl cellulose as substrate. The reaction mixture was incubated in water bath at 90°C for 30 min. The production of monomeric sugars was estimated by DNS method. One unit of enzyme was the amount of enzyme required to liberate 1 µmol of reducing sugars under the assay conditions (Miller, 1959).

In order to explore the effect of inducer on the production of recombinant protein, the BL21-CodonPlus (DE3) cells were induced by varying the IPTG concentration from 0.1 to 1 mM and cellulase production was recorded at each IPTG concentration (Pereira et al., 2010).

The production of recombinant cellulase was analyzed at 20, 25 and 37°C. For this purpose, the cells were shifted to respective temperature before induction after attaining the OD660 to 0.4 and were induced with 0.5 mM IPTG. The cellulase production was recorded at 20, 25 and 37°C. Regarding the optimization of incubation time, the sample after induction was withdrawn after every hour and was utilized for the determination of enzyme activity (Oelschlagel et al., 2015).

The overnight grown recombinant BL21-CodonPlus (DE3) cells were diluted to 1% with fresh LB broth prepared in 50 mM of each of sodium acetate buffer (3-5), Sodium phosphate buffer (5-7) and Tris HCl buffer (7-9). The cells were incubated again at 37°C till the achievement of OD660 to 0.4 and were induced with 0.5 mM IPTG followed by further incubation at 25°C for 22 h. The enzyme activity was recorded at each pH after losing the cells (Oelschlagel et al., 2015).

Various carbon sources including glucose, sucrose, lactose, starch, fructose & maltose and nitrogen sources including yeast extract, tryptone, peptone, urea, glycine and inorganic nitrogen sources including ammonium chloride and ammonium sulphate were utilized for the supplementation of LB medium at a final concentration of 1%. The overnight grown recombinant BL21-CodonPlus (DE3) cells having recombinant pET28a were diluted to 1% with the LB medium supplemented with additional carbon and nitrogen sources separately. The cells were induced with 0.5 mM IPTG and the expression of recombinant protein was analyzed at 25°C after 22 h of incubation (Zhang et al., 2009).

Prokaryotic expression system is being preferred over eukaryotic expression system due to its low cost, rapid growth rate, flexibility and ease to scale up for higher level production of proteins which don’t require post translational modifications (Porowinska et al., 2013). More over optimization of conditions is another strategy along with the suitable expression system for eminent production of recombinant proteins. IPTG is an inducer molecule, commonly utilize for production of recombinant protein being produced under the lac operon. Current study demonstrated the enhanced production of recombinant protein with the increase in IPTG concentration. Maximal cellulase production was achieved at 0.5 mM IPTG while further increase in IPTG resulted in reduced enzyme production (Fig. 1). The reduced production of recombinant cellulase at higher concentration of IPTG is due to its toxicity to cell and its ability to diminish the rate of synthesis of ribosomal RNA (Rizkia et al., 2015). These results are in agreement with the previous report for the production of cellulases from E. cellulosolvens (Yoda et al., 2005), C. saccharolyticus (Park et al., 2011) and T. maritima (Pereira et al., 2010) whereas in contrast to this, maximal cellulase production from P. furiosus (Kataoka and Ishikawa, 2014) was recorded at 0.1 mM IPTG.

Optimization of post induction incubation temperature is important for the higher level production of recombinant protein in soluble form. The recombinant protein was produced mainly as insoluble inclusion bodies at 37°C and the expression of recombinant cellulase was quite low at 20°C whereas the maximal soluble production of recombinant protein was achieved at 25°C (Fig. 2) after 22 h of incubation (Fig. 3). We could produce maximum of 18, 28 and 08 U/mL of recombinant cellulase at 20, 25, 37°C, respectively. This strategy of expressing protein at low temperature with extended incubation time was previously reported by Rincon et al. (2017), Tayyab et al. (2011) and Zhang et al. (2009). Cellulases from T. maritima (Pereira et al., 2010), Acidothermus cellulolyticus (Wang et al., 2015), Eubacterium cellulosolvens (Yoda et al., 2005) and Fervibacterium nodosum (Wang et al., 2010) showed their optimal production under low temperature between 16 to 28°C whereas cellulase from Caldicellulosiruptor saccharolyticus (Park et al., 2011) showed its high level production at 30°C with overnight incubation.

Change in pH always shows significant influence on the growth of bacteria. No significant bacterial growth or cellulase production was recorded at pH 3 or 4. However the cellulase production was increased with the increase in pH beyond 5 and maximal cellulase production was recorded at pH 7 while further increase in pH resulted in reduced enzyme production (Fig. 4). These findings are in agreement with the findings of Oelschlagel et al. (2015) while according to other reports optimal production of recombinant proteins was recorded at pH 7.5 (Wang et al., 2014; Zhang et al., 2009; Mendoza et al., 2014).

Medium supplementation with various carbon and nitrogen sources showed the enhanced production of recombinant cellulase. The high level production of recombinant protein was achieved when the medium was supplemented with glucose or maltose at a final con centration of 1%. The results depicted that glucose or maltose are carbon sources being preferred by BL21-CodonPlus (DE3) cells for their growth and for the production of recombinant protein (Table I). This is might be due to their monomeric nature and ease in absorption as compared to lactose or sucrose (disaccharides) and starch (polysaccharide). Similarly, aldo-sugars are being preferred over keto-sugars by BL21-CodonPlus (DE3) cells for their growth. These results are in agreement with previous reports by Nur et al. (2016), Bren et al. (2016), Wang et al. (2015) and Bettenbrock et al. (2007). Production of recombinant cellulase was also enhanced when the LB medium was supplemented with yeast extract or tryptone as additional nitrogen sources whereas the rest of selected nitrogen sources could not contribute significantly in the production of recombinant protein (Table I). The results demonstrated that BL21-CodonPlus (DE3) cells preferred organic nitrogen sources over inorganic nitrogen sources for the growth (Table I). These results are in agreement with previous reports by Fu et al. (2006), Zhang et al. (2009), Mahmoudi et al. (2012) and Lee and Chang (1994), who observed the higher level cellular growth and the enhanced production of recombinant protein when the medium was supplemented with yeast extract and tryptone, respectively.

This study demonstrated the optimization of conditions for the enhanced production of recombinant thermostable cellulase using a prokaryotic expression system. Optimization studies revealed the highest cellulase activity when recombinant cells were induced with 0.5 mM IPTG with post induction incubation of 22h at 25°C (28 U/mL) in the presence of 50 mM sodium phosphate buffer pH 7 (37 U/mL) and when the LB medium was supplemented with glucose and yeast extract as carbon and nitrogen sources (48 U/mL). Current work was a foundation study that will act as milestone for the industrial scale production of recombinant cellulase at domestic level and for the fulfilment of local industrial requirement of the enzyme in Pakistan.

This work was supported by Higher Education Commission of Pakistan.

The authors have no conflict of interest.