Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725

We cloned the gene ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polygalacturonic acid (PGA), and released monogalacturonic acid as its sole product. The Vmax and Km of CbPelA were 384.6 U·mg−1 and 0.31 mg·mL−1, respectively. CbPelA was also able to hydrolyze methylated pectin (48% and 10% relative activity on 20%–34% and 85% methylated pectin, respectively). The high thermo-activity and methylated pectin hydrolization activity of CbPelA suggest that it has potential applications in the food and textile industry.


Cloning, Expression, and Purification of CbPelA
We extracted the genomic DNA of C. besciiand amplified ACM61449 using PCR. The amplified gene was inserted into a pET20b (+) vector and transformed into E. coli origami (DE3). The recombinant protein, CbPelA, was expressed in soluble form following induction with 0.5 mM isopropyl β-D-1-thiogalactoside (IPTG). The cell-free supernatant was incubated at 70 °C for 30 min and then centrifuged. The supernatant was further purified on a Ni-NTA agarose affinity column to obtain the desired protein. Purity was confirmed using examining the protein on a 12% SDS-PAGE gel ( Figure 3). The molecular mass of the purified protein was 50 kD, which is congruent with the predicted molecular weight of the mature protein (51 kD).

Enzyme Properties of CbPelA
We examined the enzyme properties of CbPelA using polygalacturonic acid (PGA) as a substrate (Table 1; Figure 4).

Effect of pH on CbPelA Activity
The effect of pH on CbPelA activity was determined in 50 mM sodium-acetate buffer with different pHs (3-6.5). We found that the pH-activity curve was narrow ( Figure 4A). CbPelA retained approximately 80% of its relative activity between pH 5.0-5.5, which suggests that the activity of CbPelA is also very sensitive to pH. The results also demonstrate that the optimal pH was 5.2. In fact, all reported EPGs exhibit their highest activities in acidic or neutral conditions (Table 1). This may be due to the fact that acidic or neutral conditions are favorable for the enzyme-substrate interactions, and thus increase enzyme activity [35].

Effect of Temperature on CbPelA Activity
We examined the effect of temperature on CbPelA, and found that its activity increased as temperature increased up to 72 °C, with activity then rapidly decreasing ( Figure 4B). The CbPelA presented over 90% relative activity in the range of 65 to 75 °C, and the optimal temperature was around 72 °C. To date, only two thermophilic EPGs have been characterized (Table 1). It is known that elevated temperatures are often needed in some industrial processes, for example in the clarification or color extraction steps of fruit juice production, and in textile or plant fiber processing. Thus, the thermophilic EPG we characterized here may be compatible with these needs.

Thermo-Stability of CbPelA
The half-life of CbPelA (1 mg/mL) at 70 and 80 °C was 14 h and 90 min, respectively ( Figure 4C). CbPelA showed higher thermal-stability than most reported EPGs ( Table 1). The half-life of PecJKR01 at 60 °C is 5 h [29]. The bacterial Pgu B [8] retained about 20% of its activity at 60 °C for less than 10 min. The other EPGs (PG [13] or PGC2 [34]) isolated from eukaryotes showed low thermo-stabilities (Figure 1). The higher thermo-stability of CbPelA that we characterized may be attributed to its low molecular mass (Table 1), which may decrease the solvent-exposed hydrophobic surface area [15]. Furthermore, the C-terminal may play a significant role in enzyme thermo-stability [18]. According to our sequence alignment results (Figure 2), CbPelA has an extended C-terminus. This may be another reason that CbPelA has a high thermo-stability. We examined the effect of metal ions on CbPelA activity under optimal temperature and pH conditions. The results indicate that monovalent cations (1 mM) had no obvious effect on enzyme activity ( Figure 4D). These results were similar to those reported for other EPGs [8,13]. In contrast, the enzyme activity of CbPelA was highly inhibited by most divalent metal cations we examined. For example, the enzyme activity decreased by 68% and 48% with the addition of Ba 2+ and Ca 2+ , respectively. CbPelA was completely inhibited when Cu 2+ and Cd 2+ were added. However, the effect of divalent metal cations varies greatly among the reported EPGs. For example, Pgu B is completely inhibited by Cu 2+ [8], while PG is able to retain 50% of its activity [13]. Moreover, Mg 2+ or Co 2+ enhances the activity of PG, while Ca 2+ has no significant effect on both PG and PecJKR01 [13,29]. Therefore, the interaction between the EPGs and divalent metal cations is complex, and further investigation is needed to clarify the mechanism.

Analysis of the Degradation Products of CbPelA
The degradation products were analyzed using thin-layer chromatography (TLC) ( Figure 5). It was found that galacturonic acid was the only product of degredation, which indicates that CbPelA is an EPG. This result verified another of our analyses (see Section 2.1 above). Until now, only two bacterial EPGs (PelB [27] and Pgu B [8]) had been reported in the literature, while other EPGs were isolated from eukaryotes [6,16] or soil metagenome samples [29]. This, the CbPelA obtained in this study, is only the third EPG to be isolated from bacteria.

Substrate Specificity
CbPelA showed high activity on PGA but had no activity (about 0 U·mg −1 ) on other polysaccharides we tested (such as Avicel, CMC, Xylan, glucan, and soluble starch). These results suggest that CbPelA was specifically active towards the α-1,4-galacturonic acid linkages of galactopolysaccharides. We determined the kinetic characteristics of CbPelA for PGA with a Lineweaver-Burk plot. The K m and V max were calculated as 0.3 mg·mL −1 and 386.8 U·mg −1 , respectively. The V max of CbPelA was lower than that of PelB [27] and PgaX [33], but higher than that of other EPGs (Table 1). We also examined the effect of the degree of pectin methylation on CbPelA activity ( Figure 6). We found that a higher degree of pectin methylation corresponded to a lower activity of CbPelA. The activity of CbPelA towards PGA was much higher (over five-fold) than that towards high-methylated (>85%) pectin. This result indicates that CbPelA is a polygalacturonase rather than a pectinase, because polygalacturonases typically have unmethylated pectin.
Generally speaking, the bacterial EPGs we examined showed low or no activity towards methylated pectin. For example, Bacterial PelB from T. maritima showed low activity on a highly methyl-esterified substrate [27]. PecJKR01 showed no activity on 9% methylated pectin [29]. On the contrary, fungal EPGs commonly exhibit high activity towards methylated pectin. For example, fungal PG [13] isolated from A. giganteus showed high activity on 34% methyl-esterified pectin. However, in our study, CbPelA isolated from Caldicellulosiruptor bescii bacteria showed high activity on methylated pectin ( Figure 4A). The catalytic behavior of this protein was similar to that of EPGs reported from fungi [13,33,34]. The segments of Caldicellulosiruptor bescii CbPelA that form the active-site cleft are different than those reported from other bacterial EPGs (Figure 2). This may be one reason why CbPelA's demonstrates unique methylated pectin hydrolysis. Furthermore, the subtle changes around active-site and/or substrate-binding regions of CbPelA may also contribute to its substrate recognition. Figure 6. Effect of the degree of pectin methylation on CbPelA activity. Mean (SD) was calculated from three independent replicates.

Recombinant DNA Techniques
Caldicellulosiruptor bescii DSM 6725 was grown anaerobically at 70 °C as described by Yang [36]. The genome of C. bescii was isolated following Kataeva et al. [30]. The open reading frame of polygalacturonase (ACM61449) was amplified by PCR with PrimeSTAR ® HS DNA Polymerase (TAKARA, Dalian, China) and sequence-specific primers (sense 5'-GGAGATATACAT ATGAGAATAATTGTAACTGACT-3' and antisense 5'-GTGGTGGTGCTCGAGTAGCTACTTTTC TAA-3'; Nde I and Xho I sites in bold). The following PCR program was used for amplification: 5 min of denaturing at 98 °C, followed by 30 cycles of: denaturing (10 s at 98 °C), annealing (15 s at 58 °C), and polymerization (1.5 min at 72 °C). The program was completed by a final polymerization step Relative activity (%)

Degree of pectin methylation (%)
(10 min at 72 °C). The PCR product and plasmid pET20b were digested separately with Nde I and Xho I (TAKARA, Dalian, China) restriction enzymes. Following agarose gel purification, the digested fragments were ligated with T4 DNA ligase. The recombinant plasmid, pCbPelA, was then transformed into E. coli origami (DE3) (Novagen, Madison, WI, USA) in LB with 50 μg·mL −1 ampicillin.  [37]. Molecular masses were estimated with a Protein MW Marker (TAKARA, Dalian, China). The protein concentration was defined using a Bradford assay, with bovine serum albumin as the standard [38].

Enzyme Activity Assay
For assaying enzyme activity, CbPelA solution was added to PGA (0.2%, w/v) in sodium acetate buffer, 50 mM, pH 5.2 and incubated at 70 °C for 5 min. The reaction was stopped by adding 3,5-dinitrosalicylic acid reagent (DNS). Following centrifugation for removing the undissolved substrate, reducing sugars were measured using Miller's method [39], and galacturonic acid was used as the standard. One unit of activity of polygalacturonase was defined as the amount of enzyme that catalyzed the liberation of 1 μmol galacturonic acid per minute under optimal conditions.

Enzyme Characterization of CbPelA
The effect of temperature on CbPelA activity was carried out by incubating the enzyme with PGA (pH 5.2) at different temperatures ranging from 30 to 95 °C for 5 min. For the determination of optimal pH, PGA was first dissolved in 50 mM of sodium-acetate buffer with varying pHs, and CbPelA was then incubated with substrate solution at 70 °C for 5 min. The influence of metal ions on enzyme activity was tested using above methods with metal ions added. The final concentration of metal ions in the reaction mixture was 1 mM. For thermal-stability determination, CbPelA (1 mg/mL) in 50 mM sodium-acetate buffer was incubated at 70 and 80 °C, respectively. A volume of 20 µL of enzyme solution was removed at the indicated time points and cooled on ice. The residual activity was determined as described above.
The kinetic constants K m and V max of CpPelA for PGA were determined using Lineweaver-Burk double-reciprocal plots, which were generated by plotting the reciprocal of reaction velocity (1/V) against the reciprocal of the corresponding substrate concentrations (1/S).

Thin Layer Chromatography
We examined degradation products by thin-layer chromatography (TLC). CpPelA was incubated with PGA and stopped by boiling for 5 min. Samples were spotted on silica gel plate and chromatography was performed with 1-butanol:distilled water:acetic acid (5:3:2 (v/v)) as the solvent [40]. The desiccated plate was then sprayed with 0.5% orcinol (v/v) dissolved in 5% (v/v) sulfuric acid in ethanol followed by a 5-min heat treatment at 105 °C. Galacturonic acid (G 1 ), digalacturonic acid (G 2 ), and trigalacturonic acid (G 3 ) (Sigma Aldrich, St. Louis, MO, USA) were used as standards.

Conclusions
In this paper, the ACM61449 was cloned from anaerobic thermophilic Caldicellulosiruptor bescii and then expressed in E. coli origami (DE3). We purified CbPelA, characterized its properties, and identified it as a new thermophilic exo-PGase (EPG; EC 3.2.1.67). The recombinant enzyme exhibited high thermo-stability and achieved maximum activity when using polygalacturonic acid (PGA) as substrate. Furthermore, CbPelA showed strong activity on methylated pectin. The catalytic behavior of the protein was quite different from most bacterial exo-PGases, but was similar to fungal EPGs. These properties suggest that this novel enzyme is a promising candidate for industrial processes, such as bioenergy and food production.