Synergistic properties of cellulases from Clostridium cellulovorans in the presence of cellobiose
© Yamamoto and Tamaru. 2015
Received: 29 October 2015
Accepted: 11 December 2015
Published: 4 January 2016
An anaerobic mesophile, Clostridium cellulovorans, produces a multienzyme complex called the cellulosome and actively degrades polysaccharides in the plant cell wall. C. cellulovorans also changes cellulosomal subunits to form highly active combinations dependent on the carbon substrate. A previous study reported on the synergistic effects of exoglucanase S (ExgS) and endoglucanase H (EngH) that are classified into the glycosyl hydrolase (GH) families 48, and 9, respectively. In this study, we investigated synergistic effects of ExgS and EngK, a GH9 cellulase different from EngH. In addition, since EngK was known to produce cellobiose as its main product, the inhibition on cellulase activity of EngK with cellobiose was examined. As a result, the effect of cellobiose inhibition on EngK coexistent with ExgS was found to be much lower than that with EngH. Thus, although EngH and EngK are in the same GH9 family, enzymatic activity in the presence of cellobiose was significantly different.
KeywordsClostridium cellulovorans Glycoside hydrolase family 9 Glycoside hydrolase family 48 Synergistic effect Product inhibition
In this study, the synergistic effects of ExgS (GH48) and EngK (GH9) or EngH (GH9) in the presence of cellobiose were compared.
Bacterial strains and media
C. cellulovorans (ATCC 35296) was used as the source of chromosomal DNA. Escherichia coli HST08 (TaKaRa) and origami (Novagene) were used for the construction of plasmids and cloning host for the production of recombinant proteins, respectively.
Plasmid construction and expression of recombinant proteins
Designs of primers used in this study
Purification of recombinant proteins
The cultured E. coli cells were harvested by centrifugation, and were washed and disrupted by sonication. Cell debris was removed by centrifugation. The cell-free extracts were centrifuged (for 30 min at 4 °C at 20,000g) and separated from the supernatant and the pellets, respectively. TF-EngH was purified from the supernatant. EngK and ExgS were purified from the pellets. The supernatant (for TF-EngH) was applied onto HisTrap HP (GE healthcare) and eluted by 20 mM phosphate buffer (pH 7.4) containing 500 mM NaCl and 500 mM imidazole. The trigger-factor (TF) tag was removed from TF-EngH by HRV-3C protease (Novagen). The pellets (for EngK or ExgS) were solubilized with 8 M urea and renatured essentially as described previously (Liu and Doi 1998). The purified enzymes containing the fractions were dialyzed against 50 mM acetate buffer (pH 6.0). The concentration of purified proteins was measured by protein assay kit from Bio-Rad, using bovine serum albumin as the standard.
Enzyme activities were assayed in the presence of 0.5 % (wt/vol) concentration of acid-swollen cellulose at 37 °C in 50 mM acetate buffer (pH 6.3) containing 2.5 mM CaCl2, 0.08 mg/ml tetracycline and 0.06 mg/ml cycloheximide. Final enzyme concentration was prepared at 20 nmol/ml. Samples were collected and immediately boiled for inactivation of the enzymes. 5 or 10 mg/ml of cellobiose were added to the enzyme assay mixture for inhibition of synergistic activities among EngH, EngK, and ExgS. The reducing sugars were determined by the DNS method, as d-glucose equivalents. Activities were expressed in units, 1 U defined as the amount of enzyme releasing 1 µmol of reducing sugar per min.
Synergy effect on acid swollen cellulose between recombinant proteins EngK, EngH and ExgS
Synergy degrees and the inhibition of cellulases activity on cellulose by cellobiose
Molar percentage of enzyme (%)
Inhibition rate (%)
Inhibition of synergistic activities among EngH, EngK, and ExgS by cellobiose
Enzymatic activities of all recombinant enzymes and their synergistic activities were inhibited by 5 mg/ml cellobiose (Fig. 3; Table 2). The inhibition rates of ExgS, EngH or EngH were 98.5, 90.1 or 98.7 %, respectively. The highest specific activity of the mixture of EngH and ExgS was 0.251. The activity was inhibited to 0.017, that is, the inhibition rate was 93.4 %. In contrast, the inhibition rate of the mixture of EngK and ExgS was 56.1 %, when the molar ratio of EngH to ExgS was 25:75 %. The synergistic activity of EngK and ExgS containing 5 mg/ml cellobiose was more than twice the synergistic activity of EngH and ExgS. No activities were detected in each reaction mixture in presence of 10 mg/ml cellobiose.
Synergistic effects with either EngK or EngH and ExgS were detected in the assay against acid-swollen cellulose (Fig. 3; Table 2). These synergies were lower than the synergy between EngH and ExgS that has been reported in a previous study (Murashima et al. 2002). In addition, the inhibition of synergistic effect by cellobiose was different between EngH and EngK (Fig. 3; Table 2). The inhibition of EngK with ExgS by cellobiose was lower that of EngH with ExgS. These results indicated that the difference between EngH and EngK is not only with their enzymatic properties but also with their synergistic effects.
The importance of EngE (GH5), ExgS (GH48) and EngH (GH9) which are main subunits in the C. cellulovorans cellulosome has been reported by a number of studies. Synergistic effects between those cellulases were demonstrated by many enzymatic studies. Some of these studies have found that the enzymatic property of the cellulosome changes depending on the subunit composition of the cellulosome. On the other hand, new insights of synergistic effects between EngK (GH9) and ExgS under the inhibition by cellobiose were shown in this study. Complexation of cellulosomal enzymes perhaps change their inhibition by cellobiose. These results supported previous studies on the cellulosome of C. cellulovorans and the other clostridia.
KY carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. KY and YT contributed to the interpretations of the data. Both authors read and approved the final manuscript.
This research was part supported by Japan Society for Bioscience, Biotechnology, and Agrochemistry, the Sumitomo Foundation for Environmental Research, and New Energy and Industrial Technology Development Organization (NEDO).
The authors declare that there is no conflict of interests regarding the publication of this paper.
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