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Purification and characterization of a novel cellobiohydrolase (PdCel6A) from Penicillium decumbens JU-A10 for bioethanol production
Bioresource Technology 102 (2011) 8339–8342
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Purification and characterization of a novel cellobiohydrolase (PdCel6A) from Penicillium decumbens JU-A10 for bioethanol production Le Gao a, Fenghui Wang a, Feng Gao a, Lushan Wang a, Jian Zhao a, Yinbo Qu a,b,⇑ a b
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, PR China National Glycoengineering Research Center, Shandong University, Jinan 250100, PR China
a r t i c l e
i n f o
Article history: Received 5 April 2011 Received in revised form 7 June 2011 Accepted 8 June 2011 Available online 14 June 2011 Keywords: Cellobiohydrolase Cel6A Endo-action Penicillium decumbens
a b s t r a c t An acidic Cel6A, cellobiohydrolase (CBH) II, was purified from Penicillium decumbens and designated as PdCel6A. The deduced internal amino acid sequence of the novel CBH has a high degree of sequence identity with the CBH II from Aspergillus fumigatus. Surprisingly, PdCel6A exhibits characteristics comparable to that of CBH I, as well as CBH II. Similar to CBH I, the novel CBH has a specific activity of 1.9 IU/mg against p-nitrophenyl-b-D-cellobioside. The enzyme retains about 80% of its maximum activity after 4 h of incubation at pH 2.0. Using delignified corncob residue as the substrate, ethanol concentration increased by 20% during simultaneous saccharification and fermentation when supplemented with low doses of PdCel6A (0.2 mg/g substrate). To our knowledge, this is the first report involving a CBH I-like CBH II. The present paper provides new insight into the role of CBH II in cellulose degradation. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Cellulose is the primary structural component of plant cell walls, which can be fermented into ethanol and other organic chemical products (Han and Chen, 2007). A complete cellulase system consists of three classes of enzymes, namely cellobiohydrolase (CBH; EC 3.2.1.91), endoglucanase (EG; EC 3.2.1.4), and b-glucosidase (EC 3.2.1.21). The enzymatic process can be accomplished through the synergism of these three enzymes. CBHs are key components of cellulase complexes that will play an important role in the large-scale bioconversion of lignocellulose into liquid fuels and other useful products in the near future (Gusakova et al., 2005). Cellulolytic fungi usually produce two kinds of CBHs, namely CBH I and CBH II, most of which belong to families 6 and 7 of the glycoside hydrolases (GHs). Lee et al. (2011) reported a CBH I with a high specific activity of 10.8 U/mg toward p-nitrophenyl-b-D-cellobioside. Moreover, cellobiose competitively inhibits CBH I activity but has no effect on CBH II even up to 100 mM. Therefore, CBH II is a more interesting enzyme than CBH I because it presents significant advantages in improving cellulose hydrolysis (Limam et al., 2005). In industrial applications, the enzymatic saccharification of cellulose and the production of ethanol usually occur at low pH. However, CBHs usually retain only 50–60% of their maximum activity at pH 4.0 (Han and Chen, 2007; Limam et al., 1995; Okada et al., 1998), restricting their application in biomass bioconversion. ⇑ Corresponding author at: National Glycoengineering Research Center, Shandong University, Jinan 250100, PR China. Tel.: +86 531 8836 5954; fax: +86 531 8856 5234. E-mail address: [email protected] (Y. Qu). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.06.033
In the present study, the purification of a novel Cel6A from Penicillium decumbens JU-A10 using two-step purification is first described. PdCel6A is very stable at acidic pH, even at pH 2.0, and displays an apparent synergism with different commercial cellulases during the simultaneous saccharification and fermentation (SSF) of lignocellulosics into ethanol. This enzyme can be used for designing highly effective multi-enzyme mixtures for lignocellulose hydrolysis. CBH II reacts at the non-reducing ends of the cellulose chain. Considering that p-nitrophenol (pNP) connects to the reducing end of cellobiose, theoretically, CBH II cannot hydrolyze pNPC. The enzymatic activity of CBH II is very low when pNPC is used as the substrate (Limam et al., 1995). However, the present paper describes a new CBH I-like Cel6A, which exhibits high enzymatic activity against pNPC. The present paper may provide new insights into the mechanism of cellulose degradation by CBH.
2. Methods 2.1. Microorganism and culture condition P. decumbens JU-A10, a catabolite repression–resistant mutant strain, was obtained by physical and chemical mutagenesis in our laboratory (Sun et al., 2008). This mutant has been used for cellulolytic enzyme production at the industrial scale since 1996. The medium for cellulase production contains the following: 3% wheat bran, 0.6% microcrystalline cellulose, 3% corncob residue, 0.2% (NH4)2SO4, 0.1% urea, 0.3% KH2PO4, 0.05% MgSO4, 0.03% CaCl2, 0.4% peptone, and 0.1% Tween-80 (Cheng et al., 2009). The spore
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suspension was inoculated into 50 mL of the liquid medium in a 300 mL conical flask and grown at 30 °C with agitation at 180 rpm. 2.2. Enzyme assay CBH activity was assayed with pNPC (Sigma, USA) as the substrate (Deshpande et al., 1984). The substrate solution was 1.0 mg/mL pNPC, which also contained 1.0 mg/mL D-glucono-1,5lactone (Sigma, USA) to inhibit the hydrolysis of the substrate by b-glucosidase. The reaction mixtures contained 50 lL of the substrate solution and 100 lL of the enzyme fraction. After incubation at 50 °C for 30 min, the reaction was terminated by adding 150 lL of 10% Na2CO3.The amount of pNP released from the enzymatic reaction was estimated based on the absorbance at 420 nm. The control used was the inactive enzyme which was boiling at 100 °C for 10 min. 2.3. Purification of PdCel6A from P. decumbens JU-A10 by cellulose affinity chromatography and diethylaminoethyl (DEAE) fast flow chromatography The sample was loaded onto a cellulose affinity column and a DEAE Fast Flow column (1.6 20 cm; GE Healthcare, USA), equilibrated with 20 mM sodium acetate buffer (pH 4.8). The column was washed with 20 mM sodium acetate buffer and then eluted with a linear gradient of 0–1 M NaCl in the equilibration buffer. The CBH activity of the fractions was assayed. 2.4. Internal amino acid of PdCel6A by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry The internal amino acid sequence of Cel6A was tested by in-gel digestion of the protein and sequencing of the different peptides by mass spectrometry. The analysis was performed at Tianjin Biochip Corporation, Tianjin. 2.5. Characterization of PdCel6A 2.5.1. Effects of temperature and pH on CBH activity and stability The optimum temperature and optimum pH for purified CBH was measured in 50 mM sodium acetate buffer (pH 4.8) at various temperatures from 30 to 70 °C and at pH 1.0–8.0, respectively. Stability was investigated by incubating the enzyme without substrate in a water bath from 30 to 70 °C and at various pH levels, respectively. Samples were taken at different time intervals, and the residual activity was assayed. The control used was the inactive sample taken at different time intervals. The sample was inactivated by boiling at 100 °C for 10 min. 2.5.2. Inhibition of cellobiohydrolase activity by cellobiose Inhibition of cellobiohydrolase by cellobiose was determined with pNPC at concentrations ranging from 0.125 to 1 mg/mL. Cellobiose was present at 0, 20, and 50 mM concentrations, respectively. Inhibition constants (Ki) were determined from corresponding Lineweaver–Burk plots using standard linear regression techniques.
JAN-A; Novozymes, USA) at a dosage of 20 FPU/g, supplemented with 0.2 mg/g PdCel6A. The initial pH was adjusted to 5 and the temperature was maintained at 35 °C. Ethanol concentration was determined by SBA-4 biological sensor analyzer (Biological Institute of Shandong Academy of Science, China) (Liu et al., 2010). 3. Results and discussion 3.1. Purification of a CBH from P. decumbens The results of the CBH purification from P. decumbens are summarized in Table 1. The extracellular proteins of P. decumbens were concentrated and subjected to cellulose affinity chromatography. The major active peak of CBH activity was not absorbed onto the affinity column, whereas the minor active peak produced a band on the affinity column and it was eluted with 0–1 M NaCl. The mixture of the second active peak fractions was concentrated and loaded onto a 1.6 20 cm DEAE Fast Flow column (GE, Healthcare, USA), equilibrated with 20 mM sodium acetate buffer (pH 4.8). The enzyme was strongly absorbed by the column, and was eluted with 0–1 M NaCl. The purified protein with activity against pNPC was classified as CBH. This protein was detected as a single protein band in the SDS–PAGE analysis, which corresponded to the molecular mass of 60 kDa (Fig. 1A). The enzyme was purified 38-fold and it had a specific activity of 1.9 IU/mg against pNPC (Table 1). 3.2. Identification of the internal amino acid of PdCel6A by MALDI-TOF (MS/MS) The protein was digested with trypsin and sequenced with MALDI-TOF. Two peptide sequences, NAGFDAHFIMDTSR and VPSFVWLDTAAK, were isolated. The peptide sequence (NAGFDAHFIMDTSR) has the highest identity (100%) with CBH II from Neosartorya fischeri, whereas the other peptide (VPSFVWLDTAAK) shared 99.95% identity with CBH II from Aspergillus niger. This result shows that the protein belonging to GH family 6 should be classified as Cel6A. The enzyme was designated as PdCel6A. The gene and protein sequences of PdCel6A were then obtained from the recently sequenced genome data of P. decumbens (unpublished data). The protein sequence was compared with existing sequences using the BLAST search protocol. The protein has 78% similarity with the CBH II from A. fumigatus, 66% identity with CBH II from Humicola insolens, and 57% identity with EG from Gibberella zeae (Fig. 1B). The CBH II from H. insolens could not recognize the reducing and non-reducing ends of cellulose but it attacks interlinkages (Boisset et al., 2000). The amino acid sequence of PdCel6A shares 55% identity with the exoglucanase 3 from Agaricus bisporus, which could hydrolyze CMC and glucan (Yagüe et al., 1996). Comparison of the sequence alignment of PdCel6A with different Cel6A variants reveals that it has a surprisingly longer linker than other enzymes, with 11 additional amino acids (Fig. 1B). 3.3. Characterization of PdCel6A PdCel6A has some enzymatic characteristics in common with the well-characterized CBH I, as demonstrated by its activity on
2.6. Ethanol production by SSF with PdCel6A supplementation SSF was performed on the delignified corncob residue (provided by Longlife Ltd., Yucheng, Shandong, China) with a nutrient composition of 10 g/L yeast extract, 5 g/L peptone, 0.5 g/L CH3COONa, 0.2 g/ L MgSO4, 0.2 g/L ZnSO4, 0.01 g/L NaCl, and 0.01 g/L FeSO4. Ethanol production by SSF was performed using instant active dry yeast (Saccharomyces cerevisiae; Angel, China). SSF experiments were done by adding different commercial enzyme preparations (NS50013 and
Table 1 Purification table of PdCel6A from P. decumbens. Purification
Total activity (IU)
Total protein (mg)
Specific activity
Purification fold
Crude enzyme Ultrafiltration Cellulose affinity DEAE FF
120 97 28 1.2
2210 1780 23 0.63
0.05 0.055 1.2 1.9
1 1.1 24 38
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Fig. 1. (A) Proteins detected by SDS–PAGE. Lane 1, supernatant of the crude enzyme; Lane 2, active fraction of cellulose affinity column; Lane 3, purified PdCel6A; and Lane M, protein molecular weight markers. (B) Multiple-sequence alignment of P. decumbens PdCel6A, A. fumigatus CBH II, Humicola Insolens cel6A, A. bisporus exoglucananse 3, and G. zeae endoglucanase. The longer linker of P. decumbens PdCel6A is shown in the box. The peptides isolated from MALDI-TOF were highlighted with red underline.
2.5
B
30 40
2
50 60
1.5
70
1 0.5 0
pH=1
2.1
Cellobiohydrolase activity (IU/mg)
Cellobiohydrolase activity (IU/mg)
A
pH=2
1.8
pH=3 pH=4
1.5
pH=5
1.2
pH=6
0.9
pH=7 pH=8
0.6 0.3 0
0
2
1
C
3 Time (h)
4
5
0
1
3 4 Time (h)
2
PdCel6A supplementation
6
7
NS-50013
3.5
A10
3
Ethanol (mg/ml)
5
JAN-A
2.5
NS-50013+
2 1.5
A10+
1
JAN-A+
0.5 0 0
10
20
30
40
50
60
70
80
Time (h)
Fig. 2. (A) Thermal and (B) pH stability of PdCel6A from P. decumbens. (C) SSF with different commercial cellulase preparations. The arrow corresponds to the time when PdCel6A was supplemented with 0.2 mg/g of substrate.
pNPC. PdCel6A could also hydrolyze barley b-D-glucan (Sigma, USA) with a specific activity of 4 IU/mg protein, which is in agree-
ment with the results of CBH II. A published paper reported that CBH II hydrolyzes barley b-D-glucan unlike CBH I (Henriksson
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et al., 1995). PdCel6A has characteristics similar to both CBH I and CBH II. Hydrolysis of barley b-D-glucan and pNPC by PdCel6A would provide strong evidence of the endo-action of Cel6A. 3.4. Effects of temperature and pH on CBH activity and stability The optimum temperature for PdCel6A was 50 °C. About 80% of the maximum activity was retained from 30 to 60 °C. The enzyme retained about 90% of its maximum activity after 2 h incubation at 50 °C, whereas about 80% of the maximum activity was retained after 4 h incubation at 60 °C (Fig. 2A). The optimum pH of the purified PdCel6A was 5.0. About 90% of its maximum activity was retained within the pH range from 2 to 6. Interestingly, the enzyme could retain 90% of its maximum activity at pH 2. The enzyme retained about 80% of the maximum activity after incubation for 4 h at pH 2 (Fig. 2B). 3.5. Inhibition of cellobiohydrolase activity by cellobiose Cellobiose, the main reaction product, is a potent competitive inhibitor of PdCel6A. Inhibition constant for cellobiose (Ki values), estimated from the Lineweaver–Burk plot, was 44 mM for PdCel6A with pNPC as substrate. The inhibition was the competitive type. In general, cellobiose is a strong inhibitor of CBH I and weaker against CBH II. Inhibition constant values for CBH I-A and CBH I-B from Talaromyces emersonii were 2.5 mM and 0.18 mM respectively with pNPC as substrate (Tuohy et al., 2002). PdCel6A, like CBH II, is less sensitive to inhibition by cellobiose compared with CBH I. 3.6. Ethanol production by simultaneous SSF with PdCel6A supplementation After 16 h of SSF, glucose almost disappeared (data not shown) and ethanol concentration sharply increased. Enzymatic hydrolysis became the limiting step. The decrease in pH during the SSF affected cellulase activity. CBHs can usually retain only 60% of their maximum activity at pH 4 (Lahjouji et al., 2007). The final ethanol concentrations produced from the delignified corn with 2% substrate concentration (w/w) and 20 FPU/g of NS-50013, A10, and JAN-A were 1.22, 1.93, and 2.60 g/L, respectively. After the addition of 0.2 mg/g PdCel6A into the various enzyme preparations (8– 25 mg/g substrate), the ethanol concentration increased by 23.5%, 19.2%, and 19.2% (Fig. 2C). PdCel6A can potentially be used in cellulose saccharification under acidic conditions because of its acid tolerance. 4. Conclusions The purified PdCel6A from P. decumbens JU-A10 is very stable under acidic conditions and exhibits characteristics similar to both CBH I and CBH II. Ethanol concentration increases by about 20% if supplemented with low doses of the acid-tolerant PdCel6A. The
application of the enzyme in SSF potentially reduces the bioconversion cost of lignocellulose into ethanol. Further work is needed to determine whether the longer linker of PdCel6A induces the cellulolytic activity of the novel PdCel6A, and to improve PdCel6A expression to increase the efficiency of cellulose hydrolysis. Acknowledgements This study was supported by grants from National Basic Research Program of China (Grant No. 2011CB707403) and the National Natural Sciences Foundation of China (Grant Nos. 31030001 and 30970096). References Boisset, C., Fraschini, C., Schülein, M., Henrissat, B., Chanzy, H., 2000. Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A. Appl. Environ. Microbiol. 66, 1444–1452. Cheng, Y.F., Song, X., Qin, Y.Q., Qu, Y.B., 2009. Genome shuffling improves production of cellulase by Penicillium decumbens JU-A10. J. Appl. Microbiol. 107, 1837–1846. Deshpande, M.V., Eriksson, K.E., Pettersson, L.G., 1984. An assay for selective determination of exo-1,4-b-glucanases in a mixture of cellulolytic enzymes. Anal. Biochem. 138, 481–487. Gusakova, A.V., Sinitsyna, A.P., Salanovicha, T.N., Bukhtojarova, F.E., Markova, A.V., Ustinova, B.B., Zeijlb, C.V., Puntb, P., Burlingame, R., 2005. Purification, cloning and characterisation of two forms of thermostable and highly active cellobiohydrolase I (Cel7A) produced by the industrial strain of Chrysosporium lucknowense. Enzyme Microb. Technol. 36, 57–69. Henriksson, K., Teleman, A., Suortti, T., Reinikainen, T., Jaskari, J., Teleman, O., Poutanen, K., 1995. Hydrolysis of barley (1–3),(1–4)-b-D-glucan by a cellobiohydrolase II preparation from Trichoderma reesei. Carbohydr. Polym. 26, 109–119. Han, Y.J., Chen, H.Z., 2007. Synergism between corn stover protein and cellulase. Enzyme Microb. Technol. 41, 638–645. Lahjouji, K., Storms, R., Xiao, Z.Z., Joung, K.B., Zheng, Y., Powlowski, J., Tsang, A., Varin, L., 2007. Biochemical and molecular characterization of a cellobiohydrolase from Trametes versicolor. Appl. Microbiol. Biotechnol. 75, 337–346. Lee, K.M., Joo, A.R., Jeya, M., Lee, K.M., Moon, H.J., Lee, J.K., 2011. Production and Characterization of Cellobiohydrolase from a Novel Strain of Penicillium purpurogenum KJS506. Appl. Biochem. Biotechnol. 163, 25–39. Limam, F., Chaabouni, S.E., Ghrir, R., Marzouki, N., 1995. Two cellobiohydrolases of Penicillium occitanis mutant Pol 6: Purification and properties. Enzyme Microb. Technol. 17 (7), 340–346. Liu, K., Lin, X.H., Yue, J., Li, X.Z., Fang, X., Zhu, M.T., Lin, J.Q., Qu, Y.B., Xiao, L., 2010. High concentration ethanol production from corncob residues by fed-batch strategy. Bioresour. Technol. 101, 4952–4958. Okada, H., Sekiya, T., Yokoyama, K., Tohda, H., Kumagai, H., Morikawa, Y., 1998. Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products. Appl. Microbiol. Biotechnol. 49, 301–308. Sun, X.Y., Liu, Z.Y., Zheng, K., Song, X., Qu, Y.B., 2008. The composition of basal and induced cellulase systems in Penicillium decumbens under induction or repression conditions. Enzyme Microb. Technol. 42, 560–567. Tuohy, M.G., WalshD, J., Murray, P.G., Claeyssens, M., Cuffe, M.M., SavageA, V., Coughlan, M.P., 2002. Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii. Biochim. Biophys. Acta 1596, 366–380. Yagüe, E., Chow, C.M., Challen, M.P., Thurston, C.F., 1996. Correlation of exons with functional domains and folding regions in a cellulase from Agaricus bisporus. Curr. Genet. 30, 56–61.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Purification and characterization of a novel cellobiohydrolase (PdCel6A) from Penicillium decumbens JU-A10 for bioethanol production Le Gao a, Fenghui Wang a, Feng Gao a, Lushan Wang a, Jian Zhao a, Yinbo Qu a,b,⇑ a b
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, PR China National Glycoengineering Research Center, Shandong University, Jinan 250100, PR China
a r t i c l e
i n f o
Article history: Received 5 April 2011 Received in revised form 7 June 2011 Accepted 8 June 2011 Available online 14 June 2011 Keywords: Cellobiohydrolase Cel6A Endo-action Penicillium decumbens
a b s t r a c t An acidic Cel6A, cellobiohydrolase (CBH) II, was purified from Penicillium decumbens and designated as PdCel6A. The deduced internal amino acid sequence of the novel CBH has a high degree of sequence identity with the CBH II from Aspergillus fumigatus. Surprisingly, PdCel6A exhibits characteristics comparable to that of CBH I, as well as CBH II. Similar to CBH I, the novel CBH has a specific activity of 1.9 IU/mg against p-nitrophenyl-b-D-cellobioside. The enzyme retains about 80% of its maximum activity after 4 h of incubation at pH 2.0. Using delignified corncob residue as the substrate, ethanol concentration increased by 20% during simultaneous saccharification and fermentation when supplemented with low doses of PdCel6A (0.2 mg/g substrate). To our knowledge, this is the first report involving a CBH I-like CBH II. The present paper provides new insight into the role of CBH II in cellulose degradation. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Cellulose is the primary structural component of plant cell walls, which can be fermented into ethanol and other organic chemical products (Han and Chen, 2007). A complete cellulase system consists of three classes of enzymes, namely cellobiohydrolase (CBH; EC 3.2.1.91), endoglucanase (EG; EC 3.2.1.4), and b-glucosidase (EC 3.2.1.21). The enzymatic process can be accomplished through the synergism of these three enzymes. CBHs are key components of cellulase complexes that will play an important role in the large-scale bioconversion of lignocellulose into liquid fuels and other useful products in the near future (Gusakova et al., 2005). Cellulolytic fungi usually produce two kinds of CBHs, namely CBH I and CBH II, most of which belong to families 6 and 7 of the glycoside hydrolases (GHs). Lee et al. (2011) reported a CBH I with a high specific activity of 10.8 U/mg toward p-nitrophenyl-b-D-cellobioside. Moreover, cellobiose competitively inhibits CBH I activity but has no effect on CBH II even up to 100 mM. Therefore, CBH II is a more interesting enzyme than CBH I because it presents significant advantages in improving cellulose hydrolysis (Limam et al., 2005). In industrial applications, the enzymatic saccharification of cellulose and the production of ethanol usually occur at low pH. However, CBHs usually retain only 50–60% of their maximum activity at pH 4.0 (Han and Chen, 2007; Limam et al., 1995; Okada et al., 1998), restricting their application in biomass bioconversion. ⇑ Corresponding author at: National Glycoengineering Research Center, Shandong University, Jinan 250100, PR China. Tel.: +86 531 8836 5954; fax: +86 531 8856 5234. E-mail address: [email protected] (Y. Qu). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.06.033
In the present study, the purification of a novel Cel6A from Penicillium decumbens JU-A10 using two-step purification is first described. PdCel6A is very stable at acidic pH, even at pH 2.0, and displays an apparent synergism with different commercial cellulases during the simultaneous saccharification and fermentation (SSF) of lignocellulosics into ethanol. This enzyme can be used for designing highly effective multi-enzyme mixtures for lignocellulose hydrolysis. CBH II reacts at the non-reducing ends of the cellulose chain. Considering that p-nitrophenol (pNP) connects to the reducing end of cellobiose, theoretically, CBH II cannot hydrolyze pNPC. The enzymatic activity of CBH II is very low when pNPC is used as the substrate (Limam et al., 1995). However, the present paper describes a new CBH I-like Cel6A, which exhibits high enzymatic activity against pNPC. The present paper may provide new insights into the mechanism of cellulose degradation by CBH.
2. Methods 2.1. Microorganism and culture condition P. decumbens JU-A10, a catabolite repression–resistant mutant strain, was obtained by physical and chemical mutagenesis in our laboratory (Sun et al., 2008). This mutant has been used for cellulolytic enzyme production at the industrial scale since 1996. The medium for cellulase production contains the following: 3% wheat bran, 0.6% microcrystalline cellulose, 3% corncob residue, 0.2% (NH4)2SO4, 0.1% urea, 0.3% KH2PO4, 0.05% MgSO4, 0.03% CaCl2, 0.4% peptone, and 0.1% Tween-80 (Cheng et al., 2009). The spore
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suspension was inoculated into 50 mL of the liquid medium in a 300 mL conical flask and grown at 30 °C with agitation at 180 rpm. 2.2. Enzyme assay CBH activity was assayed with pNPC (Sigma, USA) as the substrate (Deshpande et al., 1984). The substrate solution was 1.0 mg/mL pNPC, which also contained 1.0 mg/mL D-glucono-1,5lactone (Sigma, USA) to inhibit the hydrolysis of the substrate by b-glucosidase. The reaction mixtures contained 50 lL of the substrate solution and 100 lL of the enzyme fraction. After incubation at 50 °C for 30 min, the reaction was terminated by adding 150 lL of 10% Na2CO3.The amount of pNP released from the enzymatic reaction was estimated based on the absorbance at 420 nm. The control used was the inactive enzyme which was boiling at 100 °C for 10 min. 2.3. Purification of PdCel6A from P. decumbens JU-A10 by cellulose affinity chromatography and diethylaminoethyl (DEAE) fast flow chromatography The sample was loaded onto a cellulose affinity column and a DEAE Fast Flow column (1.6 20 cm; GE Healthcare, USA), equilibrated with 20 mM sodium acetate buffer (pH 4.8). The column was washed with 20 mM sodium acetate buffer and then eluted with a linear gradient of 0–1 M NaCl in the equilibration buffer. The CBH activity of the fractions was assayed. 2.4. Internal amino acid of PdCel6A by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry The internal amino acid sequence of Cel6A was tested by in-gel digestion of the protein and sequencing of the different peptides by mass spectrometry. The analysis was performed at Tianjin Biochip Corporation, Tianjin. 2.5. Characterization of PdCel6A 2.5.1. Effects of temperature and pH on CBH activity and stability The optimum temperature and optimum pH for purified CBH was measured in 50 mM sodium acetate buffer (pH 4.8) at various temperatures from 30 to 70 °C and at pH 1.0–8.0, respectively. Stability was investigated by incubating the enzyme without substrate in a water bath from 30 to 70 °C and at various pH levels, respectively. Samples were taken at different time intervals, and the residual activity was assayed. The control used was the inactive sample taken at different time intervals. The sample was inactivated by boiling at 100 °C for 10 min. 2.5.2. Inhibition of cellobiohydrolase activity by cellobiose Inhibition of cellobiohydrolase by cellobiose was determined with pNPC at concentrations ranging from 0.125 to 1 mg/mL. Cellobiose was present at 0, 20, and 50 mM concentrations, respectively. Inhibition constants (Ki) were determined from corresponding Lineweaver–Burk plots using standard linear regression techniques.
JAN-A; Novozymes, USA) at a dosage of 20 FPU/g, supplemented with 0.2 mg/g PdCel6A. The initial pH was adjusted to 5 and the temperature was maintained at 35 °C. Ethanol concentration was determined by SBA-4 biological sensor analyzer (Biological Institute of Shandong Academy of Science, China) (Liu et al., 2010). 3. Results and discussion 3.1. Purification of a CBH from P. decumbens The results of the CBH purification from P. decumbens are summarized in Table 1. The extracellular proteins of P. decumbens were concentrated and subjected to cellulose affinity chromatography. The major active peak of CBH activity was not absorbed onto the affinity column, whereas the minor active peak produced a band on the affinity column and it was eluted with 0–1 M NaCl. The mixture of the second active peak fractions was concentrated and loaded onto a 1.6 20 cm DEAE Fast Flow column (GE, Healthcare, USA), equilibrated with 20 mM sodium acetate buffer (pH 4.8). The enzyme was strongly absorbed by the column, and was eluted with 0–1 M NaCl. The purified protein with activity against pNPC was classified as CBH. This protein was detected as a single protein band in the SDS–PAGE analysis, which corresponded to the molecular mass of 60 kDa (Fig. 1A). The enzyme was purified 38-fold and it had a specific activity of 1.9 IU/mg against pNPC (Table 1). 3.2. Identification of the internal amino acid of PdCel6A by MALDI-TOF (MS/MS) The protein was digested with trypsin and sequenced with MALDI-TOF. Two peptide sequences, NAGFDAHFIMDTSR and VPSFVWLDTAAK, were isolated. The peptide sequence (NAGFDAHFIMDTSR) has the highest identity (100%) with CBH II from Neosartorya fischeri, whereas the other peptide (VPSFVWLDTAAK) shared 99.95% identity with CBH II from Aspergillus niger. This result shows that the protein belonging to GH family 6 should be classified as Cel6A. The enzyme was designated as PdCel6A. The gene and protein sequences of PdCel6A were then obtained from the recently sequenced genome data of P. decumbens (unpublished data). The protein sequence was compared with existing sequences using the BLAST search protocol. The protein has 78% similarity with the CBH II from A. fumigatus, 66% identity with CBH II from Humicola insolens, and 57% identity with EG from Gibberella zeae (Fig. 1B). The CBH II from H. insolens could not recognize the reducing and non-reducing ends of cellulose but it attacks interlinkages (Boisset et al., 2000). The amino acid sequence of PdCel6A shares 55% identity with the exoglucanase 3 from Agaricus bisporus, which could hydrolyze CMC and glucan (Yagüe et al., 1996). Comparison of the sequence alignment of PdCel6A with different Cel6A variants reveals that it has a surprisingly longer linker than other enzymes, with 11 additional amino acids (Fig. 1B). 3.3. Characterization of PdCel6A PdCel6A has some enzymatic characteristics in common with the well-characterized CBH I, as demonstrated by its activity on
2.6. Ethanol production by SSF with PdCel6A supplementation SSF was performed on the delignified corncob residue (provided by Longlife Ltd., Yucheng, Shandong, China) with a nutrient composition of 10 g/L yeast extract, 5 g/L peptone, 0.5 g/L CH3COONa, 0.2 g/ L MgSO4, 0.2 g/L ZnSO4, 0.01 g/L NaCl, and 0.01 g/L FeSO4. Ethanol production by SSF was performed using instant active dry yeast (Saccharomyces cerevisiae; Angel, China). SSF experiments were done by adding different commercial enzyme preparations (NS50013 and
Table 1 Purification table of PdCel6A from P. decumbens. Purification
Total activity (IU)
Total protein (mg)
Specific activity
Purification fold
Crude enzyme Ultrafiltration Cellulose affinity DEAE FF
120 97 28 1.2
2210 1780 23 0.63
0.05 0.055 1.2 1.9
1 1.1 24 38
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Fig. 1. (A) Proteins detected by SDS–PAGE. Lane 1, supernatant of the crude enzyme; Lane 2, active fraction of cellulose affinity column; Lane 3, purified PdCel6A; and Lane M, protein molecular weight markers. (B) Multiple-sequence alignment of P. decumbens PdCel6A, A. fumigatus CBH II, Humicola Insolens cel6A, A. bisporus exoglucananse 3, and G. zeae endoglucanase. The longer linker of P. decumbens PdCel6A is shown in the box. The peptides isolated from MALDI-TOF were highlighted with red underline.
2.5
B
30 40
2
50 60
1.5
70
1 0.5 0
pH=1
2.1
Cellobiohydrolase activity (IU/mg)
Cellobiohydrolase activity (IU/mg)
A
pH=2
1.8
pH=3 pH=4
1.5
pH=5
1.2
pH=6
0.9
pH=7 pH=8
0.6 0.3 0
0
2
1
C
3 Time (h)
4
5
0
1
3 4 Time (h)
2
PdCel6A supplementation
6
7
NS-50013
3.5
A10
3
Ethanol (mg/ml)
5
JAN-A
2.5
NS-50013+
2 1.5
A10+
1
JAN-A+
0.5 0 0
10
20
30
40
50
60
70
80
Time (h)
Fig. 2. (A) Thermal and (B) pH stability of PdCel6A from P. decumbens. (C) SSF with different commercial cellulase preparations. The arrow corresponds to the time when PdCel6A was supplemented with 0.2 mg/g of substrate.
pNPC. PdCel6A could also hydrolyze barley b-D-glucan (Sigma, USA) with a specific activity of 4 IU/mg protein, which is in agree-
ment with the results of CBH II. A published paper reported that CBH II hydrolyzes barley b-D-glucan unlike CBH I (Henriksson
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et al., 1995). PdCel6A has characteristics similar to both CBH I and CBH II. Hydrolysis of barley b-D-glucan and pNPC by PdCel6A would provide strong evidence of the endo-action of Cel6A. 3.4. Effects of temperature and pH on CBH activity and stability The optimum temperature for PdCel6A was 50 °C. About 80% of the maximum activity was retained from 30 to 60 °C. The enzyme retained about 90% of its maximum activity after 2 h incubation at 50 °C, whereas about 80% of the maximum activity was retained after 4 h incubation at 60 °C (Fig. 2A). The optimum pH of the purified PdCel6A was 5.0. About 90% of its maximum activity was retained within the pH range from 2 to 6. Interestingly, the enzyme could retain 90% of its maximum activity at pH 2. The enzyme retained about 80% of the maximum activity after incubation for 4 h at pH 2 (Fig. 2B). 3.5. Inhibition of cellobiohydrolase activity by cellobiose Cellobiose, the main reaction product, is a potent competitive inhibitor of PdCel6A. Inhibition constant for cellobiose (Ki values), estimated from the Lineweaver–Burk plot, was 44 mM for PdCel6A with pNPC as substrate. The inhibition was the competitive type. In general, cellobiose is a strong inhibitor of CBH I and weaker against CBH II. Inhibition constant values for CBH I-A and CBH I-B from Talaromyces emersonii were 2.5 mM and 0.18 mM respectively with pNPC as substrate (Tuohy et al., 2002). PdCel6A, like CBH II, is less sensitive to inhibition by cellobiose compared with CBH I. 3.6. Ethanol production by simultaneous SSF with PdCel6A supplementation After 16 h of SSF, glucose almost disappeared (data not shown) and ethanol concentration sharply increased. Enzymatic hydrolysis became the limiting step. The decrease in pH during the SSF affected cellulase activity. CBHs can usually retain only 60% of their maximum activity at pH 4 (Lahjouji et al., 2007). The final ethanol concentrations produced from the delignified corn with 2% substrate concentration (w/w) and 20 FPU/g of NS-50013, A10, and JAN-A were 1.22, 1.93, and 2.60 g/L, respectively. After the addition of 0.2 mg/g PdCel6A into the various enzyme preparations (8– 25 mg/g substrate), the ethanol concentration increased by 23.5%, 19.2%, and 19.2% (Fig. 2C). PdCel6A can potentially be used in cellulose saccharification under acidic conditions because of its acid tolerance. 4. Conclusions The purified PdCel6A from P. decumbens JU-A10 is very stable under acidic conditions and exhibits characteristics similar to both CBH I and CBH II. Ethanol concentration increases by about 20% if supplemented with low doses of the acid-tolerant PdCel6A. The
application of the enzyme in SSF potentially reduces the bioconversion cost of lignocellulose into ethanol. Further work is needed to determine whether the longer linker of PdCel6A induces the cellulolytic activity of the novel PdCel6A, and to improve PdCel6A expression to increase the efficiency of cellulose hydrolysis. Acknowledgements This study was supported by grants from National Basic Research Program of China (Grant No. 2011CB707403) and the National Natural Sciences Foundation of China (Grant Nos. 31030001 and 30970096). References Boisset, C., Fraschini, C., Schülein, M., Henrissat, B., Chanzy, H., 2000. Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A. Appl. Environ. Microbiol. 66, 1444–1452. Cheng, Y.F., Song, X., Qin, Y.Q., Qu, Y.B., 2009. Genome shuffling improves production of cellulase by Penicillium decumbens JU-A10. J. Appl. Microbiol. 107, 1837–1846. Deshpande, M.V., Eriksson, K.E., Pettersson, L.G., 1984. An assay for selective determination of exo-1,4-b-glucanases in a mixture of cellulolytic enzymes. Anal. Biochem. 138, 481–487. Gusakova, A.V., Sinitsyna, A.P., Salanovicha, T.N., Bukhtojarova, F.E., Markova, A.V., Ustinova, B.B., Zeijlb, C.V., Puntb, P., Burlingame, R., 2005. Purification, cloning and characterisation of two forms of thermostable and highly active cellobiohydrolase I (Cel7A) produced by the industrial strain of Chrysosporium lucknowense. Enzyme Microb. Technol. 36, 57–69. Henriksson, K., Teleman, A., Suortti, T., Reinikainen, T., Jaskari, J., Teleman, O., Poutanen, K., 1995. Hydrolysis of barley (1–3),(1–4)-b-D-glucan by a cellobiohydrolase II preparation from Trichoderma reesei. Carbohydr. Polym. 26, 109–119. Han, Y.J., Chen, H.Z., 2007. Synergism between corn stover protein and cellulase. Enzyme Microb. Technol. 41, 638–645. Lahjouji, K., Storms, R., Xiao, Z.Z., Joung, K.B., Zheng, Y., Powlowski, J., Tsang, A., Varin, L., 2007. Biochemical and molecular characterization of a cellobiohydrolase from Trametes versicolor. Appl. Microbiol. Biotechnol. 75, 337–346. Lee, K.M., Joo, A.R., Jeya, M., Lee, K.M., Moon, H.J., Lee, J.K., 2011. Production and Characterization of Cellobiohydrolase from a Novel Strain of Penicillium purpurogenum KJS506. Appl. Biochem. Biotechnol. 163, 25–39. Limam, F., Chaabouni, S.E., Ghrir, R., Marzouki, N., 1995. Two cellobiohydrolases of Penicillium occitanis mutant Pol 6: Purification and properties. Enzyme Microb. Technol. 17 (7), 340–346. Liu, K., Lin, X.H., Yue, J., Li, X.Z., Fang, X., Zhu, M.T., Lin, J.Q., Qu, Y.B., Xiao, L., 2010. High concentration ethanol production from corncob residues by fed-batch strategy. Bioresour. Technol. 101, 4952–4958. Okada, H., Sekiya, T., Yokoyama, K., Tohda, H., Kumagai, H., Morikawa, Y., 1998. Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products. Appl. Microbiol. Biotechnol. 49, 301–308. Sun, X.Y., Liu, Z.Y., Zheng, K., Song, X., Qu, Y.B., 2008. The composition of basal and induced cellulase systems in Penicillium decumbens under induction or repression conditions. Enzyme Microb. Technol. 42, 560–567. Tuohy, M.G., WalshD, J., Murray, P.G., Claeyssens, M., Cuffe, M.M., SavageA, V., Coughlan, M.P., 2002. Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii. Biochim. Biophys. Acta 1596, 366–380. Yagüe, E., Chow, C.M., Challen, M.P., Thurston, C.F., 1996. Correlation of exons with functional domains and folding regions in a cellulase from Agaricus bisporus. Curr. Genet. 30, 56–61.