Elsevier

Bioresource Technology

Volume 102, Issue 17, September 2011, Pages 8339-8342
Bioresource Technology

Short Communication
Purification and characterization of a novel cellobiohydrolase (PdCel6A) from Penicillium decumbens JU-A10 for bioethanol production

https://doi.org/10.1016/j.biortech.2011.06.033Get rights and content

Abstract

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-β-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.

Highlights

► At pH 2.0, from Penicillium decumbens retained 90% maximum activity (80% after 4 h). ► PdCel6A increased ethanol yield by 20% during SSF, with delignified corncob residue as substrate. ► This is the first report concerning CBHI-like CBH II with high activity against pNPC. ► PdCel6A has longer linkages than other Cel6As, which could be induced to express full cellulolysis.

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 β-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-β-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. 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.

Section snippets

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

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,

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

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).

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