Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner

Journal of Biotechnology 214 (2015) 77–82

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Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner Huili Yu 1 , Haoran Li 1 , Dongfang Gao, Cuijuan Gao, Qingsheng Qi ∗ State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 28 July 2015 Received in revised form 9 September 2015 Accepted 14 September 2015 Available online 18 September 2015 Keywords: Antimicrobial peptide Recombinant expression Secretory production Cellulase Escherichia coli

a b s t r a c t Antimicrobial peptides (AMPs) are small molecules which serve as essential components of the innate immune system in various organisms. AMPs possess a broad spectrum of antimicrobial activities. However, the scaled production of such peptides in Escherichia coli faces many difficulties because of their small size and toxicity to the host. Here, we described a new fusion strategy to extracellularly produce significant amounts of these antimicrobial peptides in recombinant E. coli at significant amount. Employing the catalytic domain of a cellulase (Cel-CD) from Bacillus subtilis KSM-64 as the fusion partner, five recombinant antimicrobial peptides were confirmed to accumulate in the culture medium at concentrations ranging from 184 mg/L to 297 mg/L. The radical diffusion experiment demonstrated that the released model antimicrobial peptide, bombinin, had antibacterial activities against both E. coli and Staphylococcus aureus. This strategy will be suitable for the production of antimicrobial peptides and other toxicity proteins. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Antimicrobial peptides (AMPs) are small molecular endogenous peptides existing in biological natural immune systems which fight against foreign pathogen infections (Boman, 1995a; Dubin et al., 2005; Yang et al., 2001). Antimicrobial peptides not only possess a broad spectrum of antibacterial activity, but also have a strong killing effect on fungi, protozoa, viruses, and cancer cells (Bals, 2000; Sharma et al., 2000). The killing of microbes by AMPs is related to their cationic charge and the structure of these peptides (Kagan et al., 1994). These evolutionarily conserved peptides are usually positively charged and have both a hydrophobic and hydrophilic side that enables the molecule to be soluble in aqueous environments, yet are also able to enter lipid rich membranes (Izadpanah and Gallo, 2005). Once in a target microbial membrane, the peptide kills target cells through diverse mechanisms (Boman, 1995b). Thus, antimicrobial peptides are widely applied in the fields of medicine, agriculture, aquaculture and food industry because of their small molecular weight and good thermal stability (Jung et al., 2008; Sallum and Chen, 2008; Sudagidan and Yemenicioglu, 2012). Besides AMPs generated from eukaryotic origin, bacteriocins, ribo-

∗ Corresponding author. Fax: +86 531 88565610. E-mail address: [email protected] (Q. Qi). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jbiotec.2015.09.012 0168-1656/© 2015 Elsevier B.V. All rights reserved.

somally produced by bacteria are also AMPs (Ghequire and De Mot, 2014). Currently, antimicrobial peptides are mainly synthesized and produced from animals, plants, insects, yeasts and Escherichia coli (Lamberty et al., 2001; Yamada et al., 1990). Among these, E. coli is the most commonly used host for protein production because it is the best characterized strain with many available expression and regulation tools (Geng et al., 2010; Lee et al., 1998; Nuc and Nuc, 2006). However, the production yields of antimicrobial peptides in E. coli are poor because of their toxicity towards the host and their sensitivity to proteases. Many researchers have tried to fuse antimicrobial peptides to other partner proteins to solve those problems (Cabral et al., 2003; Kim et al., 2008; Lee et al., 2000; Zorko and Jerala, 2010). For example, Kim et al., 2008 constructed a fusion protein of histonin and a truncated fragment of PurF to avoid proteolytic degradation and to decrease the toxicity of histonin. By a multimeric design of the histonin gene in recombinant E. coli, the tandem repeats of histonin reached 12-mer, which resulted in a high expression level of the recombinant proteins. They also designed a furin-mediated cleavage site (RLKR residues) at the C terminus of histonin. Furin cleavage of the expressed multimeric histonin generated an intact, natural histonin. Another deficiency of the E. coli expression system is the poor secretory ability of proteins under normal culture conditions. The intracellular production of antimicrobial peptides will increase the complexity and cost of the protein purification process, which

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is not conducive to the mass production of antimicrobial peptides. Recently, it was reported that a proline-rich peptide from Drosophila, metchnikowin, which fused with a PelB sequence, was able to accumulate in the periplasmic space of E. coli in large quantities (Wu et al., 2013). This strategy greatly simplified the downstream process, but to release the periplasmic proteins to obtain the target antimicrobial peptide, adding cold sucrose solution together with EDTA for treatment after the cells are collected is still required. We previously found that the catalytic domain of a cellulase (Cel-CD) from Bacillus sp. can be secreted into the medium in large quantities. In this study, we describe a new strategy for the extracellular production of antimicrobial peptides. Using the catalytic domain of a cellulase from Bacillus sp. KSM-64 as a fusion partner, we were able to produce antimicrobial peptides extracellularly in high amounts. The extracellular expressed recombinant antimicrobial peptides had almost no effect on the growth of E. coli, which means that the toxicity of the antimicrobial peptides was veiled by Cel-CD. These results suggested that this system has potential application in the industrial production of antimicrobial peptides or other toxicity proteins. 2. Materials and methods 2.1. Bacterial strains, vectors and reagents E. coli DH5␣ and E. coli BL21 (DE3) were used for plasmids and protein expression, respectively. The plasmid pET28a was chosed as the expression vector. The restriction enzymes, DNA ladder, pre-stained protein marker were purchased from Fermentas (MBI, Canada). T4 DNA ligase was purchased from NEB (Beverly, MA, USA). PCR polymerase Primerstar was ordered from TaKaRa (Japan). The DNA sequence coding of bombinin and other antimicrobial peptide were synthesized by GenScript (USA). Primers used in this study are listed in Table 1 and ordered from BGI (Beijing, China). All other chemical reagents were of analytical grade and commercially available. 2.2. Construction of the expression plasmids The digestion and ligation method was used to construct plasmid. The cel-cd gene (Accession: M84963, 1494 to 2618) containing NcoI and BamHI restriction site were amplified by PCR with primer pairs Cel-CD-F (NcoI) and Cel-CD-L10-R (BamHI) using the Bacillus sp. KSM-64 genomes PCR template. The PCR fragments and intact pET28a vector were digested by NcoI and BamHI at 37 ◦ C for 30 min and purified using the gel extraction kit (OMEGA, China). The ligation process was performed under the function of T4 DNA ligase at 25 ◦ C for 30 min and the recombinant plasmid pET28a-Cel-CD-L10 was then transformed into E. coli DH5␣. BamHI restriction site with enterokinase cleavage recognition sequence were incorporated at the 5’end of bombinin. The digested fragment was ligated into plasmid pET28a-Cel-CD-L10 to construct pCel-bombinin plasmid containing the fusion gene. The positive transformants were verified by DNA sequencing (BGI). The other four antimicrobial peptides from different origins were also fused with Cel-CD-L10, respectively. The codonoptimized DNA sequence coding for the four antimicrobial peptides were synthesized from GenScript (Table 2). Four recombinant plasmids pCel-histatin5, pCel-magainin, pCel-melittin and pCel–cecropin were constructed (Table 1). 2.3. Expression of the recombinant antimicrobial peptide The recombinant E. coli was inoculated into 3 mL LB medium containing 25 ␮g/mL kanamycin at 37 ◦ C, 250 rpm for 12 h, then

diluted into 50 mL fresh medium in 1% inoculum size and cultivated until the optical density at 600 nm reached 0.6–0.8. IPTG was added at final concentration of 0.5 mM for the induction of the fusion genes. Samples were taken at different times and centrifuged at 6000 rpm for 10 min. The supernatant and the pellets were analyzed by SDS-PAGE and detected by Coomassie brilliant blue staining. The protein concentration was measured using Easy protein quantitative kit (TransGen) and 0.22 mg/mL Bovine serum albumin (BSA) was used as a standard. 2.4. Purification of recombinant antimicrobial peptide To purify the recombinant proteins, the supernatant was collected and exchanged using binding buffer (50 mM sodium phosphate, pH 8.0; 0.3 M sodium chloride; 20 mM imidazole) using protein superfilter (Amicon Bioseparations, Millipore NMWL 10,000) at 4 ◦ C. The recombinant protein Cel-bombinin was then purified by Profinity IMAC Ni-Charged Resin(Bio-Rad) as we mentioned before (Wang et al., 2009). The column was washed with 5 columns of binding buffer and the protein was eluted with elution buffer (50 mM sodium phosphate, pH 8.0; 0.3 M sodium chloride; 250 mM imidazole). The eluted recombinant protein was then transferred into a 10 KD Ultra centrifugal filter (Millipore) to remove the high concentration imidazole at 6000 rpm for 30 min at 4 ◦ C. 2.5. Cleavage of the recombinant antimicrobial peptide The full length recombinant antimicrobial peptide was cleaved using enterokinase. The enterokinase cleavage reaction was carried out in cleavage buffer (20 mM Tris, 100 mM sodium chloride, pH 8.0) with 5 ␮L enterokinase (4U/␮L) and 100 ␮L purified 6 × HisCel-bombinin (0.5 mg/mL). The incubation was performed at 25 ◦ C for 9 h and analyzed by Tricine-SDS-PAGE (Schagger and von Jagow, 1987). 2.6. Congo red staining An agarose plate-based assay was used to determine the hydrolytic activity of the cleaved Cel-CD. 1% (wt/vol) Carboxymethyl cellulose (CMC-Na) as a substrate was added into the agarose (0.8%, wt/vol) plate. The plate containing the activated peptide and Cel-CD was incubated at 37 ◦ C for 2 h and then flooded with 0.2% (wt/vol) Congo red solution and stained for 30 min. Then the Congo red solution was removed and 5 mL of water was used to wash the plate. Finally, 5 mL 0.9% NaCl solution was applied for 15 min, and the plates were then dried and photographed. 2.7. Antimicrobial activity assay The antimicrobial activity was assayed by radial diffusion method using Gram-negative E. coli TOP10 and Gram-positive Staphylococcus aureus ATCC 6538p (Wei et al., 2005). Briefly, bacteria were cultivated in Luria–Bertani (LB) at 37 ◦ C with 250 rpm for 12 h. One hundred microliter of inoculums was mixed with 10 mL of melt LB agar and poured into the sterile Petri-dishes. The melt agar was allowed to solidify for 30 min and then punctured with the sterile ring. The recombinant antimicrobial peptide was cleaved by enterokinase and incubated at 25 ◦ C for 8 h. 50 ␮L reaction mixtures was transferred into the hole on plate at 37 ◦ C for 12 h and the diameter of the clear zone surrounding each well was measured. Enterokinase cleavage buffer and uncleaved recombinant antimicrobial peptide was applied as negative control and synthesis bombinin was used as positive control.

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Table 1 Primers and plasmids used in this study. Primers/plasmids

Sequence/information

Bombinin-F (BamHI) Bombinin-R (XhoI) Histatin5-F (BamHI) Histatin5-R(XhoI) Melittin-F(BamHI) Melittin-R(XhoI) Cecropin A-F(BamHI) Cecropin A-R(XhoI) Magainin II-F(BamHI) Magainin II-R(XhoI) Cel-CD-F(NcoI) Cel-CD-F(NdeI) Cel-CD-L10-R(BamHI) pET28a-Cel-CD-L10 pCel-bombinin pCel- histatin5 pCel- melittin pCel- cecropin A pCel- magaininII p6His-Bombinin

5-TTTTGGATCCGATGACGATGACAAAGGCAT-3 5-TTTTCTCGAGTTAATTCGCAAAATGTTCCGCCA-3 5-TTTTGGATCCGATGACGATGACAAAGATAG-3 5-TTTTCTCGAGTTAGTAACCACGGTGAGAGTGAT-3 5-TTTTGGATCCGATGACGATGACAAAGGCAT-3 5-TTTTCTCGAGTTATTGCTGGCGCTTACGCTTCA-3 5-TTTTGGATCCGATGACGATGACAAAAAATG-3 5-TTTTCTCGAGTTAGCCTTTCGCAATCGCGGTCG-3 5-TTTTGGATCCGATGACGATGACAAAGGTAT-3 5-TTTTCTCGAGTTAGGAGTTCATGATTTCACCAA-3 5-GGAATTCCCATGGGAAGGAAACACTCGTGAAGAC-3 5-GGAATTCCATATGGAAGGAAACACTCGTGAAGAC-3 5-TTTTGGATCCAAGTACTTTCGTGTATTTTGTA-3 pET28a containing Cel-CD and L10 flexible peptide pET28a containing Cel-CD,L10 flexible peptide and bombinin antimicrobial peptide pET28a containing Cel-CD,L10 flexible peptide and histatin5 antimicrobial peptide pET28a containing Cel-CD, L10 flexible peptide and melittin antimicrobial peptide pET28a containing Cel-CD, L10 flexible peptide andcecropin A antimicrobial peptide pET28a containing Cel-CD,L10 flexible peptide and magainin II antimicrobial peptide pET28a containing 6-His tag,Cel-CD,L10 flexible peptide and bombinin antimicrobial peptide

Underline represents the restriction site. Black bold represents the L10 flexible peptide. Black bold italic represents the enterokinase cleavage site.

Table 2 Secretionof antimicrobial peptides from different sources. AMPs

Bombinin

Magainin II

Histatin 5

Melittin

Cecropin A

Origin UniProtKB Length (aa) pI MW(Da) Secretion amount (mg/L) OD600 (g/L) Wet weight

Bombina variegata P01505 24 9.70 2294.68 295 ± 20.80 2.61 ± 0.24 8.35 ± 1.03

Xenopus laevis P11006 23 10.00 2466.93 238 ± 33.00 2.69 ± 0.32 8.41 ± 0.79

Human P15516 24 10.28 3036.33 297 ± 14.09 3.85 ± 0.13 11.74 ± 1.31

Apis mellifera P01501 26 12.02 2847.49 184 ± 11.45 2.31 ± 0.27 6.89 ± 1.14

Hyalophora cecropia P01507 38 10.30 4023.82 229 ± 8.70 3.29 ± 0.19 10.01 ± 1.63

MW and PI were calculated by Compute pI/Mw tool. The protein concentration was determined by the Easy protein quantitative kit (TransGen). The UniProtKB No. is from the UniProt (http://www.uniprot.org/).

3. Results 3.1. Design of fusion system for extracellular bombinin production In a previous study, we found that the catalytic domain of a cellulase (Cel-CD) from Bacillus sp. KSM-64 can be secreted in large amounts when overexpressed in recombinant E. coli BL21(DE3) and the Cel-CD can be employed as a carrier for the extracellular production of recombinant proteins(Gao et al., 2015). Thus, in this study, we tried to produce antimicrobial peptides extracellularly in recombinant E. coli by fusion with Cel-CD. Bombinin from Bombina variegata was initially used as an example to explore the feasibility of this system. The 72 bp coding region of bombinin genes were cloned to the 3 end of the cel-cd gene into pET28a/cel, resulting in pCel-Bombinin. Between Cel-CD and bombinin, a protease digestion site (DDDDK) of enterokinase was designed for the enzymatic release of the antimicrobial peptides in the subsequent downstream process. The digestion site of enterokinase was after amino acid sequence DDDDK and therefore did not affect the following AMP. A flexible peptide containing 10 amino acid residues was added ahead of the enterokinase digestion site to avoid the influence of Cel-CD on the digestion of enterokinase. The recombinant E. coli BL21 (DE3) harboring the Cel-Bombinin expression vector was cultivated in LB medium for overexpression. Samples were taken at time intervals of 0, 0.5, 1, 2, 4, 6, 8 and 16 h after IPTG induction for the analysis of expression and secretion process (Fig. 1). After 0.5 h of induction, the recombinant protein Cel-Bombinin was easily detected using the Coomassie staining inside the cells and the intracellular protein accumulation reached

Fig. 1. Secretory expression and purification of Cel-bombinin. M, protein molecular weight marker; (A) the percent of the expressed Cel-bombinin of the whole-cell proteins; (B) time course analysis of the expressed Cel-bombinin; (C) time course analysis of the secreted Cel-bombinin;

its maximum after 8 h (Fig. 1A and B). The extracellular recombinant protein was only detected after 2 h of induction and progressively accumulated in the medium with the growth of the cells. The final extracellular concentration of Cel-bombinin reached 297 mg/L. The content of the extracellular accumulated Cel-Bombinin was around 90% (Fig. 1C). After one step purification of Profinity IMAC NiCharged Resin, the purity of recombinant protein reached 95.5%

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Fig. 3. Cleavage of Cel-bombinin recombinant protein by enterokinase. Lane C represents the uncleaved Cel-bombinin.

Fig. 2. Expression and solubility analysis of the antimicrobial peptides. M, protein molecular weight marker; lane 1, protein Cel-CD; lane 2, protein Cel-histatin; lane 3, protein Cel-bombinin; lane 4, protein Cel-magainin; lane 5, protein Cel-melittin; lane 6, protein Cel-cecropin; ‘a’ indicates the soluble protein and ‘b’ indicates the insoluble fractions.

(data not shown). These results demonstrated that the extracellular production of bombinin in recombinant E. coli is feasible. High amounts of extracellular recombinant protein can be obtained with extended cultivation.

and the cytoplasmic protein GroEL (Horwich et al., 1993), to exclude the possibility that the expression of Cel-melttin caused cell lysis. The Cel-CD was detected in the culture medium at every time point, whereas MBP and GroEL were not detected (data not shown). These results indicated that the expression of recombinant antimicrobial peptides does not affect the stability of the host and the Cel-CD can be used as a general carrier protein for the extracellular production of various antimicrobial peptides. 3.3. Activity detection of the cleaved recombinant protein

3.2. Cel-CD as a general carrier for other antimicrobial peptides production To further investigate whether Cel-CD could be used as the fusion partner for extracellular production of other antimicrobial peptides, four other antimicrobial peptides from different sources, including magainin II, histatin 5, melittin and cecropin were investigated. The expression and solubility of these recombinant proteins were analyzed by SDS-PAGE (Fig. 2). All fusion proteins were expressed with high solubility and were detected in the medium after 24 h of cultivation; however, the expression and secretion levels varied (Table 2). The recombinant Cel-histatin5 had the highest expression level, which accounted for 67.5% of the soluble proteins (determined by software image J). The expression amounts of other recombinant proteins were also more than 52.4%. The lowest extracellular production was the fusion of Cel-magainin, which was 184 mg/L in the medium. Other fusions can be exported in the amounts ranging from 229 to 297 mg/L. To verify whether the expression of these five recombinant antimicrobial peptides inhibited the growth of cells, we also measured the growth of the expression strains. Compared with the control group, the Cel-histatin5 expression strain grew the best of all, with an OD600 reading which reached 3.85 after 16 h of cultivation. The OD600 of the Cel-melttin expression strain was the lowest, at a value of 2.31 (Table 2). We then performed western blot analysis against Cel-CD together with the control proteins, the periplasmic maltose binding protein (MBP) (Shuman and Panagiotidis, 1993)

As the secreted recombinant antimicrobial peptides had almost no antimicrobial activity, we tried to remove Cel-CD from the secreted recombinant protein to obtain the active antimicrobial peptides. Recombinant Cel-Bombinin was chosen as an example again. After purification, enterokinase (4 U/␮L) was added to the reaction system and incubated at 25 ◦ C. The cleavage process of the recombinant Cel-bombinin was monitored by SDS-PAGE (Fig. 3). After 2 h of digestion, more than 70% Cel-bombinin was cleaved. The bombinin was able to be completely cut off from the recombinant protein after 8 h of digestion. The antimicrobial activity of bombinin was then analyzed by spreading 5 ␮L reaction mixture on plates inoculated with the relevant bacteria, E. coli and S. aureus, without isolation and purification (Fig. 4A&B). The concentration of bombinin was about 0.10 mg/mL after 8 h of digestion. Clear inhibition zones were observed after incubation, which demonstrated that it had antimicrobial activity towards both gram-positive and gram-negative bacteria. The control group which contained the undigested Cel-bombinin did not show any activity. Then, we applied the enterokinase reaction system with Cel-bombinin directly on the plates. Clear inhibition zones were also observed (data not shown). This experiment proved that the direct cleavage of Cel-CD and bombinin recombinant protein in the plates also worked. The cellulase activity analysis indicated that both of the recombinant Cel-bombinin and the cleaved Cel-CD are cellulolytic (Fig. 4C). This indicated that Cel-bombinin may be used as a bifunctional protein and the Cel-CD fusion system can

Fig. 4. Antibacterial activities of bombinin against E. coli (A) and Staphylococcus aureus (B). Zone 1, enterokinase cleavage buffer (50 ␮L); zone 2, recombinant protein before cleavage; zone 3, recombinant protein after enterokinase cleavage; zone 4, synthesized bombinin (0.05 mg/mL). (C) Cellulolytic activity analysis of recombinant protein and cleaved Cel-CD. Zone 1, enterokinase cleavage buffer (50 ␮L); zone 2, recombinant protein before cleavage; zone 3, recombinant protein after enterokinase cleavage.

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be used as a platform for the extracellular production of bioactive antimicrobial peptides.

4. Discussion Antimicrobial peptides (AMPs) are small molecules that serve as essential components for the innate immune system in a wide variety of organisms and have broad spectrum antimicrobial activity. However, the scaled production of the antimicrobial peptides is still a challenge because of their small size, toxicity to the host cell, and complicated isolation and purification processes (Monincova et al., 2010). In this study, by fusion with the secretory catalytic domain of a cellulase, we were able to efficiently produce antimicrobial peptides outside the recombinant E. coli. This is the first report that antimicrobial peptides can be produced outside the cells of E. coli in large amounts. The extracellular accumulation amount reached 184–297 mg/L in batch cultivation before optimization. We believe that cultivation in a precisely controlled fermentor at optimized conditions will further improve the production yield. Many researchers have tried to transport the antimicrobial peptides into the periplasmic space by employing the transmembrane signal sequences to avoid the disadvantages of intracellular accumulation and the correct formation of disulfide bonds, such as PelB. Compared with the periplasmic accumulation, Cel-CD can carry the antimicrobial peptides into the culture medium, which enabled the scaled production by overcoming the spatial limitation of periplasmic space. In addition, the secretion of Cel-CD and its recombinant proteins was a two-step process, and the oxidizing environment and Dsb system of periplasm benefits the formation of disulfide bonds (de Marco, 2012). Compared with the most used lactic acid bacteria (LAB) for the production of antimicrobial substances, which is commonly applicable for the food industry, the E. coli strain are more widely used for the production of proteins because of the best characterized expression and regulation systems (Choi and Lee, 2004; Zacharof and Lovitt, 2012). And the antimicrobial peptides produced in LAB are bioactive that may also inhibit the growth of the host strain in industry production when the concentration of antimicrobial substances beyond the scope of the bacteria-resistant(Kong and Lu, 2014). For example, the yield of nisin in LAB fermentation systems is limited to 100–200 ␮g/L and the high production and purification costs of nisin are the main factors limiting its use in industrial production (Simsek, 2014). However, the recombinant antimicrobial peptides fused with Cel-CD did not show any antibacterial activity or inhibition to cell growth, which indicated their low toxicity to the host. It may also be one of the reasons why recombinant E. coli can produce high amounts of antimicrobial peptides. The extracellular production of antimicrobial peptides fusion with Cel-CD provided a cheap and fast method with an easy purification process, and will have huge potentials in scale up. This strategy can also be used to produce proteins other than antimicrobial peptides if it is applicable. Because Cel-CD is the catalytic domain of a cellulase, it also provides additional cellulosic function besides its carrier role. In the feed industry, cellulase can be used in fodder to promote the digestion of cellulose in the livestock (Lynd et al., 2002). Antimicrobial peptides are normally used in the feed industry instead of antibiotics which may induce antibiotic resistance (Hall et al., 2015). In these recombinant proteins, an enterokinase digestion site between Cel-CD and antimicrobial peptides was designed in the recombinant protein, which can be easily cleaved by the enterokinase to generate antimicrobial peptide and catalytic cellulase inside the stomach of livestock. Therefore, the recombinant fusion proteins can be used as bifunctional molecules, of which the cellulosic activity provides a possibility to digest the cellulose in the fod-

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