**4. Recombinant fungal Cellulases produced by different expression systems**

Numerous study techniques have been improved in recent years for cloning, heterologous expression and characterization of cellulases (**Table 1**). Several studies show efficient technologies to produce endoglucanases and β-glucosidases cloned in *E. coli* strains.

The Fungus *Trichoderma virens* ZY-01 expressed an endoglucanase cloned in a vector of expression pET-32-EG, being successfully elaborated, and expressed, in a heterologous way, in *Escherichia coli* and the target protein presented a weight of 39 kDa by electrophoresis SDS-PAGE [36].

*A. fumigatus* gene encoding endo-1,4-β-glucanase (Afu6g01800), studied by Bernardi et al. [46], was cloned in the vector pET-28a (+) and expressed in the strain of *E. coli R*osetta ™ (DE3). The research results showed that the afegl7 enzyme belonged to the GH7 family, in which the Af-egl7 gene encodes the protein comprising 460 amino acids with a CBM1 domain in the 424–460 residues and molecular mass of 52 kDa.


#### *Biodegradation Technology of Organic and Inorganic Pollutants*


#### *Recombinant Fungal Cellulases for the Saccharification of Sugarcane Bagasse DOI: http://dx.doi.org/10.5772/intechopen.98363*


**Table 1.**

*Recombinant fungal Cellulases used in the Saccharification of sugarcane bagasse.*

#### *Biodegradation Technology of Organic and Inorganic Pollutants*

**372**

#### *Recombinant Fungal Cellulases for the Saccharification of Sugarcane Bagasse DOI: http://dx.doi.org/10.5772/intechopen.98363*

RNA-Seq of β-glucosidase and genomic data are instruments that can be used to express *T. harzianum* genes for the deterioration of lignocellulosic biomass. The target gene of the recombinant protein (rThBgl) cloned and expressed heterological in *Escherichia coli* Rosetta was purified with high yields. The results showed a significant increase in the activity of β-glicosidase and in the filter paper cellulase (FPA). The cellulase produced by the transformers reached higher hydrolysis yield, with less enzymatic load during the saccharification of pretreated corn straw [41].

Delabona et al. [55] reported that *Trichoderma harzianum* overexpressed the methyltransferase of the global regulator - LAE1, in order to improve the production of cellulases, considering that the evaluation of the impact of LAE1 to induce cellulases made use of soluble carbon sources and lignocellulose and low cost in an agitated bioreactor. Using sugarcane bagasse with sucrose, the overexpression of lae1 culminated in a significant increase in the expression of the gh61b (31x), cel7a (25x), bgl1 (20x) and xyn3 (20x) genes. As a result, reduction of sugar released from the pretreated sugarcane bagasse, hydrolyzed by the recombinant crude enzymatic cocktail, obtained 41% cloned improvement through plasmid in *Escherichia coli*.

Two new genes of the β-glucosidases of *Aspergillus niger* 321 were successfully cloned in the pGEM-T vector [37]. *Aspergillus fresenii* (JCM 01963) *Escherichia coli* TOP 10 and *K. phaffii* X-33 (Invitrogen, USA) were used as host strains. The bgl T2-opt gene was synthesized according to the *K. phaffii* codon trend and constructed in the (pPICZαA vector Invitrogen, USA) with the sites of the restriction enzymes EcoRI and XbaI. This article effectively discovered a new β-1,4-glucosidase bgl T2 and its *Aspergillus fresenii* ORF, under the help of high-yield sequencing of the mRNA technique. Such a method is more convenient than the traditional of obtaining a new enzyme and its genetic information, as deluded in the discussion section. The bgl T2 gene was expressed by *K. phaffii* X33. The properties of bgl T2 were tested, including pH and temperature optimums for catalysis, pH tolerance, thermostability, effects of unusual chemicals and kinetic properties against pNPG [56].

The gene of *M. thermophila* coding for endoglucanase (EG) was isolated from fungal genomic DNA and then cloned and amplified in *E. coli* strains and, finally, expressed heterologic in *P. pastoris* and two basic strategies were followed for the production of EG. These strategies include controlling proteolysis through low temperature and adding numerous amino acid supplements to the culture medium. The enzyme presented high thermostability and was able to hydrolyze several natural substrates, cellobiose as the main product, characteristics that reflect its potential use in different biotechnological applications [38].

The study by Bernardi et al. [48] used the vector *Pichia pastoris* X-33 on to improve the characterization of an endo-1,4-β-glucanase, thermostable GH7 of *Aspergillus fumigatus* (Af EGL7). The kinetic parameters Km and Vmax were estimated and evidenced a robust enzyme which provided an improved hydrolysis of sugarcane bagasse "in natura", exploded sugarcane bagasse, corn cob, rice straw and bean straw [48]. A recombinant thermoalkalin endoglucanase of *Myceliopthor thermophila* BJA (rMt-egl) was used in the application and enzymatic saccharine of agro residues. The gene of this codon-optimized endoglucanase (Mt-egl) was expressed, constitutively in *Pichia pastoris* under the regulation of the GAP promoter. It was confirmed that recombinant endoglucanase (rMt-egl), efficiently hydrolyzed industrial agro residues, which were tested, and wheat bran. The effort aims to improve the production of rMt-egl by various approaches to molecular biology and cultivation [39].

Zhao et al. [42] investigated the fungi *T. reesei* and *Aspergillus niger* with the intention of further improving cellulase production and performance in enzymatic hydrolysis. For this study, *Escherichia coli* DH5a was used for plasmid propagation. *Agrobacterium tumefaciens* AGL-1 was used for the transformation of recombinant *T. reesei*, constructed, and transformed into a pCAMBIA1300-PsCT vector. The vector *Pichia pastoris* was used for the production of recombinant CBH II by Zhao et al. [42] using a cloning vector of pUCm-T (Sangon, Shanghai, China) to obtain the *T. reesei* Rut-C30 and *A. niger* NL02. A vector pUC18-PsT containing fragments of 1.6 kb of Pcbh1-ss and 1.4 kb of Tcbh1 was used as vector structure to construct the set of DNA sequences with the expression information. As a result, a binary vector pCAMBIA1300-hph, in which it was added to this hygromycin gene, culminating in a final expression vector pCAMBIA1300-hph-PsCT. The results confirmed that the BG and CBHII genes provide a good performance in the hydrolysis of steam exploded corn pomace.

The recombinant endoglucanase (EG I) gene of *Trichoderma reesei* was successfully expressed in *Pichia pastoris*, with the objective of producing oligosaccharides from various biomass-derived substrates. Recombinant endoglucanase I (ReEG I) showed catalytic activity in relation to cellulose and xylan hydrolysis. Among several glucan and xylan substrates (paper pulp, carboxymethylated cellulose, oat xylan, birch xylan), birch xylan exhibited higher yield of xylooligosaccharides (XOS) [54].

An endoglucanase gene (ctendo7) of the fungus *Chaetomium thermophilum* was expressed in *Pichia pastoris*. The recombinant enzyme was purified by affinity chromatography with Ni2+ and subsequently characterized, through this analysis it was possible to conclude that the enzyme belongs to the family of glycosides hydrolase 7 and exhibited considerable activity against carboxytyl sodium cellulose (CMC-Na) and xylan of 1.91 IU / mg and 3.05 IU/mg in the ideal reaction condition of 55°C, pH 5.0, respectively, showed high hydrolytic efficiency in multiple lignocellulosic substrates at high temperatures [43].

The fungus *T. aurantiacus* RCKK was cloned in *P. pastoris* X-33 for overexpression. After the expression of recombinant endoglucanase (rEG), of molecular size of ~33 kDa confirmed by SDS-PAGE and western blotting, followed by determination of gel activity by zymogram analysis, the recombinant was successfully expressed in *P. pastoris* X-3 and bioreactor tests demonstrated that the enzyme is suitable for industrial applications [44].

An endoglucanase (TmEgl) was isolated from the solid-state culture of the ectomycorrhizal fungus *Tricholoma matsutake* (TmE-gl5A) cultivated in barley and vermiculite, which purified by fractionation of ammonium sulfate, ionic exchange, hydrophobic and gel filtration. TmEgl5A showed a molecular mass of approximately 40 kDa, as determined by SDS-PAGE. The gene encoding TmEgl was cloned and expressed in *Pichia pastoris* KM71H. These results suggested that *T. matsutake* produces a typical endoglucanase in solid state culture. *T. matsutake* presents itself as a strong candidate for the production of enzymes that degrade the cell wall of plants [52].

The unique candidate for GH5 cellulase of *A. glaucus* produced an endoglucanase called AgCMCase, which was cloned and expressed in the *Pichia pastoris* system [47]. The purified AgCMCase degraded the CMC-Na and was also able to hydrolyze the corn straw and rice to release sugar. The study showed that AgCMCase activity was retained by more than 95% after 4 h of incubation in the presence of NaCl 4 M, suggesting that it is a halotolerant enzyme. Thus, the interesting properties of AgCMCase can make it a potential candidate for industrial applications.

Recombinant β-glucosidase (EC 3.2.1.21) of *Aspergillus nidulans* AN2227 was expressed using buffered methanol complex medium (BMMY). Purification was performed using precipitation with ammonium sulfate and anionic exchange chromatography in the DEAE-Sephadex A-50 column. The enzyme was purified 2.58

#### *Recombinant Fungal Cellulases for the Saccharification of Sugarcane Bagasse DOI: http://dx.doi.org/10.5772/intechopen.98363*

times from the crude extract. The β-glucosidase was purified for electrophoretic homogeneity, containing a relative molecular weight of 100 kDa, as determined by electrophoresis in polyacrylamide gel, with sodium dodecile sulfate (SDS-PAGE). In the study, β-glucosidase was purified for electrophoretic homogeneity from the crude extract. The characteristics expressed from *P. pastoris* X33, with high-level expression, were described. The study suggests that the protein may be present in the monomeric form, with the enzyme having a good pH and temperature stability, making it an excellent candidate for cellulose hydrolysis [35].

A new bgl1 gene, which encodes a GH3 family of β-glucosidase of *Penicillium verruculosum* (PvBGL) was cloned and expressed heterological in the strain of *P. canescens* RN3–11–7 (niaD-) under the control of the xylAgene promoter. After the construction of the rPvBGL vector its properties were studied and compared with those of rAnBGL of *Aspergillus niger*, previously expressed in the same fungal host. It was observed that rPvBGL had an observed molecular mass of 90 kDa (SDS-PAGE data). It was possible to verify that rPvBGL converted polymeric substrates into glucose much faster than the recombinant BGL of *A. niger* (rAnBGL). Thus, this study showed the possibility of using rPvBGL for the construction of complex and balanced enzymatic preparations of cellulase based on the fungus *P. verruculosum* [49].

A β-glucosidase (BGL) of *Hypocrea sp.* W63 was cloned and expressed in *Pichia pastoris* and recombinant enzyme after purification presented a specific activity of 194.25 IU/mg. This study used *C. autoethanogenum* and *A. succinogenes* for the co-production of ethanol and succinic acid, using sugarcane bagasse as a source of fermentable sugars. The good conversion of epB-BGL suggests a great potential for the biorefining of cellulosic material [53].

Other study produced an exoglucanase (Cel6A) cloned in *Pichia pastoris*. The Cel6A gene was derived from *Trichoderma reesei* was produced synthetically, and the codon optimized for better expression in yeast *P. pastoris*. The gene was placed under the regulation of the GAP promoter, and the recombinant plasmid, called pLIPI-TrCel6A, inserted with the *T. reesei* Cel6A gene (TrCel6A), integrated into the genome of *P. pastoris* SMD1168H. The recombinant enzyme was successfully expressed by *P. pastoris*, with a main product that shows a molecular size of about 50 kDa. The recombinant Plasmid Cel6A selected was linearized with the enzyme BamHIO recombinant plasmid, pLIPI-TrCel6A, carrier of the *T. reesei* Cel6A gene (TrCel6A) integrated into the genome of *P. pastoris* SMD1168H [50].

## **4.1 Recombinant endoglucanases**

Generoso et al. [27] using the expression system in *Pichia pastoris*, obtained a β-1,4-endoglucacase belonging to Glycosil hidrolases 12 (cel12a), cloned in the vector pPICZαA, isolated from the filamentous fungus *Trichoderma harzianum* IOCzianum-3844. The recombinant enzyme rThEGIII presented a molecular mass of 25 kDa, which is similar to the predicted mass, which is 24.6 kDa, as demonstrated by the authors. A large amount of rThEGIII was produced after 24 h of methanol induction, where in approximately 48 h, 300 mg of the purified enzyme was obtained from 1 L of medium. The optimum pH and temperature for rThEGIII activity were 5.5 and 48.2°C, respectively, similar to other EGIII already described. These characteristics indicate that rThEGIII is promising for simultaneous saccharification and fermentation, since the authors showed that the enzyme presented stability at temperatures close to ideal, lasting several days with acceptable activity.

Another recombinant endoglucanase was reported by Quay et al. [10], from the fungus *Aspergillus niger* ATCC 10574. The coding gene for the enzyme (EglA) was cloned in a pPICZαC vector and expressed in recombinant form in *P. pastoris* X-33.

After purification, the recombinant protein obtained presented a mass of ~30 kDa. Based on biochemical characterization, EglA had excellent activity at 50°C and ideal pH of 4.0, with a high stability at temperatures between 30 and 50°C and pH between 2.0 and 7.0. EglA showed greater affinity in the presence of β-glucan followed by carboxymethylcellulose (CMC) with a specific activity of 63.83 and 9.47 U/mg, respectively. Significant increase in activity was also observed with the presence of metal ions (Mn2 , Co2+, Zn2+, Mg2+, Ba2+, Fe2+, Ca2+ and K+ ). Based on these attributes, this enzyme can be signaled in order to be explored for enzymatic hydrolysis of agro-industrial residues.

A recombinant endoglucanase (MtEG7a), belonging to the family of glycosides hydrolase 7, was obtained by Karnaouri et al. [28], isolated from the fungus *Myceliophthora thermophila*; cloned in a pPICZαC vector and functionally expressed in the yeast *Pichia pastoris*. The purified recombinant enzyme (MtEG7a) was tested for its activity in relation to different substrates; where the enzyme showed high activity for β-glucan of barley (298 U/mg) and carboxymethylcelllulase (177 U/mg), also presenting activity for xylan-containing substrates, such as wheat arabinoxylan (5 U/mg). The highest activity levels were verified at pH 5.0 and the ideal activity temperature was 60°C, rapidly losing its activity at temperatures above 65°C. This study shows that the primary enzymatic activity of MtEG7 a hydrolysis the β-1.4 bonds of substrates because the activity of MtEG7a in β-1,3-glucan bonds was completely inhibited. In addition, the characteristics in terms of catalytic efficiency and thermostability of MtEG7a, makes it a good candidate for industrial applications, including the saccharification of lignocellulosic materials [28].

Rubini et al. [57], reported the isolation and cloning of the first cDNA of *P. echinulatum* (Pe-egl1) that encodes a supposed endoglucanase. This cDNA was expressed in a system of heterologous expression based on the methyl yeast trophic *Pichia pastoris*. *P. echinulatum* EGL1 secreted in the culture supernatant of a recombinant strain of *Picchia pastoris* revealed several characteristics of industrial interest, such as an optimal activity at 60°C and in a wide pH range. Recombinant *P. echinulatum* EGL1 is also interesting for its high thermostability.

Lahjouji et al. [58] described a cDNA of celobiohydrolase Tvcel7a de *Trametes versicolor* cloned and expressed in *Aspergillus niger*. The biochemical properties of purified TvCel7a obtained from both peaks were studied in detail. The optimum pH and temperature were 5.0 and 40°C, respectively. The enzyme is stable in a pH range extending from 3.0 to 9.0 and at temperatures below 50°C. Kinetic parameters with the p-nitrophenyl substrate β-D-cellobioside (pNPC) were 0.58 mM and 1.0 μmol/ min/mg of protein for purified TvCel7a found in peaks 1 and 2. TvCel7a catalyzes the hydrolysis of pNPC, filter paper, β-glucan and avicel in several degrees, but no detectable hydrolysis was observed when the substrates carboxymethylcellulose, laminarin and pNPG were used.

Nakazawa et al. [59] attempted to increase the specific activity of *T. reesei* EG III in *E. coli* by random gene mutagenesis using error-prone PCR followed by plateassay activity screening. They reported that the yield in the active form of EG III was improved in transforming and the specific activity of their mutant (2R4) was increased. In addition, the stability in the pH and heat of these mutants increased unexpectedly.

Koseki et al. [60] produced an endoglucanase of the glycosyl hydrolase family 61 of *Aspergillus kawachii* (AkCel61) and a truncated enzyme only with the catalytic domain (rAkCel61ΔCBM) in *Pichia pastoris* and analyzed its biochemical properties. The proteins rAkCel61 and rAkCel61ΔCBM produced small amounts of oligosaccharides from soluble carboxymethylcellulose. They also exhibited a slight hydrolytic activity in relation to laminarin. However, they showed no detectable activity in relation to microcrystalline cellulose, arabinoxylan and pectin. Both

#### *Recombinant Fungal Cellulases for the Saccharification of Sugarcane Bagasse DOI: http://dx.doi.org/10.5772/intechopen.98363*

recombinant enzymes also showed no detectable activity for p-nitrophenyl-β-Dglucosides, p-nitrophenyl-β-D-cellobiosides and p-nitrophenyl-β-D-celotriosides.

Igarashi et al. [61] report the identification of the gene encoding the endoglucanase (EG) of the family 45 (GH) of *Phanerochaete chrysosporium*, cloning the cDNA, determining its heterologous expression in the methylotrophic yeast *Pichia pastoris* and characterizing the recombinant protein. The recombinant protein showed hydrolytic activity in relation to amorphous cellulose, carboxymethylcellulose, liquena, barley-glucan and glucomannan, but not xylan. In addition, a synergistic effect was observed with cellobiohydrolase of the recombinant GH 6 family of the same fungus for amorphous cellulose as substrate, indicating that the enzyme can act together with other cellulolytic enzymes to hydrolyze cellulosic biomass in nature.

A new β-1,3-1,4-glucanase gene (designated as PtLic16A) of *Paecilomyces thermophila* was successfully cloned and expressed in *Pichia pastoris* as β-1,3- 1,4-active extracellular glucanase. The purified enzyme had a molecular mass of 38.5 kDa in SDS-PAGE. It was optimally active at pH 7.0 and at a temperature of 70°C. In addition, the enzyme exhibited strict specificity for β-1,3-1,4-D-glucans. This was the first report on cloning and expression of a β-1,3-1,4-glucanase gene of *Paecilomyces sp* [62].

The gene encoding an endoglucanase of the glycosyl hydrolase (GH) family 45 (Cel45A) was cloned from *P. decumbens* and expressed in *Pichia pastoris* [63]. As far as we know, this is the first report of characterization of a protein of the GH 45 family in *Penicillium* species. The purified recombinant enzyme showed higher activity on glucomannan konjac (KGM) than on sodium carboxymethylcellulose (CMC-Na) or phosphoric acid cellulose (PASC). The highest hydrolytic activity was detected at pH 5.0 in KGM and pH 3.5 in CMC-Na, indicating that the mode of action in both substrates may be different for Cel45A. The optimum temperatures in both substrates were 60°C and about 90% of the relative activities were retained at 70° C. Products released from PASC and CMC-Na were mainly cellobiose, cellotriose. The protein with the highest glucomannanase activity can aid in the efficient degradation of lignocellulose by *P. decumbens* in the natural state.

### **4.2 Recombinant Exoglucanases**

In a study conducted by Li et al. [29], a gene (cbh1) encoding a cellobiohydrolase (CBH) was isolated from the fungus *Aspergillus niger* NL-1. The cellobiohydrolase gene (cbh1) was successfully expressed in *Pichia pastoris* KM71H, presenting molecular mass of approximately 60 kDa. The amino acid sequence encoded by cbh1 shows high homology with the glycoside hydrolase sequence family 7. The recombinant cbh1 exhibited ideal activity at 60°C and pH 4.0 with Km and Vmax for CMC-Na of 13.81 mM and 0.269 μmol/min, respectively. When submitted to 2 h of incubation at 90°C, the enzyme retained more than 80% of its activity and was stable in the pH range 1.0 ± 10.0; due to moderate to high temperature stability and a wide pH range, the authors point out that this enzyme has potential in several industrial applications.

Taipakova et al. [26], obtained the cellobiohydrolase coding gene (Cel6B), belonging to the glycosyl hydrolase 6B family, lentinula edodes isolate cloned in vector pET11d and transformed into *E. coli* (Rosetta DE3). The recombinant protein obtained presented a mass of 46.4 kDa. However, there was the formation of an insoluble inclusion body, preventing enzymatic activity. Such a feature has been observed before, according to Chiang et al. [64], overexpressed proteins in *E. coli* can lead to the formation of the inclusion body. To obtain the recombinant protein in the active form, Taipakova et al. [26], denaturated with 6 M guanidine chloride.

After this stage, the enzyme showed activity of 0.12 U/min, being considered much lower when compared to other celobiohidolases, however, an optimization of this expression system in *E. coli* has a great possibility of obtaining that of active cellulases.

The genome of the basidiomycete *Phanerochaete chrysosporium* contains sequences encoding at least 166 putative hydrolase glycosides, many of which are predicted to β-1,3-glucanases [65]. Kawai et al. [66], cultivated *P. chrysosporium* with laminarin as the only carbon source and found that several β-1,3-glucanases were secreted in the medium. The cDNA encoding a new β-1,3-glucanase with molecular mass of 36 kDa was cloned and expressed in a heterologous way in the methylotrophic yeast *Pichia pastoris*. Based on the catalytic activity of the recombinant enzyme in relation to various substrates β-1, 3-glucan, the recognition pattern for the branched structure of β-1,3/16-glucan is discussed: Lam16A generates nonbranched oligosaccharide from branched β-1,3/1,6-glucan.

Voutilainen et al. [67] characterized three new cellobiohydrolases originated from thermophilic ascomycetes fungi. The properties of these three cellobiohydrolases were compared to one of the best characterized celobiohydrolases, *T. reesei* Cel7A. *C. thermophilum* Cel7A showed the highest specific activity and optimum temperature in soluble substrates and these properties also correlate well with its high activity in polymeric substrates.

A gene(cel4) encoding for a cellobiohydrolase II (Ex-4) Ex-4 has been isolated from the basidiomycete of the white rot strain *Irpex lacteus* MC-2 and successfully expressed in yeast *Pichia pastoris*. The recombinant Ex-4 showed endo-processive degradation activity for cellulosic substrates and a synergistic effect on Avicel degradation was observed when the enzyme acted together with cellobiohydrolase I (Ex-1) or endoglucanase (En-1) produced by *I. lacteus* MC-2 [68].
