Raw material processing generates tons of agricultural and industrial waste every year. Unfortunately, biomass accumulation and misuse of such materials cause serious environmental issues. In addition, increased energy consumption, depletion of fossil fuel sources, and the need to abate global warming have drawn special attention to a new generation of renewable energy [1
]. In this scenario, reusing lignocellulosic residues to synthesize chemical compounds and high-value products like biofuels and other green chemicals has emerged as one of the most potential strategies to overcome environmental problems [2
Lignocellulose consists mainly of cellulose (35–50%), hemicellulose (20–30%), and lignin (20–30%) [5
]. Cellulases produced by fungi and bacteria can degrade cellulose [6
]. In fact, cellulases are considered to be some of the most important industrial enzymes as they can convert cellulose to sugars that can be further fermented to generate bioethanol and biobased products [6
]. The classic mechanism of substrate degradation by cellulases involves the synergistic action of three types of hydrolytic enzymes: endo-1,4-β-glucanases (EC 184.108.40.206), cellobiohydrolases/exoglucanases (EC 220.127.116.11 and EC 18.104.22.168), and β-glucosidades (EC 22.214.171.124) [8
Endo-1,4-β-glucanases catalyze the initial attack on the cellulose fibrils and randomly cleave the β-1,4-glycosidic bonds present in the amorphous regions of the cellulose chain. The other two cellulases then act to convert the oligosaccharides released from this hydrolysis into glucose [10
]. According to the carbohydrate-active enzymes (CAZymes) database, endo-1,4-β-glucanases are widespread and are classified into 16 glycosyl hydrolase (GH) families. Enzymes belonging to the GH7 family can act on a broad range of substrates, including cellulose, β-glucan, lichenin, laminarin, and even xylan. This lack of specificity makes the endo-1,4-β-glucanases of this family very attractive for various industrial applications [9
Because enzymes need to be exposed to extreme conditions during several industrial processes, stability to high temperatures within a wide pH range, as well as tolerance to inhibition by reaction products, are the most desired enzymatic properties at the industrial level [9
]. Biomass saccharification at elevated temperatures reduces polysaccharides viscosity and bacterial contamination risks and allows enzymes to be directly used after steam pre-treatment, thereby shortening process duration and saving energy [11
, a thermophilic fungus, secretes several CAZymes that can be further used in enzymatic cocktails. Therefore, this fungus plays an important role in biomass deconstruction [4
]. In our previous work, we expressed recombinant Af-EGL7 in E. coli
and characterized its function [4
]. Compared to the prokaryotic system, heterologous expression in the methylotrophic yeast P. pastoris
offers many advantages, especially in terms of recombinant protein processing, folding, and post-translational modifications, which can influence enzyme functioning and even stability [15
This paper reports on the successful gene Af-egl7 cloning and expression in P. pastoris X-33 and on the characterization of the purified Af-EGL7. It shows that a recombinant GH7 endo-1,4-β-glucanase, with higher pH and thermal stabilities, is more active on complex biomasses. Our results highlight the industrial potential of the A. fumigatus recombinant endoglucanase Af-EGL7.
3. Materials and Methods
3.1. Strains, Culture Conditions and Vector
Aspergillus fumigatus Af293, kindly donated by Prof. Dr. Sérgio Akira Uyemura (University of São Paulo, Ribeirão Preto, Brazil), was grown in yeast extract-agar-glucose (YAG) medium (2.0% (w/v) dextrose, 2.0% (w/v) agar, 0.5% (w/v) yeast extract, and 0.1% (v/v) trace elements) at 37 °C for two days to obtain a fresh conidium suspension. The conidia were inoculated to a final concentration of 2 × 106 per mL of YNB minimal medium (1× salts solution, 0.1% (v/v) trace elements, and 0.05% (w/v) yeast extract) containing 1% (w/v) fructose and incubated at 37 °C and under shaking at 200 rpm for 16 h. Next, the mycelia were harvested, washed, and transferred to YNB medium containing 1% (w/v) sugarcane exploded bagasse (SEB) at 37 °C and 200 rpm for 24 h. Then, the mycelia were harvested for RNA extraction.
The plasmid pPICZαA (Invitrogen, Carlsbad, CA, USA) was used for gene cloning, sequencing, and expression. Escherichia coli DH10β grown at 37 °C and 200 rpm in low salt Luria–Bertani medium supplemented with zeocin (50 µg·mL−1) was used to propagate the recombinant vector pPICZαA/Af-egl7. Pichia pastoris strain X-33 (Invitrogen, Carlsbad, CA, USA) cells harboring the recombinant expression vector pPICZαA/Af-egl7 were used to produce heterologous protein. The employed growth conditions are described in the EasySelect™ Pichia Expression Kit manual (Invitrogen, Carlsbad, CA, USA).
The low-viscosity substrate CM-Cellulose was purchased from Sigma (Sigma–Aldrich, St. Louis, MO, USA). Medium-viscosity Barley β-glucan and Xyloglucan from tamarind seed were acquired from Megazyme (Megazyme International, Bray, Co. Wicklow, Ireland). The natural substrate Sugarcane Exploded Bagasse (SEB) was kindly provided by Prof. Dr. João Atílio Jorge (University of São Paulo). Sugarcane bagasse “in natura” was provided by Prof. Dr. Delia Rita Tapia Blácido (University of São Paulo, Ribeirão Preto, Brazil). Rice straw, bean straw, barley bagasse, and corncob were provided by Prof. Dr. Maria de Lourdes Teixeira de Moraes Polizeli (University of São Paulo, Ribeirão Preto, Brazil).
3.2. RNA Extraction, cDNA Synthesis, and Gene Amplification
A. fumigatus mycelia were collected after growth, as described above. After freezing in liquid nitrogen and grinding into a fine powder, total RNA was isolated by using the Direct-zolTM RNA MiniPrep kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions. cDNA was synthesized by using SuperScript® II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA).
Specific primer sequences containing overlapping regions between the vector and the insert were employed (F: 5′-GAGAAAAGAGAGGCTGAAGCTGAATTCCAACAACCCGCCGCG-3′ and R: 5′-ATCCTCTTCTGAGATGAGTTTTTGTTCTAGCAGACACTGAGAGTA-3′; overlapping sites are underlined). The amplification reaction was performed with Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MS, USA) by using the follow thermocycling conditions: 98 °C for 30 s; 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 1 min; and 72 °C for 10 min. The PCR product was analyzed by electrophoresis and purified from 1% (w/v) agarose gel by using the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany).
3.3. Cloning, Transformation of P. pastoris, and Screening of Recombinant Transformants
The ORF, without predicted signal peptide, was cloned into the vector pPICZαA (previously digested with the restriction enzymes Eco
RI and Xba
I) by the Circular Polymerase Extension Cloning (CPEC) method [17
]. The CPEC reaction was performed with Phusion High-Fidelity DNA Polymerase
(Thermo Fisher Scientific, Waltham, MS, USA); the thermocycling conditions used were as follows: 98 °C for 30 s; 35 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 2 min 30 s; and 72 °C for 10 min. The cloning product was transformed into E. coli
DH10β, and the resistant transformants were selected by zeocin (50 µg·mL−1
). The recombinant expression vector pPICZαA/Af-egl7
was linearized with the restriction enzyme Pme
I and transformed into P. pastoris
X-33 competent cells by electroporation, according to the EasySelect™ Pichia
Expression Kit manual (Invitrogen, Carlsbad, CA, USA).
Recombinant transformants with high-level endoglucanase expression were screened in yeast extract-peptone-dextrose (YPD) plates containing 1% (w/v) low-viscosity carboxymethylcellulose (CM-Cellulose), zeocin (100 µg·mL−1), and 1% (v/v) methanol for induction. After incubation at 30 °C for 3 days, the plates were stained with 0.1% (w/v) Congo red solution for 20 min and de-stained with 1 M NaCl until pale orange hydrolysis zones appeared against an orange background (approximately 20 min).
3.4. Recombinant Af-EGL7 Heterologous Expression in P. pastoris
A single colony of recombinant P. pastoris X-33 harboring the vector pPICZαA/Af-egl7 was inoculated in buffered glycerol-complex medium (BMGY) (2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base, 1% (w/v) yeast extract, 1% (v/v) glycerol, 4 × 10−5 % (w/v) biotin, 100 mM potassium phosphate buffer, pH 6.0) and grown at 30 °C and 240 rpm until the culture reached log phase growth (O.D.600nm = 2–6). Then, the cells were harvested by centrifugation at 3000× g for 5 min and resuspended in buffered methanol-complex medium (BMMY) (2% (w/v) peptone, 1.5% (v/v) methanol, 1.34% (w/v) yeast nitrogen base, 1% (w/v) yeast extract, 4 × 10−5 % (w/v) biotin, 100 mM potassium phosphate buffer, pH 6.0) to an O.D.600nm = 1. Af-EGL7 expression was induced at 30 °C and 240 rpm for 6 days (optimal time) under the control of the AOX1 promoter. Additional 1.5% (v/v) final concentration methanol was added to the medium every 24 h to maintain the expression levels. Once the Af-egl7 gene was fused with the α-factor signal sequence, the recombinant protein was obtained extracellularly.
3.5. Recombinant Af-EGL7 Purification
After 6-day culture, the culture supernatant was collected by centrifugation at 3000× g for 5 min and concentrated to the maximum by using an Amicon Ultra-15 Centrifugal Filter—10 kDa cutoff (Millipore, Burlington, MS, USA). Next, the concentrate was resuspended in 20 mM sodium phosphate buffer containing 500 mM NaCl (pH 7.4) and loaded onto Ni Sepharose 6 Fast Flow resin (Ge Healthcare, Little Chalfont, United Kingdom) pre-equilibrated with the same buffer. After incubation at 4 °C for 1.5 h under stirring, a linear gradient from 0 to 500 mM imidazole in 20 mM sodium phosphate buffer containing 500 mM NaCl (pH 7.4) was applied to the column to elute the His6-tagged recombinant endoglucanase. All the fractions were collected, and protein was analyzed by 10% (w/v) SDS-PAGE, stained with Comassie Brilliant Blue R-250 (Sigma–Aldrich, St. Louis, MO, USA).
The fractions containing purified Af-EGL7 were mixed and submitted to buffer-exchange by using Amicon Ultra-15 Centrifugal Filter—10 kDa cutoff to remove excess imidazole prior to the subsequent enzymatic assays.
3.6. Endoglucanase Activity Assay
Af-EGL7 activity was determined by measuring reducing sugars from the reaction by the 3,5-dinitrosalicylic acid (DNS) method [41
]. The enzymatic reactions were performed as described by Bernardi et al., 2018 [4
]. Briefly, the reaction mixture consisting of 1% (w
) CM-Cellulose in 50 mM sodium acetate buffer (pH 5.0) was incubated at 55 °C for 10 min. The enzyme action was stopped by adding an equal volume of the DNS reagent. The mixture was boiled for 5 min and cooled down, and the absorbance was measured at 540 nm. One unit of endoglucanase activity was defined as the amount of enzyme that released 1 µmol of reducing sugar from the substrate per minute. Each assay was carried out in triplicate. Protein concentration was determined by the Greenberg method [18
The effect of different Cu2+ concentrations (from 0 to 15 mM) on the Af-EGL7 activity was also tested. The reactions were performed as described above.
3.7. Af-EGL7 Deglycosylation by Endo H
Purified Af-EGL7 deglycosylation was accomplished by Endoglycosidase H (Endo H, New England Biolabs, Ipswich, MA, USA) in both denaturing and non-denaturing conditions, according to the manufacturer’s instructions. In the first case, the recombinant protein was initially denatured at 100 °C for 10 min. Then, the deglycosylation reaction was performed at 37 °C for 24 h, as described by Meleiro et al. (2017) [34
]. Proteins were further analyzed by SDS-PAGE.
3.8. Af-EGL7 Stability Assays
Thermostability was determined by pre-incubating 0.05 µg of purified Af-EGL7 in 50 mM sodium acetate buffer (pH 5.0) without substrate, at temperatures ranging from 55 to 90 °C for durations ranging from 30 min to 72 h. The residual activities were measured under standard conditions (pH 5.0, 55 °C, 10 min), as described in Section 3.6
. The pH stability was estimated by measuring the residual enzymatic activity under standard conditions after incubation of the enzyme without substrate in Mcllvaine (citrate–phosphate) buffers pH 3.0–8.0 at 4 °C for up to 72 h [4
3.9. Determination of Kinetic Parameters
The Af-EGL7 kinetic parameters (KM, Vmax, and kcat) were determined when CM-Cellulose (2.5–30 mg·mL−1), β-glucan (0.5–15 mg·mL−1), or xyloglucan (1.0–8.5 mg·mL−1) were used as substrates. The reactions were performed in 50 mM sodium acetate buffer (pH 5.0) as previously described. The parameters were calculated by linear regression, by using the Lineweaver–Burk graphical method.
3.10. Glucose and Cellobiose Effect on Af-EGL7 Activity
The glucose and cellobiose effect on Af-EGL7 activity was determined in the presence of increasing concentrations (0.5–50 mM) of both sugars by using the chromogenic substrate Azo-Xyloglucan from Tamarind (Megazyme International, Bray, Co. Wicklow, Ireland). The enzymatic assays were performed in 85 mM sodium acetate buffer (pH 4.5) at 40 °C for 10 min, according to the manufacturer’s instructions, with slight modifications. The reactions were stopped by adding 1.7 volumes of absolute ethanol. The supernatants were harvested by centrifugation at 1000× g for 10 min, and the absorbances were measured at 590 nm. Enzyme activity without glucose or cellobiose addition was considered 100%.
3.11. Lignocellulosic Biomass saccharification
Lignocellulose enzymatic hydrolysis was carried out as described previously by Bernardi et al. (2018) with some modifications [4
]. The saccharification was accomplished in 50 mM sodium acetate buffer (pH 5.0) containing 1% (w
) of one of the following biomasses: sugarcane bagasse “in natura”, SEB, rice straw, corncob, barley bagasse, or bean straw.
The reactions consisted of 0.009 FPU Celluclast® 1.5L (Sigma–Aldrich, St. Louis, MO, USA) and 10 µg of Af-EGL7, added per 10 mg of each biomass. The reactions were conducted at 55 °C and 1000 rpm for up to 72 h in a final volume of 1 mL. DNS was added to stop the reactions and to measure the released reducing sugars. Control experiments were conducted in the same way, in the absence of Af-EGL7. The reported results represent the means ± SD calculated from at least three experimental replicates.
3.12. Statistical Analysis
Data are expressed as the mean of replicates ± SD. Significant differences between the treatment groups were analyzed by using Tukey’s test (significance, p < 0.05).
3.13. Reproducibility of the Results
All the data are the mean of at least three independent experiments and show consistent results.