Purification and enzymatic properties of a new thermostable endoglucanase from Aspergillus oryzae HML366

Aspergillus oryzae HML366 is a newly screened cellulase-producing strain. The endoglucanase HML ED1 from A. oryzae HML366 was quickly purified by a two-step method that combines ammonium sulfate precipitation and strong anion exchange column. SDS-PAGE electrophoresis indicated that the molecular weight of the enzyme was 68 kDa. The optimum temperature of the purified endoglucanase was 60 ℃ and the enzyme activity was stable below 70 ℃. The optimum pH was 6.5, and the enzyme activity was stable at pH between 4.5 and 9.0. The analysis indicated that additional Na+, K+, Ca2+, and Zn2+ reduced the catalytic ability of enzyme to the substrate, but Mn2+ enhanced its catalytic ability to the substrate.The Km and Vmax of the purified endoglucanase were 8.75 mg/mL and 60.24 μmol/min·mg, respectively. In this study, we report for the first time that A. oryzae HML366 can produce a heat-resistant and wide pH tolerant endoglucanase HML ED1, which has potential industrial application value in bioethanol, paper, food, textile, detergent, and pharmaceutical industries.

Cellulose can be fermented to produce ethanol and other types of energy and chemicals. Concerns about global climate change, increased demand for energy, and reduced oil supply have prompted people to develop renewable energy to replace fossil fuels. The production of ethanol mediated by cellulose hydrolysis has become a subject of great interest (Chandrasekhar et al. 2021;Srivastava et al. 2021).
The endoglucanase (EC 3.2.1.4) acts on the non-crystalline areas inside the cellulose molecule to randomly hydrolyze the β-1,4-glycosidic bond in the cellulose molecule to produce short cellulose chains, thereby playing an important role at the initiation step of reaction. Endoglucanases provide raw materials that can be used in industrial applications. Endoglucanases is used to remove fluff fibers on the surface of cellulose to enhance the softness and brightness of cotton. Endoglucanases are also used to Yongling Qin, Baoshan Qin, Jian Zhang, Yue Fu, and Qiqian Li contributed equally to this work. promote the soil removal from fabrics. It can be added to detergent products to increase color and soften fabrics. As a feed additive, it can improve the absorption rate of starch and vegetable oil by animals. In food industry, endoglucanases can increase the yield of vegetable juices. It can also increase the strength of pulp. Thus, endoglucanases have been widely used in pulp, textiles, bioethanol, washing, wine and beer, food processing, animal feed, agriculture, biomolecular chemical products, and the pharmaceutical industry (Araújo et al. 2021;Haq et al. 2021;Aich and Datta 2020;Li et al. 2021;Pereira et al. 2021;Ibrahim et al. 2021;Petrova et al. 2009;Zhang et al. 2021).
Our group previously collected samples from the National Nature Reserve of Huanjiang County, Guangxi, and screened a cellulase-producing fungus HML366, which was identified as Aspergillus oryzae by phenotype and ITS-rDNA sequence analysis (Qin et al. 2011). We have completed the purification studies of partial cellulases from A. oryzae HML366 and performed tandem time-offlight mass spectrometry detection. Relying on the fulllength sequencing data of A. oryzae chromosome genes performed by Machida et al. (2005). We first reported of production of hypothetical protein XP_001816831 and β-glucosidase with high transglycosylation activity in A. oryzae HML366 (He et al. 2013). A. oryzae HML366 can simultaneously produce a new 33.6 kDa xylanase (He et al. 2015). An extracellular enzyme HML CBH1 with a molecular weight of 48 kDa can also be isolated from the enzyme solution of A. oryzae HML366. This enzyme belongs to glycoside hydrolase family 7, and has both exoglucanase and endoglucanase activities (Qin et al. 2020).
In this study, our group aimed to complete the purification of endoglucanase from this strain, and provided experimental basis for the comprehensive application of cellulase.

Microorganism and culture conditions
A. oryzae HML366 was grown on potato dextrose agar (PDA) slants and stored at 4 °C in Guangxi Colleges Universities Key Laboratary of Exploitation and Utilization of Microbial and Botanical Resources. A. oryzae HML366 was originally isolated from the soil beneath the rotten wood in Mulun Forestry Center, Huanjiang County, Guangxi, China (Qin et al. 2011) and deposited in the Chinese Center for Type Culture Collection (Accession No. CCTCC AF 2021152).
Cellulase re-screening solid medium Ten-gram bagasse and 6-g bran were mixed well with 30 mL Mandels nutrient salt solution in a 500 mL Erlenmeyer flask (Eveleigh et al. 2009). The cultures were turned twice a day and incubated at 30 °C for 5 days. 200 mL sterile ddH 2 O was added to the culture, and extracted in a constant temperature water bath at 40 °C for 1 h before filtering with four layers of gauze. The solution was centrifuged at 6000 r/min for 10 min to obtain the crude enzyme solution. The supernatant was collected and stored at 4 °C for future use (He et al. 2013).

Endoglucanase rapid identification plate
One percent sodium carboxymethyl cellulose made with sodium acetate buffer (pH5.0) was added to the plate with 1.5% agarose, followed by dropping 100 μL enzyme solution to the plate, and reacted for 30 min at 30 ℃. The plate was stained with 0.2% Congo red for 30 min, then decolorized with three times volume of 1 mol/L NaCl. The presence of transparent circle on the plate indicated that there was endoglucanase activity in the sample (He et al. 2013;Sugimura et al. 2003).

Determination of enzyme activity and protein concentration
One percent (w/v) carboxymethyl cellulose (CMCNa, Fluka) dissolved in 2% (w/v) sodium citrate (50 mM/pH 4.8) was used as a substrate, and the 3,5-di Nitrosalicylic acid (DNS) method was used. Enzyme activity (U) is defined as the amount of enzyme needed to catalyze the production of 1 μmol glucose per minute (Miller 1959). The protein concentration was measured at 595 nm according to the Bradford method (1976) by using the Bradford Protein Assay Kit (Beyotime Institute of Biotechnology (China)). All assays were performed in triplicate.

Purification of A. oryzae HML366 endoglucanase
All purification steps were performed at 4 °C Seven aliquots of crude enzyme solution were prepared and 100 mL for each aliquot. The proteins in each sample were precipitated with 30%-90% relative saturation (10% concentration gradient increase) of ammonium sulfate, respectively. After precipitation, the samples were incubated for 12 h at 4 ℃. The enzyme solutions were centrifuged for 20 min at 4 ℃ with a speed of 8000 r/min. The precipitates were collected after centrifugation and dissolved in citric acid buffer solution (pH 4.8), and made up to 1/5 of the original volume. Sephadex G-25 gel column was used to quickly desalt from enzyme solution, and the enzyme activity of endoglucanase was identified with endoglucanase quick identification plate to determine the best conditions for ammonium sulfate precipitation and salting-out. The crude enzyme products were stored at 4 °C.
The crude enzymes were further purified by using MonoQ10/100GL (Amersham Biosciences, Sweden) strong anion exchange chromatography column (Petrova et al. 2009;Zhang et al. 2021;Qin et al. 2011) with a BioLogic Duo-Flow protein purifier (Bio-Rad) (pressure 530 psi). 0.01 mol/L Tris-HCl (pH 8.3) solution was used as the starting buffer, and 1 mol/L NaCl in 0.01 mol/L Tris-HCl (pH 8.3) was used as the elution buffer, and flow rate was 1 mL/ min. The enzyme was eluted using 60 mL of 0 to 0.3 M linear gradient of NaCl, and collected 0.5 mL per tube. The purified enzyme was stored at 4 °C. The enzyme activity of endoglucanase was determined, and the protein purity was detected by SDS-PAGE.

SDS-polyacrylamide gel electrophoresis analysis
According to Laemmli (1970) method, the enzyme products were subjected to separation with 12% SDS-PAGE gel in Tris-glycine buffer (pH 8.3) at a voltage of 120 V. The gel was stained with coomassie brilliant Blue R 250, and the molecular weight of enzymes was evaluated by comparing the relative mobility of purified protein with low molecular weight protein standard.

Zymogram analysis
The purified enzyme was subjected to non-denaturing protein gel electrophoresis at 4 ℃ with pH 8.3 electrophoresis buffer and 50 V constant voltage. The separation gel and stacking gel was made by 8% acrylamide and 4% acrylamide, respectively, and 0.1% (w/v) sodium carboxymethyl cellulose (CMCNa, Fluka) was added in the separation gel. After the electrophoresis, the separation gel was stained for 20 min with 0.2% Congo red and decolored with 1 mol/L NaCl to perform zymogram analysis (He et al. 2013).

The effect of temperature and pH on enzyme activity
The definition of relative enzyme activity: the highest enzyme activity under a certain condition of the experimental project was set to 100%, and the ratio of enzyme activity under other conditions to the highest enzyme activity was defined as relative enzyme activity.
To determine the optimal temperature of endoglucanase, the enzyme activity was measured under the conditions of 30-90 ℃ in 50 mM acetate buffer. To determine the effect of temperature on the stability of enzyme, the enzyme was incubated for 60 min in a water bath at temperatures between 40 and 90 ℃, and the residual enzyme activity was measured at 60 ℃.
To determine the optimal pH of enzyme, the enzyme was stored in following buffers with a concentration of 50 mM: disodium hydrogen phosphate-citric acid buffer, pH 3.0-7.5; Tris-HCl buffer, pH 7.5-pH 9.0; and glycine-NaOH buffer, pH 9.0-11.0. The solutions were first stored for 24 h at 4 ℃, followed by 3 h at 30 ℃. The relative enzyme activity was determined at the optimal temperature. Relative enzymatic activity was defined as follows: the maximum enzymatic activity under specific control conditions were defined as 100%, and the measured activities under varying conditions in the same experiment were normalized to derive the relative enzymatic activity in percentage. Three parallel tests were performed.

Effect of metal ions on enzyme activity
Different metal ions were added to the purified enzyme solution with a final concentration of 1 mM, 2.5 mM, and 5 mM, and the enzyme activity was tested. The enzyme activity was calculated according to the average value of data obtained from three parallel experiments.

Kinetics analysis of the purified enzyme
To determine the kinetic parameters of the enzymatic reaction of endoglucanase, CMC-Na (0.2-3 mM) was used as the substrate and the reaction was performed in optimal pH sodium acetate buffer at optimal temperature. The initial reaction rate was calculated, and the Km value and Vmax of the purified enzyme were calculated by using Lineweaver-Burk plot (Horovitz and Levitzki 1987).

Thin-layer chromatography analysis of enzymatic hydrolysis products
The CMC-Na containing 50 μL purified enzyme was dissolved in 50 mmol sodium acetate buffer (pH5.0) to make 1% cellobiose, cellotriose, and cellotetraose substrates. Onemilliliter substrate was transferred and kept it at 30 ℃ for 12 h. The hydrolyzed product was detected with silica thin plate chromatography. The extender was made by mixing of n-butanol, ethyl acetate, ammonia, and water with a ratio of 6:3:3:1 (v/v). Color developer A was made by mixing 1 g aniline with 25 mL acetone, and developer B was made by mixing 1 mL diphenylamine with 25 mL acetone. Developer A and B were mixed, followed by adding 5 mL 85% phosphoric acid and mixed well. The plate was dried off after chromatography was finished, and the developer was sprayed. The plate was dried at 120 °C for 10 min to develop the color (Jo et al. 2003).

Purification and identification of endoglucanase produced by solid-state fermentation of A. oryzae HML366
The endoglucanase activity and protein amount were increased greatly when the saturation of ammonium sulfate was between 50 and 80%, and the maximum value appeared when the saturation was 80%. The proteins were precipitated under 80% of the ammonium sulfate saturation, and redissolved in 0.1 mol/L citric acid-sodium citrate buffer solution (pH 4.8). Sephadex G-25 gel column was used to quickly desalt, and the recovered enzyme product was stored at 4 °C for further purification.

Linear elution analysis of enzyme purification
Tris-HCl elution buffer that contains 1 mol/L NaCl was used for elution at a flow rate of 1 mL/min. Enzyme was separated and purified with a continuous salt gradient from 0 to 0.5 of NaCl. It was found that the purified HML ED1 was mainly located in the No. 60 collection tube corresponding to peak 1. It was shown that 8% of elution buffer can purify endoglucanase very well (Fig. 1).
The enzyme solution in the No. 60 collection tube was concentrated and subjected to SDS-PAGE electrophoresis, and the molecular weight standard was Prestained Color Protein Molecular Weight Marker P0071 (Beyotime Biotechnology, China). HML ED1 protein was shown as a single band on the gel with the molecular weight of 68 kDa (Fig. 2).
After a two-step purification, the endoglucanase HML ED1 from A. oryzae HML366 was isolated (Figs. 2 and 3), and the purification yields were 17.6% and the purification folds were 4.8 (Table 1).

Purified HML ED1 rapid identification plate
Sodium carboxymethyl cellulose is a specific substrate for endoglucanase, which can produce a transparent circle after being hydrolyzed (He et al. 2013). The transparent circle in Fig. 3 confirmed that the enzyme solution produced by solid fermentation of A. oryzae HML366 had endoglucanase activity. After active staining with 0.2% Congo red stain solution and decolorizing with 1 M NaCl, a clear band was Fig. 1 Protein purification map of anion exchange chromatography. 1, the peak of HML ED1 protein; A the ultraviolet absorption curve of protein; B the conductivity curve. C the elution buffer concentration (%). The abscissa showed the elution time, and the left ordinate was the protein UV absorbance (AU) at 280 nm and the elution buffer concentration (%), and the right ordinate was the conductance (mS/cm) shown, indicating that HML366 can produce an endoglucanase (Fig. 4, lane 1).

The influence of temperature and pH on the activity of purified endoglucanase HML ED1
The enzyme was reacted with sodium carboxymethyl cellulose substrate at temperatures ranging from 25 to 90 ℃, and the optimal reaction temperature of HML ED1 was determined by measuring the enzyme activity. It was shown that the optimal enzymatic reaction temperature of the enzyme was 60 ℃ (Fig. 5A). The purified enzyme showed an increased enzyme activity between 25 and 60 ℃, and the relative enzyme activity was 100% at 25 ℃ for 0.5 h and 30 ℃ for 1 h, and there was almost no loss of enzyme activity. The enzyme activity at 60 ℃ for 0.5 h and 1 h still remained 97% and 96.8%, respectively. The enzyme activity was gradually decreased when the enzyme solution was incubated at temperature ranging from 70 to 90 ℃. These data indicated that the enzyme activity was relatively stable between 25 and 70 °C (Fig. 5B).
The enzyme activity of HML ED1 was gradually increased at pH between 3.0 and 6.5, and the highest enzyme activity was shown at pH 6.5 (Fig. 5C), indicating that the enzyme was acid cellulase. The enzyme activity of HML ED1 was greatly influenced at pH between 3.0 and 4.5, and the relative enzyme activity was 62.14%, 70.08%, 86.54%, and 90.56% for pH value at 3, 3.5, 4, and 4.5, respectively. The relative enzyme activity remained between 95.46 and 100% at pH 5.0 to 6.5, indicating that the enzyme activity was relatively stable in this interval. The enzyme activity loss was gradually increased over pH 9.0, and the enzyme activity was 94.46% and 64.26% at pH 9.0 and 11.0, respectively (Fig. 5D). Thus, HML ED1 was stable in a pH value ranging from 4.5 to 9. These characteristics suggested that this isolated enzyme is suitable for industrial saccharification processes for bioethanol production and other applications.

Analysis of purified endoglucanase HML ED1 hydrolysate
The CMC Na, cellobiose, cellotriose, and cellotetraose hydrolysates were analyzed by TLC (Fig. 6). Four spots were found for CMC Na hydrolyses; they were glucose, cellobiose, cellotriose, and cellotetraose; cellotriose was hydrolyzed to obtain cellobiose; cellotetraose was hydrolyzed to obtain cellobiose and cellotriose. Cellotriose and cellotetraose cannot be hydrolyzed to produce glucose. No spots were found in cellobiose (Fig. 6). It was speculated that  the randomly cleavage of internal β-1,4-glycosidic bonds by HML ED1 mainly produced cellobiose and cellotriose, but not glucose. These results indicated that HML ED1 has endoglucanase activity.

Enzymatic reaction kinetics
Based on Lineweaver-Burk plot, it was shown that Km and Vmax were 8.75 mg/mL and 60.24 μmol/min·mg, respectively ( Table 2).

The influence of metal ions on enzyme activity
Metal ions are often used as activators or inhibitors in the catalytic reaction of enzymes. Therefore, adding appropriate metal ions to the enzyme reaction system can improve the catalytic efficiency of the enzyme. When the concentration of metal ions exceeds 1 mM, the platform period will be reached, and the activity will be inhibited when the concentration rises again (Bano et al. 2019). At 1 mM ion concentration, Ag 2+ , Co 2+ , Cu 2+ , Zn 2+ , and Hg 2+ had a strong inhibitory effect on the purified endoglucanase HML ED1 of A. oryzae HML366, while Mn 2+ , Ca 2+ , and Mg 2+ had obvious activation effects on HML ED1, and Na + and K + had no significant effect (Table 3). The active site may contain sulfhydryl groups. These sulfhydryl groups participate in catalysis and are essential for maintaining the structure of the enzyme. The divalent cobalt ion forms a complex with a variety of amino acids, and the enzyme active site bound by the cobalt ion is irreversible, which completely inhibits the enzyme's activity. Other ions are the same as Mg 2+ , Mn 2+ , Ca 2+ , Na + , Cu 2+ , and Fe 3+ , and these metals also tend to form metal complexes with proteins, which ultimately affect enzyme activity by changing their structures (Patel and Shah 2021;Wang et al. 2020).

Discussion
Endoglucanases are produced by various filamentous fungi, such as Penicillium, Fusarium, Trichoderma, and Aspergillus (Hirasawa et al. 2019). Among them, Trichoderma is widely used as a cellulase producer, and Aspergillus has received more attention due to its powerful ability to secrete cellulase (Hirasawa et al. 2019;Liu et al. 2011;Tian et al. 2018).
The endoglucanase of Clostridium thermocellum has an optimal pH of 6.6 and an optimal temperature of 70 °C. It hydrolyzes carboxymethyl cellulose, cellodextrin, cellotetraose, and cellopentose at a higher rate, but does not hydrolyze crystalline cellulose (Fauth et al. 1991).
Koga et al. extracted the endoglucanase STCE1 from Staphylotrichum coccosporum NBRC 31817. STCE1 has an optimum temperature of 60 ℃ and is highly resistant to anionic surfactants and oxidants; thus, STCE1 is a universal enzyme used for laundry (Koga et al. 2008). Chaabouni et al. purified two endoglucanases EG A and EG B from Penicillium occitanis. The optimal temperature for the enzyme activity of EG A and EG B is 60 °C and   (Rawat et al. 2015). Heat-resistant enzymes have obvious advantages as catalysts. In these processes, high temperatures often promote enzymes to penetrate cell wall materials and destroy cellulose raw materials, resulting in better hydrolysis. Thermophilic fungi are now considered to be a promising source of enzymes. The thermostable cellulase used for cellulose degradation can increase the rate of hydrolysis and saccharification (Fontes et al. 1997;Lee et al. 2010;Li et al. 2006;Ghio et al. 2020;Saqib et al. 2010). Hg 2+ interacts with cysteine residues in the sulfhydryl bond, and can change the tertiary structure of the protein.
Because the active site may contain sulfhydryl groups, these sulfhydryl groups participate in catalysis and are essential for maintaining the structure of the enzyme. Since the divalent cobalt ion forms a complex with a variety of amino acids and the binding is irreversible, the enzymetic activity can be completely inhibited. Other ions are the same as Mg 2+ , Mn 2+ , Ca 2+ , Na + , Cu 2+ , and Fe 3+ , and these metals also tend to form metal complexes with proteins, which ultimately affect enzyme activity by changing their structures (Patel and Shah 2021;Wang et al. 2020).
Thermophilic fungi can produce heat-resistant enzymes. In the process of cellulose degradation, cellulose swells at a higher temperature and converts to a form that can be more easily broken down. The screening of thermophilic fungi and the application of heat-resistant enzymes are important directions for comprehensive applications of cellulose. These thermal enzymes have great application potential in the food, chemical, pharmaceutical industry, and environmental biotechnology (Araújo et al. 2021).
After hydrolysis of cellulose to produce glucose, it can be further fermentated to produce ethanol. The thermostable endoglucanase can improve the hydrolysis of cellulose and promote high-efficiency saccharification, generating more glucose than previous reports. Thus, thermostable endoglucanase can enhance the hydrolysis efficiency and catalyze the conversion of cellulosic biomass into fermentable sugars; thus, it can be used in the production of cellulosic ethanol. In addition, the thermostable endoglucanase may also be used as a robust hydrolase that can be integrated into the industrial fermentation process (Fauth et al. 1991;Koga et al. 2008;Lee et al. 2010;Tian et al. 2018).
Recent studies have shown that the addition of purified endoglucanases to commercial cellulases can cause stimulating effects on the hydrolysis of lignocellulosic biomass (Sujit et al. 2014).
A. oryzae HML366 is a cellulase-producing strain newly screened by our group from the original forest sampling. 68 kDa endoglucanase HML ED1 was isolated by the two-step rapid purification method. Javed et al. isolated a 25 kDa endoglucanase (Javed et al. 2009), and Kitamoto et al. purified and obtained 31 kDa and 53 kDa endoglucanases (Kitamoto et al. 1996), but 68 kDa endoglucanase has not been reported so far. We showed that the enzyme activity was stable below 70 ℃, and it was also stable at pH ranging from 4.5 to 9.0. Our analysis indicated that Km and Vmax of the enzyme was 8.75 mg/mL and 60.24 μmol/ min·mg, respectively. This endoglucanase has many useful features, including a wide range of pH and thermal stability. These characteristics make the enzyme very suitable for hydrolysis involved in saccharification processes, including the production of bioethanol, fabrics, food, and animal feed. In this study, we report for the first time that A. oryzae HML366 can produce heat-resistant and wide pH tolerance endoglucanase HML ED1, which has potential industrial value in bioethanol, papermaking, feed, food, textile, detergent, and pharmaceutical industries.
Funding This study was supported by the National Natural Science Foundation of Guangxi (2020GXNSFAA297218) and the National Natural Science Foundation of China (No. 31660017).
Data availability All data generated or analyzed during this study are included in this published article.

Declarations
Ethics approval and consent to participate This work did not involve the direct study of humans. All applicable international, national, and/ or institutional guidelines for the care and use of animals were followed and all. This article does not contain any studies with human participants or animals performed by any of the authors.

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The authors confirm that the work described has not been published before, that it is not under consideration for publication elsewhere, that its publication has been approved by all co-authors. The authors agree to publication in the International Microbiology Express.