# Safe production of Aspergillus terreus xylanase from Ricinus communis: gene identification, molecular docking, characterization, production of xylooligosaccharides, and its biological activities – Journal of Genetic Engineering and Biotechnology

Aug 12, 2022

### Molecular identification of xylanase producing enzyme

Aspergillus isolates were identified as A. terreus strain RGS.Eg-NRC after isolation and sequencing of the Internal transcribed spacer (ITS) region, which is 600 bp length (results are shown in Fig. 1). Then, it was deposited under accession no. MW282328. Partial nucleotide sequencing of the ITS region showed 97% identity with existing species in the GenBank database under the genus Aspergillus. However, from the blast results, the strain of RGS.Eg-NRC was closely related to only A. terreus. Subsequently, a phylogenetic tree was constructed using the isolates’ partial ITS region sequences. A phylogenetic tree includes representative strains of related species obtained using the neighbor-joining method. From the phylogenetic tree (Fig. 2), its result showed that RGS.Eg-NRC was closely related to A. terreus ATCC 1012ITS with 97% similarity.

### Cytotoxic activity tests

Cytotoxic activity tests (in vitro bioassay using human normal cell lines) were conducted to examine the safety of xylanase produced from Ricinus communis and establish its biological activities (antioxidant and antitumor). The samples were examined against the normal human epithelial cell line BJ1 (normal skin fibroblast), and the concentration range of the sample (unfermented Ricinus communis) ranged between 100 and 0.78 μg/ml. Results showed that while its LC50 was 50.9 μg/ml, its LC90 was 76.7μg/ml. Moreover, fermented Ricinus communis did not record any cytotoxic activity (Table 1), which means it is very safe.

### Optimization of xylanase production

Subsequently, the production of A. terreus xylanase was optimized using two steps:

#### One variable at a time

Different factors were used to study their effect on xylanase production from A. terrus. First, eight different agricultural wastes (garlic peels, onion peels, palm kernel wastes, pomegranate peels, potato peels, Ricinus communis, sesame waste, and wheat brane) were collected for xylanase production, after which A. terrus was grown using these agricultural wastes. Of all of these substrates, while Ricinus communis gave the highest xylanase yield (15.36 IU/g), garlic peels gave the lowest activity (0.5 U/g) (Fig. 3a). Subsequently, the time course of xylanase production by the SSF was studied. The results in Fig. 3b show that xylanase was maximally produced after 7 days of fermentation (yield of 20.23 U/g). Furthermore, results showed that substituting tap water with four different mineral salt solutions as moisturizing agents improved xylanase activity to 35.22 U/g d, and this result was obtained from MA II (Fig. 3c). Investigations also revealed that the best productivity was observed by replacing the constituent mixture of nitrogen sources with an equal value of each organic and inorganic nitrogen source constituent. Here, we observed that the productivity further increased to 41.66 U/g ds (Fig. 3d). Additionally, using varying corn concentration steps (0.2, 0.4, 0.8, 1, 1.4, and 1.6%), the productivity increased to 55.95 U/g ds (data not shown) as the concentration increased to reach (1.4%). However, after supplementing with 4.5% (w/v) of different sugar additives, significant effects on xylanase activity were further observed, with an even higher yield of 66.88 U/g ds of Glucose (data not shown).

### Statistical optimization of xylanase production

Many researchers believe that since a statistical optimization model for the fermentation process can overcome the limits of old empirical methods, it is more important for optimizing xylanase output. The statistical approaches adopted for xylanase optimization are as follows:

#### The Plackett–Burman design

PBD was used to investigate the relative interaction and the variables of different parameters for culture processing. Eleven trials for seven variables (Table 2) were used to first clarify the wide variation in the production of xylanase from 66.88 to 116.95 U/g. Results showed the great influence of the different factors in the fermentation process, with the highest value of the xylanase (116.95 U/g) being produced in trial 1, comprising the following: Ricinus communis waste (3 g/flask), corn steep (1%), KH2PO4 (6.5 g/l), glucose (4%), moistening agent (9 ml), inoculum (3 ml), and an incubation period of 9 days. Contrastively, the lowest value of xylanase (24.79 U/g) was produced in trial 6, comprising Ricinus communis waste (7 gm), corn steep (1%), KH2PO4 (8.5 gm/l), glucose (4%), moistening agent (7 ml), inoculum (3 ml), and an incubation period of fi5ve days.

The main effects of the investigated parameters on xylanase production are estimated and visually depicted in Fig. 4. Investigations revealed that while the examined factors, carbon, KH2PO4, and glucose, had negative effects, the nitrogen source, time, inoculum, and moistening agent (moisture) had positive effects. The confidence level, P-effect, and t-test of the statistical analysis of the PBD are indicated in Table 3. Therefore, since the P-value for the variables, carbon, time, and the moistening agent showed a high significant level, they were selected for further optimization.

The equation below shows a first-order model that describes the relationship between the seven components and xylanase activity:

$${Y}_{mathrm{activity}}=119.5084119-14.58268826 {mathrm{X}}_1+10.14870896 {mathrm{X}}_2-2.63264 {mathrm{X}}_3+3.521503 {mathrm{X}}_4+5.949054 {mathrm{X}}_5+3.718916414 {mathrm{X}}_6-22.14766313 {mathrm{X}}_7$$

#### Central composite design

The most effective factors’ optimal amounts (carbon, moistening agent, and time) arising from PBD were further analyzed by applying RSM, involving CCD in 20 trials (Table 4). The culture medium contained Ricinus communis waste (3 g/flask), corn steep (1%), KH2PO4 (6.5 g/l), glucose (4%), moistening agent (9 ml), and inoculum 3 ml. Experiments were conducted for an incubation period of 9 days as the central point of the CCD3.

Table 4 lists the independent variables, their coded matrices, and replies, including the experimental and projected values for xylanase activity. We observed a variation in enzyme yield over time, ranging from 5 to 245 IU/g during the 20 runs of the experiments. Notably, the highest level of the produced xylanase was 245 IU/g obtained in run 9, which indicated that the optimal levels of the tested variable were as follows: Ricinus communis waste (1 g/flask), moistening agent (3 ml), and incubation period (11 days).

Furthermore, the model’s accuracy’s coefficient (R2) was 0.9862, indicating that the independent variables accounted for 98.62% of the response variability. As a result, the current R2 value confirmed the validity of the existing model for xylanase production, including a good connection between the experimental and theoretical values (Table 4).

The equation below was used to analyze the association between variables and response using a second-order polynomial equation:

$${Y}_{mathrm{Activity}}=243.0467-121.621 {mathrm{X}}_1+13.42214 {mathrm{X}}_2+6.255342 {mathrm{X}}_3+14.60489 {{mathrm{X}}_1}^2+0.052727 {{mathrm{X}}_2}^2-0.07528 {{mathrm{X}}_3}^2-4.42794 {mathrm{X}}_1{mathrm{X}}_2+1.749367 {mathrm{X}}_1{mathrm{X}}_3-0.18782 {mathrm{X}}_2{mathrm{X}}_3$$

where Y represents the response and xylanase yields were represented using X1, X2, and X3.

Subsequently, the P-value was employed to determine each coefficient’s magnitude, which revealed the pattern of the factors’ interactions. The statistical analysis of data (Table 5) indicated a high significant effect on xylanase production and smaller P-values (P < 0.05). Furthermore, the residual analysis (Fig. 5), which involved plotting the observed-predicted values (residuals) vs. the response (optimization process), revealed that the residuals formed a symmetrical pattern and were evenly distributed across the range, indicating that the average model was correct for all observed results.

### Validation of the model

The proposed model’s validity was evaluated by predicting A. terreus xylanase production for each trial of the matrix. The experimental results in Table 4 show that the maximally observed xylanase production (245 U/gds) was very close to the predicted value (236 U/gds). Investigations also revealed that the optimum xylanase production reached 245 U/gds (12.1-fold) by applying OVAT, followed by statistical optimization for the SSF of Ricinus communis waste.

### Biochemical characterization of A. terreus strain RGS.Eg-NRC xylanase

#### Optimal pH and stability

According to the data in Fig. 6, the optimum pH of the A. terreus xylanase was six. Figure 7 shows the pH stability of the enzyme under assay. Investigations revealed that while the enzyme was almost stable at pH 6 for 2 h, it was extremely stable at a pH range of 5 to 6. Following a 2-h incubation at this pH range, the enzyme lost 12.77% of its activity.

### Influence of temperature on the activity of enzymes

The temperature dependency of A. terreus xylanase activity at temperatures ranging from 40 to 60 °C and at pH 6 is shown in Fig. 8. We observed that the activity of xylanase was stable at 60 °C, indicating that its optimum temperature was 55 °C. Therefore, a high temperature is recommended from a biotechnological standpoint because it improves conversion rates, reduces microbial contamination, and allows for increased substrate solubility.

#### Thermal stability

The Arrhenius equation is useful for calculating the activation energy and the efficiency of chemical reactions. When a reaction obeys the Arrhenius’ equation, a linear relationship is shown after graphing the log of residual activity against time, displaying a first-order kinetic reaction of the enzyme, whose gradient and intercept can then be used to calculate the activation energy (Ea). Xylanase formed a similar graph, which was consistent with this equation.

Subsequently, Arrhenius plots were used to calculate the catalysis’s activation energy (Ea) for the A. terreus xylanase (Fig. 9). For the Arrhenius plots of xylanase, the regression equation was y = −2.8772 + 10.901.

The results showed that the Ea of xylanase from A. terreus was 23.919 kJmol. The lower the value of Ea, the less energy it takes to conform to the enzyme-substrate complex’s active site. Therefore, because of these characteristics, and since it requires a low activation energy value, which will impact the total cost of industrial processing, A. terreus xylanase was considered more suitable for industrial applications.

Additionally, Fig. 10 reveals that while the enzyme did not lose any activity after 120 min of incubation at 40 °C and 45 °C, 85.6% activity was preserved after 120 min of incubation at 50 °C. However, it lost roughly 13.5% of its activity after 120 min incubation at 60 °C.

### Hydrolysis property (production of xylooligosaccharides)

Subsequently, the hydrolysis period of beech wood xylan by A. terreus xylanase was studied. The amount of reducing sugars released is shown in Fig. 11. Results showed that while the highest reducing sugar (21.08 mg/ml) was achieved after 28 h, other oligosaccharides of various sizes were detected by thin-layer chromatography (Fig. 12). Accordingly, FTIR was obtained from the hydrolysis of xylan. The applicability of the FTIR spectroscopy spectra to identify different functional groups in oligosaccharide samples has been proven. Hence, Fig. 13 shows the FTIR spectra of freeze-dried XOS products. The results showed that while the wideband near 3274 cm1 corresponded to the hydrogen-bonded OH group than water, all main carbohydrate components, the band detected at 2921 cm1 was attributed to the stretching vibrations of C–H bonds (CH2 symmetric stretching) [38]. Results also showed that the signals in 1417, 1382, 1246, and 1212 cm−1 were related to acetyl groups. Therefore, these bands were allocated to the single-bonded oxygen (C–O) stretching and symmetric CH3 bending vibrations. Notably, the region between 1150 and 920 was where xylan’s fingerprint was located. The C–O and C–C stretching and/or CO–H bending, including the glycosidic linkage (C–OC) contributions, was attributed to the region between 1150 and 920 cm1 in the xylan fingerprint. However, the greatest band at 1034 cm1 was assigned to the C–O and C–C stretching and/or CO–H bending and glycosidic linkage (C–OC) contributions [39], suggesting the preponderance of xylan oligosaccharides. Finally, the dominant glycosidic connections between xylose units in hemicelluloses were linked to the band at 897 cm1.

### Biological activity of xylooligosaccharides

#### Antioxidant capacity

Additionally, our study evaluated the antioxidant capacity of different XOS concentrations (0.5, 0.75, 1.00, 1.25, 1.50, and 2.00 mg/ml) using different assays (DPPH, reducing power ability, ABTS, and FRAP). BHT was used as a standard antioxidant agent. Results illustrated in Table 6 show that all investigated assays were similarly dose dependent, increasing gradually with increasing concentration. DPPH radical scavenging activity (%) ranged from 17.37 ± 0.14 to 82.79 ± 014% at concentrations 0.5 and 2.00 mg/ml, respectively. However, reducing power ability was evaluated by reading the absorbance at 700 nm, and results varied from 0.423 ± 0.013 to 1.024 ± 0.012 for the lowest (0.5 mg/ml) and the highest (2.00 mg/ml) concentrations. After the ferric reducing power capability investigations, results showed that the highest concentration of XOS (2.00 mg/ml) exhibited 1453 ± 19.67 μm Trolox/100g and gradually decreased to 346 ± 15.52 μm Trolox/100g using the lowest concentration of XOS (0.5 mg/ml). Moreover, the ABTS radical scavenging activity followed the same trend and increased gradually from 29.77 ± 0.29 to 80.57 ± 0.19% by increasing XOS concentrations from 0.5 to 2.00 mg/ml.

#### In vitro antitumor activity

Figure 14 indicates that although different concentrations of XOS (1.00, 2.00, 3.00, and 4.00 mg/ml) exhibited a weak to moderate effect on the viability of Ehrlich ascites carcinoma cells, the activity of the cells was increased gradually by increasing the concentration of XOS. We also observed that while the dead cells (%) varied from 14.1 ± 0.79 to 35.61 ± 0.35 at 1 and 4 mg/ml, respectively, the standard antitumor drug (vincristine) recorded 90.65 ± 0.33% dead cells. Additionally, increasing the concentration of XOS to more than 4 mg/ml did not increase the dead cells of Ehrlich ascites carcinoma cells anymore. Based on these results, we surmise that the antitumor potential of XOS may likely be attributable to the efficient release of phenolic compounds, including its scavenging activity, which could alleviate the harmful effects of reactive oxygen species.

### Xylanase partial purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Ethanol, acetone, and ammonium sulfate fractional precipitation achieved the crude enzyme partial preparation. A 60–70% ammonium sulfate fraction recovered 60.88%-specific activity with 3.9 U/mg protein. Thus, enzyme purification increased the activity by 31 folds. For zymogram analysis, the native PAGE was used. A clear zone on the agar gel revealed the presence of xylanase and helped localize the xylanase when the native PAGE gel was overlayered on the agar gel seeded with its substrate (xylan) (Fig. 15). During the enzyme preparation, zymogram analysis also indicated the existence of a single xylanase. Subsequently, the enzyme’s molecular weight was determined using SDS-PAGE. Partial purified enzymes had just one band, corresponding to a molecular weight of ~36 kDa (Fig. 15).

### Isolation and molecular characterization of the xynC gene from A. terreus RGS.Eg-NRC

The designed primers for the xynC gene were successfully amplified and confirmed to be 981 bp, as expected for the molecular weight of the total length of mRNA encoding the xylanase, using cDNA as a template (Fig. 16). The assembled sequence was subsequently subjected to a BLAST search on NCBI against the available sequences deposited in the NCBI database. BLAST analysis revealed an open reading frame (ORF) comprising 981 bp in the full-length xylanase gene (xynC) that encodes a protein consisting of 326 amino acids. Therefore, the gene sequence for the xynC gene was submitted to GenBank (accession number: LC595779.1).

Next, the nucleotide sequence of xlnC was translated to its deduced amino acids (understudied protein) and then aligned against six other retrieved xylanases from the reviewed protein database, UniProt (Fig. 17). Xylanase C (understudied protein) had a 100% similarity with the A. terreus QOCMB8 strain. Furthermore, amino acid residues ranging from 11 to 326 were reported by the InterProScan server (EMBL) to belong to the glycosyl hydrolase family 10 (GH10). Subsequently, signal peptides and their cleavage sites were checked by the SignalP-6.0 server, and the results included residues ranging from 1 to 19 (Fig. 18). Therefore, we searched for O-glycosylation and N-glycosylation sites using the NetNGlyc 4.0 and GlycoEP server, but did not find any potential O-glycosylation and N-glycosylation sites. Amino acid composition and physicochemical properties are illustrated in Fig. 19. Investigations revealed the active site of XynC at two positions (Glu 156 and Glu 262), where E156 was the general acid/base residue and E262 was the catalytic nucleophile. Finally, while the theoretical molecular weight of a xyl protein was calculated as 35.3 kDa, the isoelectric pH was 8.16 (Fig. 19).

Subsequently, phylogenetic analysis was conducted to place xyl among the known xylanase family members (Fig. 20). All 21 curated xylanase proteins retrieved from the reviewed UniProt protein database were related to various organisms and were selected from the database for the phylogenetic analysis. The dataset included xylanase proteins from fungi. Final comparative analyses indicated that xylanase exhibited 91% sequence similarity to homologous fungal xylanase proteins (e.g., Aspergillus sp.)

### Secondary structure prediction of xylanase

The deduced amino acid sequence of XynC (protein ID: BCO16052.1) was aligned against the PDB using BLASTp to conduct a sequence homology search and comparative modeling. Significant sequence similarities were 78% with Aspergillus niger xylanase (PDB ID 4XUY) and 73% with Penicillium simplicissimum DSM 17393 (PDB ID 1B30). The sequence alignment of xylanase and templates in the ESPript server and secondary structure prediction of the xylanase protein are presented (Fig. 21). According to the characterization of the xylanase model by the DSSP program, the secondary structure of xylanase is composed of 13 ????-helixes and 9 ????-sheets.

### Homology modeling and validation of xylanase

Since sequence similarities were higher with Aspergillus niger, X-ray structural coordinate files of xylanase from this strain (PDB 4XUYA, identity 78%) were used to conduct homology modeling and build the 3D structure of xylanase produced from the A. terreus strain RGS.Eg-NRC. Then, best hit (E-value 0) crystal structures of the xylanase protein were used as templates to construct 3D models of xylanase based on sequence similarity, residues completeness, crystal resolution, and functional similarities. Based on the DOPE score, top-scoring models (Fig. 22) were selected for energy minimization and validation studies out of the 300 predicted xylanase models. Subsequently, the selected model was subjected to energy minimization using YASARA Server force fields of the Swiss-PdbViewer, after which the generated model was assessed using general stereochemical parameters by PROCHECK, VERIFY3D, and ERRAT of the SAVES server. As a result, the Ramachandran plot of the energy-minimized model of xylanase structures was also generated. The x-axis of the Ramachandran plot is split into four quadrants, which include the low-energy region, the allowed region, the generally allowed in region, and the disallowed region (Fig. 23). PROCHECK analysis of the 3D modeled xylanase protein revealed that while 90.2% of residues fell in the most favored regions of the Ramachandran plot, 8.3% fell in the additional allowed regions, 1.5% in the generously allowed regions, and no residue was in the disallowed regions, indicating that the generated model was of good quality. Similarly, template 4XUY of A. niger had corresponding values, with 91.3% falling in the most preferred regions, 8.7% in additionally allowed regions, and no residues in the generously allowed and disallowed regions of the Ramachandran plot. Furthermore, using VERIFY3D and ERRAT at the SAVES server, the overall quality factor and compatibility of the atomic model (3D) with amino acid sequence (3D-1D) for the model were observed as 83.02% and 78.94. Thus, the Ramachandran plot, ERRAT, VERIFY3D, and PROSA results confirm that the generated model was reliable and of good quality.

### Docking and molecular interaction studies with the three-dimensional model of xylanase

Figure 24 summarizes the docking results of xylan with the 3D xylanase model. The results showed an interaction affinity with a score of −8.7 kcal/mol that formed 10 hydrogen bonds with Tyr298, Ser299, Tyr199, Glu71, Asn72, Lys75, Asp77, Trp292, and Glu262. Furthermore, while we observed three nonhydrogen bond interactions within the activity pocket, one Pi-sigma bond (with residue Tyr199) and only one carbon-H (with Glu156) were also formed.

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