Screening of the Xylanase-producing Trichoderma Strain and the Optimization of its Enzyme Production Conditions

doi:10.21203/rs.3.rs-7863122/v1

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Screening of the Xylanase-producing Trichoderma Strain and the Optimization of its Enzyme Production Conditions

https://doi.org/10.21203/rs.3.rs-7863122/v1

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Purpose

Xylanases have attracted considerable attention due to their excellent potential industrial uses.

Results

In this study, a xylanase producing strain was isolated from soil and identified as Trichoderma semiorbis Tsejk8, and the conditions for xylanase production were optimized. Additionally, two xylanase-related genes were cloned, and their functions were analyzed. The results indicated that the optimal conditions for xylanase production included maltose as the carbon source, peptone as the nitrogen source, an optimal pH of 6.0, and an incubation time of 120 h, yielding an enzyme activity of 40.7 U/mL. Following the purification of the protein via ammonium sulfate precipitation and ion exchange chromatography, four distinct protein bands were observed. Mass spectrometry analysis of these bands identified 14 associated proteins. Bioinformatics analysis revealed that two of these proteins belongs to GH3 (Glycoside Hydrolase family 3) beta- xylosidase.

Conclusions

In summary, the newly isolated strain Tasjk8 exhibits xylanase activity, which offers an effective and eco-friendly means of converting biomass into raw materials for industrial applications.

Trichoderma semiorbis

Xylanase

Single-factor optimization

Protein sequencing

Xylan is a complex polysaccharide predominantly found in the cell walls of plants, constituting a major component of hemicellulose. Hemicellulose is an abundant natural resource, contributing approximately 35% to the dry weight of plants, and is extensively present in agricultural by-products, with its abundance in nature being second only to cellulose (Bhardwaj et al 2019). Xylanase is a complex ensemble of enzymes primarily responsible for the breakdown of xylan; it is not a single enzyme but a combination of various components, including endo-xylanases, β-xylosidases, and branching enzymes (such as α-glucuronidase, α-L-arabinofuranosidase, feruloyl esterase, and acetylxylan esterase), that collaboratively work to effectively decompose xylan (Chaudhary et al 2023). By employing enzymatic methods, xylan can be efficiently degraded into xylooligosaccharides and xylose, thereby enhancing the development and utilization of xylan.

Numerous organisms are known to produce xylanase. Recent years have seen the documentation of various bacteria, fungi, plants, and animals capable of xylanase production both domestically and internationally (Abena and Simachew 2024). The xylanase produced by fungi exhibits high activity and is primarily extracellular (Paloheimo et al 2007), facilitating easier isolation and purification. Notable xylanase-producing fungi include Aspergillus spp., Penicillium spp., and Trichoderma spp. (Haltrich et al 1996). Trichoderma are filamentous fungi capable of synthesizing a variety of enzymes, with xylanase being one of the most significant (He et al 2009). Compared to other organisms, Trichoderma exhibit a faster reproduction rate and enhanced capabilities to absorb and utilize soil nutrients, playing an integral role in soil bioremediation and promoting crop biomass, as well as providing biological control against plant diseases and pests (Druzhinina et al 2011). The application of highly active xylanase produced by Trichoderma can effectively diminish chemical pollutants in the papermaking process.

This study employs soil samples from Nanchang City, Jiangxi Province, to isolate and purify fungi, specifically targeting strains capable of xylanase production. The research focuses on optimizing the conditions for enzyme production and obtaining purified xylanase through ammonium sulfate precipitation, dialysis, and ion-exchange chromatography. Additionally, the study performs protein sequencing and gene analysis to gain deeper insights into the enzymatic characteristics. Ultimately, this research aims to provide high-quality microbial resources for future investigations, and the findings are expected to have significant practical applications.

Culture media

PDA medium (g/L) contained peeled potatoes 200, glucose 20, and agar 15.

Xylanase fermentation basic medium (g/L) contained xylan 5, NaNO₃ 3, K₂HPO₄ 1, MgSO₄·7H₂O 0.5, KCl 0.5, and FeSO₄·7H₂O 0.01, at pH 7.0.

Screening medium (g/L) contained xylan 5, K₂HPO₄ 1, MgSO₄·7H₂O 0.5, KCl 0.5, FeSO₄·7H₂O 0.01, and yeast 2, at pH 7.0.

SNA (synthetic nutrient-poor agar) (g/L) contained KH₂PO₄ 1.0, KCl 0.5, KNO₃ 1.0, MgSO₄·7H₂O 0.5, glucose 0.2, sucrose 0.2, agar powder 14.

Identification of Strain

The strain was transferred to PDA and SNA plates and colony diameters were measured every 12 h at five different points to generate a growth curve. Once conidia developed, morphological characteristics of conidiophores and conidia were observed under an optical microscope.

Genomic DNA from the target strain was extracted using the CTAB method. Primers were designed to amplify translation elongation factor 1-alpha (tef1) (Zhu and Zhuang 2015), as follows:

tef1F: CATCGAGAAGTTCGAGAAGG,

tef1R: AACTTGCAGGCAATGTGG

PCR amplification was conducted, and the products were analyzed using 0.1% agarose gel electrophoresis. Following purification, the samples were submitted to Shanghai Bio-engineering Company for sequencing. A phylogenetic tree was constructed using the neighbor-joining (NJ) method by MEGA7 software (Kumar et al 2016).

Screening of Xylanase-producing Strains

The strain was inoculated onto potato dextrose agar (PDA) plates and incubated at 28°C for 36 hours in an inverted position. Following this, agar discs, measuring 0.5 cm in diameter, were obtained using a punch, and three discs were transferred to a 250 ml Erlenmeyer flask containing 50 mL of culture medium. The flask was then incubated at 28°C with shaking at 200 rpm for 5 days. Subsequently, the culture was vacuum filtered to collect the broth, which was further filtered through a 0.22 µm-micron filter and stored at -20°C for future enzyme activity assays.

Optimization of Xylanase Production Conditions

Five distinct carbon sources—1% glucose, maltose, xylan, sucrose, and soluble starch—were assessed for their ability to support xylanase production. Fermentation was carried out at 28°C and 200 rpm for 5 days. Subsequently, five nitrogen sources—0.5% potassium nitrate, sodium nitrate, ammonium sulfate, tryptone, and yeast extract—were evaluated under identical fermentation conditions to determine the optimal nitrogen source. Using the optimal carbon and nitrogen sources, the initial pH of the medium was adjusted to 4.0, 5.0, 6.0, 7.0, and 8.0 to identify the best pH for enzyme production. Finally, the optimized conditions (carbon source, nitrogen source, and pH) were applied to evaluate the effect of fermentation time by conducting experiments over 2, 3, 4, 5, and 6 days. All experiments were performed in triplicate.

Determination of Xylanase Activity (DNS assay)

The culture solution was prepared and centrifuged at 4,000 rpm for 15 min at 4°C to collect the supernatant. This supernatant was then filtered through a 0.22 µm filter to obtain the crude enzyme solution. 1 mL crude enzyme solution was mixed with 1 mL of 0.1% xylan solution at room temperature. The mixture was incubated at 37°C for 30 min to allow the xylanase to act on the xylan. The reaction was terminated by boiling for 5 min, after which the mixture was rapidly cooled in cold water for 5 min, followed by the addition of 3 mL of DNS solution and thorough mixing. The mixture was placed in a boiling water bath for 5 min to sustain the reaction for the determination of reducing sugars, after which it was cooled in cold water for 5 min. The absorbance of the prepared control and experimental samples was measured at a wavelength (λ) of 540 nm. The amount of enzyme required to catalyze the production of 1 µg of reducing sugars per min is defined as one unit of enzyme activity (U). The calculation is shown as follow:

U = W×N×1000∕(T×V)

where U is the xylanase activity per mL, W is the glucose concentration obtained from the control glucose standard curve, N is the reaction volume in mL, T is the reaction time in min, 1000 converts mg to µg, and V is the volume of the crude enzyme solution in mL.

Xylanase Purification

Ammonium sulfate was progressively added to the crude enzyme solution while stirring at low speed at 4°C until a final concentration of 60% was reached, and stand at 4°C for 1 h. The mixture was then centrifuged at 8,000 rpm for 30 min at 4°C, discarding the supernatant and retaining the precipitate. The precipitate was dissolved in 0.2 mol/L phosphate buffer (pH 7.0). The resulting solution was transferred into a dialysis bag with a molecular weight cutoff of 3,500 Da, which was placed in a container filled with xylan buffer for dialysis at 4°C, with the buffer being replaced every 6 h for a total of six exchanges, until all ammonium sulfate was removed. The treated DEAE-cellulose was packed into a glass adsorption column, and an equal volume of 0.2 mol/L phosphate buffer (pH 7.0) was added to wash the column, designated as the loading solution. Subsequently, 2 mL of enzyme solution was added and allowed to adsorb for 30 min, followed by washing with 5 mL of buffer, designated as the post-loading liquid. Elution was performed using 10 mL of buffer containing 0.5 mol/L NaCl, with the eluted liquid collected. All solutions were stored at 4°C. The protein content was measured using the Coomassie Brilliant Blue method, and enzyme proteins were identified through SDS-PAGE analysis. After SDS-PAGE, the target band was excised and sequence by Sangon Biotech (Shanghai).

Analysis of the Enzymatic Properties of Xylanase

The purified enzyme was incubated at 20, 30, 40, 50, 60, 70, and 80°C for 30 min, and the remaining xylanase activity was measured under optimal reaction conditions to analyze the thermal stability of the enzyme. Various pH values (4.0, 5.0, 6.0, 7.0, and 8.0) were also tested to evaluate its acid-base stability.

Phylogenetic tree analysis

The phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 7.0 software, employing bootstrap analysis with 1000 repetitions. The full-length amino acid sequences of the selected genes were used to construct this tree.

Statistical Analysis

All data were analyzed using a one-way analysis of variance (ANOVA) in the R programming language (R version 4.3.1).

Strain Screening Results of High-Xylanase Producing Trichoderma Strains

A total of 117 Trichoderma strains were isolated and purified. The screening process was carried out using shake-flask fermentation, and the results are illustrated in Fig. 1. Among the tested strains, strain JK8 demonstrated the highest xylanase activity, thereby designating it as a high-xylanase-producing strain for subsequent experiments.

Identification of Xylanase-producing Strains

As illustrated in Fig. 2a, strain Tsejk8 exhibited rapid growth on PDA medium, characterized by initial white mycelium. After 4 days, it began spore production, and the aerial mycelium became well-developed. In contrast, Fig. 2b shows slower growth on SNA medium, with initial white mycelium and spore production commencing after 5 days, accompanied by less developed aerial mycelium.

Observations under a light microscope, depicted in Fig. 2c, revealed that the mycelium was white or light-colored, slender, septate, and highly branched. Conidiophores were organized into dense, hemispherical to cushion-like structures, typically displaying extensive branching at acute or nearly right angles, in whorls of 2 to 4, with conidial areas exhibiting various shades of green or gray. As indicated in Fig. 2d, the conidia were unicellular, spherical, and green. The strain showed rapid growth; as shown in Fig. 2e, it reached a diameter of 6.33 cm after 60 h of incubation at 28°C on PDA medium, while on SNA medium, it attained a diameter of 5.5 cm under the same conditions. The limited nutritional components of the SNA medium compared to PDA suggest that nutritional factors minimally impact the growth of this strain. Preliminary identification confirmed JK8 as Trichoderma semiorbis.

Phylogenetic analysis revealed that this strain shares the highest homology with Trichoderma semiorbis isolate HZA10 (OR046019.1), as depicted in Fig. 2f, where the strain is positioned in the same branch. Combining molecular biological and morphological identification, the strain was ultimately confirmed as Trichoderma semiorbis and designated as Tsejk8.

Figure 1 Screening of Trichoderma strains producing xylanase

Figure 2 Growth characteristics of strain Tsejk8. (a) Morphology on PDA medium. (b) Morphology of SNA medium. (c) Characteristics of conidiophores (scale: 10 µm). (d) Characteristics of conidia (scale of 10 µm). (e) Strain growth rate. (f) phylogenetic tree of Tef1 gene cloned from Tsejk8

The Optimal Culture Conditions for the Production of Xylanase Enzyme

In order to improve laccase production, the fermentation conditions including carbon source, nitrogen source, initial pH and incubation time were optimized (Fig. 3). Figure 3a illustrates that when xylan served as the carbon source in the culture medium, enzyme activity in the fermentation broth's supernatant was significantly elevated. In contrast, the use of sucrose, glucose, or maltose as carbon sources resulted in lower enzyme activity, suggesting that polysaccharides such as starch and xylan are more effective than monosaccharides or disaccharides in promoting xylanase production by the strain. Dhaver (Dhaver et al 2022) optimized the culture conditions and medium components for xylanase production by T. harzianum, discovering that the incorporation of xylan-rich materials, such as wheat bran, significantly improved xylanase yield.

Figure 3 Optimization of enzyme production conditions of Tsejk8. (a) Carbon sources. (b) Nitrogen sources. (c) Initial pH. (d) Fermentation time. Statistical analysis was conducted using ANOVA in R. Different letters indicate statistically significant differences (p < 0.05), whereas identical letters denote no significant difference.

With xylan established as the carbon source, potassium nitrate yielded the lowest enzyme activity in the fermentation broth's supernatant. Conversely, sodium nitrate, ammonium sulfate, peptone, and yeast extract all produced higher enzyme activity, with peptone yielding the highest enzyme activity in the fermentation broth (Fig. 3b). Organic nitrogen sources, such as tryptone, yeast extract, peptone, and soy meal, have a significant impact on the enhancement of xylanase production. Aspergillus sp. IN5 is reported to be highly productive in the presence of soybean residue (Boondaeng et al 2024), corn step liquor for T. reesi (Lappalainen et al 2000) and peptone for Pichia kudriavzevii. Although no significant differences in enzyme activity were observed among the four nitrogen sources—sodium nitrate, ammonium sulfate, peptone, and yeast extract. In consideration of prior research findings, we selected peptone as the optimal nitrogen source for strain Tsejk8.

Figure 3c illustrates that when xylan and peptone were employed as the carbon and nitrogen sources, respectively, pH values of 4 and 8 resulted in high enzyme activity in the fermentation broth. Raghukumar (Raghukumar et al 2004) also observed that the A. niger strain isolated from mangrove detritus exhibited maximum activity at pH 3.5, with an additional peak at pH 8.5. Additionally, pH 8 is more conducive to the growth of Trichoderma. therefore, it was selected as the optimal pH. Thus, a pH of 8 is concluded to be the optimal pH for xylanase production by strain Tsejk8.

Figure 3d indicates that when xylan was used as the carbon source, peptone as the nitrogen source, and a pH of 8 was maintained, a cultivation time of 120 h resulted in elevated enzyme activity in the fermentation broth. Although no significant differences in enzyme activity were observed among the 48h, 96h, 120h, and 144h time points, 120h was chosen as the optimal cultivation time based on previous research findings and the growth characteristics of Trichoderma. Following optimization, the enzyme activity of Tsejk8 reached 40.7 U/mL, indicating a 35.6% increase compared to the levels recorded before optimization.

Purification of Xylanase from Tsejk8

As presented in Table 1, the initial crude enzyme solution had a total volume of 80 mL, exhibiting a total activity 2,075.491 U/mg. Following the ammonium sulfate precipitation treatment, a clarified enzyme solution with a total volume of 8.2 mL was collected, at which point the total activity increased to 2,530.92 U/mg. Compared to the initial crude enzyme solution, the purification fold reached 1.2, and the enzyme activity recovery rate was 24.4%. Subsequently, after further purification using a DEAE-cellulose ion exchange column, the xylanase solution's total activity exhibited a remarkable increase to 25,625.82 U/mg. In comparison to the initial crude enzyme solution, the purification fold significantly increased to 12.3, and the enzyme activity recovery rate improved to 35.6%.

Table 1
Purification of Xylanase
Purification Step	Total Volume (mL)	Total Activity (U)	Total Protein (mg)	Specific Activity (U/g)	Recovery Rate (%)	Purification Fold
Crude Enzyme Solution	80 ± 4.50	1516.3 ± 107.45	0.73 ± 0.065	2.08 ± 0.041	100	1
Ammonium Sulfate Precipitation and Dialysis	8.2 ± 0.75	369.64 ± 39.65	0.15 ± 0.016	2.53 ± 0.012	24.4 ± 0.89	1.2 ± 0.029
Anion Exchange Chromatography	13 ± 0.60	539.40 ± 39.9	0.02 ± 0.004	25.63 ± 2.625	35.6 ± 2.11	12.3 ± 1.019

Table 1 Purification of Xylanase

Enzymatic Characterization

The fermentation broth of strain Tsejk8 was filtered using a 0.22 µm membrane to obtain a crude enzyme solution (corresponding to lane 1 in Fig. 4a), which initially removed large molecular impurities from the fermentation broth, establishing a foundation for the subsequent purification process. The crude enzyme solution was then processed through ammonium sulfate precipitation. This method leveraged the differences in protein solubility at varying salt concentrations to achieve preliminary enrichment of the target protein, with the resulting precipitate corresponding to lane 2 in Fig. 4a. The ammonium sulfate-precipitated sample then underwent further purification via DEAE-cellulose ion exchange chromatography. Utilizing the principle of ion exchange, the target xylanase was separated from other impurities based on the charge properties and quantities of the proteins. The treated sample post this step is presented in lane 3 of Fig. 4a. SDS-PAGE electrophoresis was performed on samples at each stage, indicate that as the purification process advances, the number of protein bands progressively decreases, ultimately revealing five principal bands with molecular weights ranging from 35 to 100 kDa.

Effect of Temperature and pH on Xylanase Activity

The enzyme activity was measured across six different temperatures. As shown in Fig. 4B, within the temperature range of 20 to 30°C, enzyme activity gradually increased with rising temperature. The xylanase activity for strain Tsejk8 peaked at 30°C after a 30-min reaction. Although elevated temperatures from 30 to 40°C also resulted in high enzyme activity after 30 min, the activity was lower compared to that observed at 30°C. Beyond 50°C, enzyme activity rapidly declined. Therefore, the optimal reaction temperature for strain Tsejk8 is approximately 30°C. Figure 4C illustrates that within a pH range of 3.0 to 4.0, enzyme activity increased with increasing pH, peaking at pH 4.0. However, in the pH range of 4.0 to 8.0, enzyme activity significantly decreased as pH continued to rise, suggesting that xylanase produced by strain Tsejk8 is an acidophilic enzyme, with an optimal pH of 4.0.

Figure 4 Properties of xylanase. Effect of temperature and pH on xylanase activity. (a) SDS-PAGE electropherogram. M, marker. Lane1, crude fermentation broth. Lane2, ammonium sulfate precipitation. Lane3, DEAE-cellulose ion-exchange chromatography. (b) Effect of temperature on xylanase activity. (c) Effect of pH on xylanase activity.

Analysis of Xylanase-related Proteins

Upon excising the protein bands and conducting sequencing, we identified 14 proteins with molecular weights ranging from 41.6 to 98.9 kDa. Domain analysis revealed that two of these proteins (Tsebxl01 and Tsebxl02) belong to the PLN03080 superfamily (Fig. 5). Clustering analysis indicated that Tsebxl01 has the highest homology with the BXL1 protein of T. guizhouense, while Tsebxl02 exhibits the highest homology with the BXL1 protein of T. simmonsii, both of which are classified within the xylanase family. Xylanolytic enzymes are classified as glycoside hydrolases (GH) based on homologies in structural elements and hydrophobic clusters, and they are organized into several families, namely 5, 7, 8, 9, 10, 11, 12, 16, 26, 30, 43, 44, 51, and 62. These enzymes are capable of hydrolyzing the β-1,4-glycosidic linkages in xylosides (Nguyen et al 2018). In this study, we cloned two β-xylosidases, Tasbxl01 and Tasbxl02, which belong to the GH3 family.

Table 2 Analysis of Proteins

Table 2
Analysis of Proteins
Accession	Sore Sequest	MW (kDa)	Calc.pI	Size of mRNA(bp)	Note
Tsejk801	2.49	61.9	6.23	1731	Isoamyl alcohol oxidase
Tsejk802	13.27	69	5.6	1989	Xyloglucanase
Tsejk803	2.35	77.2	7.23	2205	GH3 beta-glucosidase
Tsebxl01	35.1	86.7	5.55	2388	GH3 beta-xylosidase
Tsejk805	12.38	80.4	6.98	2283	Glucan endo-1,3-beta-glucosidase
Tsejk806	2.87	67.5	5.29	1902	GH15 glucamylase
Tsebxl02	10.24	81	5.45	2295	GH3 beta-xylosidase
Tsejk808	8.93	62.5	7.01	1731	Amidase
Tsejk809	82.71	83	7.4	2328	GH55 exo-1 3-beta-glucanase
Tsejk810	43.36	61.5	6.07	1728	GH71, alpha-1,3-glucanase
Tsejk811	24.46	53.9	5.47	1464	Glutaminase A
Tsejk812	2.41	41.6	5.69	1128	Actin
Tsejk813	2.33	98.9	5.74	2673	Exo-beta-D-glucosaminidase
Tsejk814	2.41	92.6	6.9	2637	Subtilisin-like protease PPRC1
Notes: Score Sequest: The sum of peptide scores reflects the overall confidence in protein identification; a higher score indicates greater reliability.

Figure 5 Analysis of the xylanase proteins. (a) Domain of the 16 proteins; (b) phylogenetic tree of two xylanase proteins

This study provides the inaugural report on the production and characterization of xylanase activity from T. semiorbis. The properties of the xylanase preparation derived from strain Tsejk8 indicate its potential application in industrial processes that require high stability and optimal activity at acidic pH, such as in animal feed. Furthermore, the identification of two xylanase genes will facilitate investigations into the molecular mechanisms governing xylanase production by this strain, thereby establishing a theoretical foundation for genetically modifying the strain to enhance xylanase production at the molecular level.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

This study focused on the analysis of fungi. The work does not describe any studies involving human participants or animals. Our manuscript complies with the Ethical Rules applicable.

Funding

This work was supported by National Natural Science Foundation of China 31660020, Science and Technology Project Founded by the Education Department of Jiangxi Province GJJ151252, GJJ161233.

Author contribution statement

Hu performed the cultivation. Liu performed DNA extraction. Fu designed the experiments, and contributed to the writing of the manuscript. Fan supervised the project and reviewed the manuscript, provided computational support and contributed to the writing of the manuscript. All authors read and approved the final manuscript.

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Screening of the Xylanase-producing Trichoderma Strain and the Optimization of its Enzyme Production Conditions

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Abstract

Purpose

Results

Conclusions

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Introduction

Materials and methods

U = W×N×1000∕(T×V)

Statistical Analysis

Result and discussion

Conclusions

Declarations

Conflict of interest

Ethical standards

Funding

Author contribution statement

References

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