Xylooligosaccharides production by an immobilized recombinant fungal xylanase

doi:10.21203/rs.3.rs-3914175/v1

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Xylooligosaccharides production by an immobilized recombinant fungal xylanase

https://doi.org/10.21203/rs.3.rs-3914175/v1

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Xylooligosaccharides (XOS) are potential prebiotic ingredients for food industries, mainly obtained after xylan hydrolysis by endoxylanases. Enzyme immobilization possibilities recovery and reuse, in addition to improving its physical-chemical characteristics, such as stability and catalytic efficiency. This work aimed to immobilize the SM2 xylanase derived from the XynA gene from Orpinomyces sp. PC-2 and to evaluate its potential for XOS production. For this, SM2 xylanase was immobilized using the cross-linking methodology. The free and immobilized enzymes were characterized regarding the effect of pH, temperature, and thermostability. The cross-linked enzyme aggregate was evaluated for reuse and storage conditions and used for xylooligosaccharides production. Both free and immobilized SM2 xylanase showed maximal activity at 60 ºC. The immobilized enzyme was more active at acidic and neutral conditions, and the free enzyme showed greater activity at basic conditions. The half-life of the free and immobilized xylanase was 30 h and 216 h, respectively. In reuse tests, enzymatic activity increased with each cycle, and there was no statistical difference in the activity of SM2 xylanase aggregate stored at 4 and 25 ºC. After saccharification, xylobiose (0.903 g/L), xylotriose (0.487 g/L), and xylohexose (0.809 g/L) were detected. As a result, immobilization enhanced thermostability, shifted the pH of maximum activity to 5, facilitated reuse, and eliminated the need for refrigerated packaging. Finally, the xylooligosaccharides produced by the SM2 xylanase are known for their prebiotic role, providing potential application of the immobilized enzyme in the food industry.

cross-linking immobilization

xylanase

enzyme stability

biocatalysis

protein engineering

Xylooligosaccharides (XOS) are xylose oligomers linked by β-1,4 xylosidic bonds obtained from the hydrolysis of xylan, the main constituent of the hemicellulosic fraction of plant biomasses [1]. Short-chain XOS, containing from 2 to 6 xylose monomers, are of great interest to the food industry due to their prebiotic action [2]. Prebiotics are nutrients resistant to the various conditions of food production processes and digestion in the upper digestive tract of mammals, which, when fermented by the intestinal microbiota, offer several benefits to the host's health, such as (i) reduction of intestinal constipation; (ii) prevention of gastrointestinal infections; (iii) improvement of nutrients digestion and absorption; (iv) inhibition of pathogenic microorganisms growth; and (v) promotion of probiotics growth, such as Lactobacillus and Bifidobacterium [3–4].

Xylanases or endo β-1,4 xylanases (EC 3.2.1.8) are enzymes that hydrolyze xylan into xylooligosaccharides [5]. The SM2 xylanase, derived from XynA xylanase from the fungus Orpinomyces sp. PC-2, previously developed to increase thermostability and overexpressed in E. coli [6–7] showed high thermal stability and efficiency in the hydrolysis of arabinoxylan compared to the wild-type xylanase, showing the possibility of application in the baking industry [8]. Studies carried out by Almeida [9] also showed the efficiency of SM2 to produce XOS from the hydrolysis of lignocellulosic biomasses such as corn cob, rice bran, soybean husk, and coffee residues.

Immobilization is a key aspect of enzyme applications in industrial processes. It enables the recovery and the reuse of enzymes in successive cycles, thereby reducing costs, promoting stabilization, and even improving catalytic efficiency [10]. Cross-linked enzyme aggregates (CLEA) immobilization is a support-free technique that utilizes a bifunctional agent to facilitate the formation of cross-linked enzyme aggregates [11]. This technique has gained attention due to its independence from physical support, preserving the original enzymatic activity, and enhancing stability [11].

Therefore, this study investigated the biochemical properties of the SM2 xylanase when immobilized by CLEA compared to the free xylanase and accessed the application potential of the resulting cross-linked aggregates for the production of xylooligosaccharides (XOS) from rice bran.

Cultivation and maintenance

Escherichia coli BL21(DE3) RIPL, previously constructed [7] for expression of the engineered xylanase SM2, was obtained from the collection of the Laboratory of Molecular Biology and Phylogeography, Universidade Federal de Viçosa. The cells were activated in LB agar medium (1.0% tryptone; 0.5% yeast extract; 0.5% NaCl; and 1.5% agar) containing 0.1 mg/mL of kanamycin for 24 h at 37 ºC. A single colony was transferred to 5 mL of liquid LB medium containing 0.1 mg/mL of kanamycin and incubated at 37 ºC, 180 rpm for 14 h.

Crude extract production

Xylanase expression was performed as described by Ventorim et al. [7], using whey as the inducing agent, replacing isopropyl-β-D-thiogalactopyranoside (IPTG). The concentration of reducing sugars in whey was determined by the dinitrosalicylic acid method [12] and adjusted for the final concentration of 5 mM.

Activity assay

Enzymatic activity of free and immobilized xylanase was performed using the 3,5-dinitrosalicylic acid reagent as described by Ventorim et al. [7]. The 250 µL reaction mixtures were composed of 1% (w/v) beechwood xylan prepared in sodium phosphate buffer, pH 6.5, and 50 µL of crude extract, as the source of the free or the immobilized xylanase (obtained from immobilization procedure - section 2.5), and carried out for 10 min at 50°C. The reaction was stopped by the addition of 250 µL of 3,5-dinitrosalicylic acid (DNS) and the tubes were placed in a boiling bath for 5 min. Finally, 500 µL of distilled water was added and 200 µL of the reaction mixture was aliquoted into a 96-well flat-bottom microplate. The absorbance reading was performed at 540 nm on the Multiskan Go spectrometer (Thermo Scientific). The amount of released reducing sugars was determined by using a D-xylose standard curve. One unit of xylanase activity (U) was defined as the amount of enzyme capable of releasing 1 µmol of xylose per minute under the assay conditions.

Protein concentration

The protein quantification was performed by the Coomassie Blue binding method using bovine serum albumin (BSA) as standard [13].

Xylanase immobilization

Acetone, isopropanol, ethanol, and ammonium sulfate were tested as precipitating agents. The precipitating agent was added to a final concentration of 80% to 50 µL of the xylanase crude extract diluted 100 times and the mixture was incubated at − 20°C for one hour. After incubation, a volume of glutaraldehyde corresponding to 1% of the total volume was added. The microtubes were agitated for 3 hours, followed by centrifugation at 10,000 g for 15 minutes. The supernatant was discarded, the pellet was washed with sodium phosphate buffer, pH 6.5, and the amount of immobilized xylanase (U) was evaluated by the activity assay (section "Activity assay").

Biochemical characterization

The effect of pH on the activity of immobilized and free xylanase was performed as the standard xylanase assay (section "Activity assay") at pH values between 2 and 11, using different buffers at a concentration of 100 mM: McIlvaine buffer (pH 2.2 to 8) and sodium carbonate buffer (pH 9, 10 and 11).

The effect of temperature on the activity of immobilized and free xylanase was performed as the standard activity assay (section "Activity assay") at temperatures ranging from 30 to 80°C, at pH 6.5.

To determine thermostability, microtubes containing immobilized and free xylanase were incubated at 50°C. The remaining activity was evaluated by performing the activity assay at intervals of 24 hours for 9 days (216 h).

Immobilized xylanase reuse tests

Xylanase aggregates were used for multiple cycles of activity assay to assess the potential for reuse. The enzymatic assay was performed as described in the section "Activity assay" and, before stopping the reaction, the mixture was centrifuged, and the supernatant recovered to stop the reaction by adding DNS. The precipitate was used in a new enzymatic test by adding a new substrate solution. The first-cycle activity was considered 100% as a basis for determining the relative activities in subsequent cycles.

Immobilized xylanase storage test

The storage stability of immobilized xylanase was evaluated for 0, 5, 10, and 15 days at 4°C and room temperature. Activity tests were conducted according to the section "Activity assay".

Enzymatic hydrolysis of NaOH-treated rice bran and evaluation of xylooligosaccharides (XOS) release

Rice bran biomass alkaline pre-treatment was performed with 10% (w/v) solids in a 1% NaOH solution in the autoclave for 1 hour at 120°C. Biomass was washed with 4 L of distilled water, until clarification, filtered in nylon fabric, and stored at − 20°C for later use. The exact mass of solids before and after the pre-treatment was measured and recorded to calculate recovery from the pre-treatment stage.

The efficiency of immobilized enzymes in XOS production was verified through saccharification of pre-treated rice bran. Enzymatic hydrolysis was performed in 100 mM sodium phosphate buffer, pH 6.5, added of 10 mM sodium azide and 40 mg/L tetracycline to avoid microbial contamination. The assays were carried out in triplicates, with 10% (m/v) solids and 1 U of immobilized xylanase per gram of pre-treated biomass, in 50 mL Falcon tubes, incubated at 50°C, under 150 rpm shaking in a M380 refrigerated shaker incubator (Marconi). The reaction progress was monitored at times 0, 24, 48, 72, and 96 h, and an independent control without enzymes for each point was performed. After each time, 1 mL of solution was collected and centrifuged at 9,300 x g for 10 min at 8°C. The supernatant was stored at – 20°C for quantification of XOS by high-performance liquid chromatography (HPLC).

The enzymatic hydrolysis samples were filtered with 0.45 µm syringe filters and subjected to the chromatograph for XOS quantification. High-performance liquid chromatography was performed on a chromatograph model CBM-20A/20Alite (Shimadzu) with a RID − 20A refractive index detector (Shimadzu). XOS quantification was performed using the Aminex 7 HPX-87P column (BioRad) at 80°C with a flow rate of 0.6 mL/min and elution with deionized water. The analytical curve was constructed with standards of xylobiose, xylotriose, xylotetrose, xylopentose, and xylohexose at concentrations of 0.1, 0.5, 1.0, 2.0, and 5.0 g/L.

Statistical analysis

All enzymatic tests were subjected to statistical Student's T-test (p < 0.05) using the Minitab 19 software.

Production and immobilization of SM2 xylanase

Heterologous expression systems have been successfully used in the production of enzymes of biotechnological interest, in addition to allowing modifications to be made to improve catalytic properties [14], as is the case of SM2 xylanase derived from XynA from Orpinomyces sp. PC-2 [7]. Heterologous systems also employ inducing agents capable of defining the most appropriate moment for expression, ensuring high levels of production and final yields. The expression system to produce SM2 xylanase uses whey as an inducing agent, an abundant and low-cost byproduct, replacing Isopropyl β-D-thiogalactopyranoside (IPTG), which is the inducer commonly used in prokaryote expression systems. This replacement brings advantages such as lower production costs and the development of more sustainable processes [15]. In this case, the SM2 xylanase enzymatic activity of 11,000 U/mL (11,393.88 ± 444.33 U/mL) was obtained from the previously constructed expression system [7].

Different precipitating agents were evaluated in the first stage of CLEA immobilization, and isopropanol stood out as the best precipitating agent for xylanase (data not shown). Activity recovery after isopropanol precipitation was approximately 37.3%. The remaining xylanase activity in the supernatant was 4.2% (Table 1). The loss in activity observed after this step may be due to conformational changes in protein molecules caused by isopropanol which resulted in loss of enzymatic activity. Other works describe protein denaturation and consequently the loss of enzymatic activity caused by organic solvents [16–17].

Table 1

Activity values and yields of free SM2 xylanase and after precipitation and immobilization steps
	U_TOTAL	Precipitation (%)	Immobilization (%)
Free SM2	11.39 ± 0.44	100	-
Isopropanol 80% precipitation	4.25 ± 0.20	37.3	100
Precipitation supernatant	0.48 ± 0.02	4.2	-
CLEA SM2	0.06 ± 0.00	-	1.5
CLEA supernatant	0.28 ± 0.03	-	6.5

The second stage of immobilization using glutaraldehyde resulted in an immobilization yield of 1.5% (Table 1). Hero et al. [17] showed similar results obtaining a yield of 1.3% of the xylanase aggregate using isopropanol as the precipitating agent and glutaraldehyde to bind the enzymatic aggregates. It is important to highlight that enzyme immobilization without support is an empirical process, in which each enzyme has its particularities and that the choice of precipitating and crosslinking agents, time, and temperature of crosslinking are factors that directly interfere with the formation of the aggregate and in its immobilization [18–19]. In this way, the functional biochemical properties of the aggregates obtained were evaluated and compared with the free enzyme to validate the applicability of the immobilization process and the aggregates in biotechnological processes.

Biochemical characterization

Immobilized xylanase showed greater activity at neutral and acidic pH values, while free xylanase was more active in the more basic pH range (Fig. 1a). This change in the pH range of the enzyme's best performance when immobilized is interesting when aiming for application in food industry processes, as they are normally carried out in more acidic to neutral pH ranges.

The greater activity of immobilized SM2 xylanase at neutral and acidic pH values may be due to the stabilization of charged regions of the molecule in the protein aggregate. Glutaraldehyde forms interactions with free basic residues on the surface of the enzyme, which promotes electrostatic changes in the molecule and modifies the microenvironment of the immobilized enzyme [16, 20].

Regarding the effect of temperature, free and immobilized xylanases showed a gradual increase in relative activity at temperatures from 30 to 60 ºC, with an optimum temperature of 60 ºC (Fig. 1b), as found in the studies with free SM2 xylanase, performed by Ventorim et al. [7] and Passarinho et al. [8]. However, the immobilized enzyme showed greater stability than the free enzyme at temperatures above 60°C, maintaining 99% of the maximum activity at 70°C and almost 50% at 80°C, suggesting that the immobilization process promoted the stabilization of the molecules and extended the range of activity of the enzyme at higher temperatures, compared to the free SM2.

Kumar et al. [21], also obtained an improvement in the enzymatic activity of immobilized xylanase compared to the free enzyme at higher temperatures, such as 70 and 80 ºC, with an optimal temperature of 60 ºC for the enzyme aggregate and 50 ºC for the free enzyme. It is suggested that the immobilization process promotes rigidity in the enzyme structure due to the strong covalent bonds formed between the enzyme aggregates and glutaraldehyde, in addition to the greater proximity between the molecules, protecting them from distortion or damage due to heat [20, 22]. The improvement observed in xylanase performance at higher temperatures appears to be due to this effect and highlights the potential for application of the aggregates in industrial processes in which the temperature undergoes large variations, such as in beer production, which varies from 48 to 78 ºC; and in the juice pulp industry that varies between 30 and 60 ºC [14, 23–24].

Thermostability is an important parameter for industrial processes, as it indicates the period of efficient activity of the enzyme at a given temperature. SM2 is a xylanase obtained by directed evolution and selected due to its greater thermostability compared to the native xylanase. This enzyme has a half-life of approximately 30 hours at 50 ºC [7]. The immobilization process, in addition to promoting better performance of SM2 xylanase at higher temperatures, also led to a significant improvement in thermostability, as the enzyme reached 216 hours at the same temperature, with an activity of 50%, which represents an increase of more than 7 times compared to the free enzyme (Fig. 2). This result reinforces that the bonds formed between the aggregates and glutaraldehyde promoted structural changes in the molecule, making the immobilized xylanase more stable at higher temperatures and prolonged periods.

The improvement in thermostability resulting from the formation of enzymatic aggregates was also documented by Hong et al. [25], in which the laccase immobilized by crosslinking using genipin as a natural cross-linker incubated at 50 ºC for 7 hours showed greater thermostability than the free enzyme. Ullah et al. [26] also obtained promising thermostability results with an α-amylase aggregate using glutaraldehyde evaluated at 40, 50, 60, and 70 ºC when compared to the free enzyme. Finally, Verma et al. [20] also obtained cross-linked xylanase aggregates, using glutaraldehyde as the cross-linker, with better performance at 60 and 70°C after 4 h of incubation.

Based on the characteristics shown by the SM2 enzymatic aggregate, such as performance in neutral and acidic pH conditions, activity, and stability at temperatures greater than 50°C, we proceeded with the application test of the immobilized enzyme in the saccharification of rice bran for XOS production.

Saccharification of rice bran to produce xylooligosaccharides (XOS)

The potential of free SM2 xylanase to produce xylooligosaccharides from pre-treated rice bran of interest to the food industry was previously accessed by Almeida [9]. The products of enzymatic hydrolysis performed with 20 U of xylanase per gram of alkaline pre-treated biomass (10% of solid load with 1% NaOH) at 50°C, for 72 h, were mainly xylobiose (X2), xylotriose (X3), xylopentose (X5), and xylohexose (X6). Based on these results, rice bran was chosen to carry out the enzymatic hydrolysis tests with the SM2 aggregates. The alkaline pretreatment of rice bran yielded 75.7% by mass. The saccharification step with 1 U of immobilized xylanase per gram of pretreated biomass proved to be effective in producing XOS with a low degree of polymerization, formed by 2 to 6 xylose residues (Fig. 3).

In the first 24 hours of saccharification, only the production of xylobiose (X2) was detected among the XOS investigated. After 48 hours it was possible to observe the production of xylotriose (X3) and xylohexose (X6), in addition to the continued release of X2. After 72 hours of enzymatic hydrolysis, the concentration of X2 was 0.616, and X3 and X6 reached their maximum values, 0.49 and 0.86 g/L, respectively. Finally, after 96 hours, there was no variation in the concentrations of X3 and X6, however, X2 continued to be released. Endo-xylanases, especially those from the GH11 family, such as SM2, which are considered true xylanases due to their high substrate specificity, randomly cleave the β-1,4-xylosidic bonds between two D-xylopyranoside residues and not at the ends [27–29]. This explains why there was no release of the xylose monomer. Furthermore, xylotetraose (X4) and xylopentose (X5) were not detected.

Manrich [30] was able to produce X2, X3, X4, and X5 from alkaline pre-treated sugarcane bagasse (10% of solid load with 1.4% NaOH) at 50°C, for 4 h using xylanase immobilized in agarose-glyoxyl, with an enzymatic load of 20 U/gram of pre-treated biomass. Ai et al. [31] used 600 U of a non-covalent immobilized xylanase in an Eudragit S-100 anionic copolymer in the saccharification of alkaline pretreated ground corn cobs (in a ratio of 6:1 kg/kg with 2% NaOH) for 24 hours, at 55 ºC. The XOS obtained were xylobiose and xylotriose. Xylobiose and xylotriose were also the only XOS obtained from the saccharification of alkaline pretreated corn cob powder (15% NaOH in a ratio of 1:20 (m/v)) at 50 ºC, 30 rpm for 24 h [32]. The authors used a xylanase from Bacillus halodurans immobilized by ionic bonding on an anion exchange resin (0.255 U of immobilized enzyme for 1 mL of 2.0% (w/v) xylan).

The variety of XOS produced in the enzymatic saccharification step directly depends on the biomass, the composition of the xylan, the type of pre-treatment used, the properties of the selected xylanase, and the reaction time [33]. Rice bran proved to be a promising biomass for hydrolysis by immobilized SM2 xylanase, even with the low enzymatic load applied. The variety of XOS released highlights their potential for application as prebiotics in the food industry. Furthermore, the use of xylanase in immobilized form makes reuse possible, reducing process costs.

Reuse

Reuse determines the number of cycles that it is possible to use the same enzyme and is essential for the industry, as with reuse process costs tend to decrease [34–35]. Usually, enzymatic activity decreases with each cycle performed. However, the activity of the immobilized SM2 xylanase gradually increased over the reuse cycles evaluated, reaching almost double the initial activity after the tenth cycle (Fig. 4).

The formation of the cross-linked enzymatic aggregate occurs first by the approximation of the molecules, in which stabilizing interactions occur between them, such as hydrogen bonds, van der Waals force, hydrophobic interactions, and ionic bonds [36]. Then, with the addition of the bifunctional agent, covalent cross-links are formed between these aggregates, which can form dense aggregates with little flexibility. Despite promoting increased thermostability, this low flexibility and the proximity between enzyme molecules by cross-links can affect the positioning of the substrate in the catalytic site, restricting enzymatic activity. We, therefore, suggest that prolonged exposure of molecules to 50°C promotes changes in the system's kinetic energy, resulting in the disruption of weak interactions between molecules and, consequently, increasing their flexibility and exposing catalytic sites that were not previously exposed. As a result, the interaction between the substrate and the enzyme is facilitated and, in this way, the enzymatic activity increases with each cycle.

The increase in activity shown by the immobilized enzyme throughout reuse cycles reinforces the potential for its application in industrial processes that require high yields, specificity, low cost, and conditions of high temperature and acidic pH.

Storage assessment

The stability of the enzyme during storage is an important criterion for the feasibility of application in industry [21]. The immobilized xylanase showed good maintenance of enzymatic activity during the evaluated period, with similar results in the two tested conditions, room temperature and under refrigeration, maintaining around 80% of its initial activity after 15 days (Fig. 5). In this sense, it is suggested that it is not necessary to spend energy on maintaining xylanase aggregates under refrigeration, thus reducing costs and care with transportation and packaging. Similar results were observed for cross-linked xylanase aggregates for 28 days of evaluation [23] and for tyrosinases also immobilized by cross-linking for 90 days [37]. Therefore, it is possible to suggest that immobilization by cross-linked aggregates favors the stabilization of molecules, not only regarding long-term storage but also in the conditions of application in industrial processes.

Immobilized enzymes are of interest to industry due to the possibility of reuse and greater stability. The cross-linked SM2 xylanase aggregates showed promising biochemical properties for industrial applications, such as optimal activity in acidic and neutral pH conditions and temperatures between 60 and 70°C, maintaining more than 50% of maximum activity at 80°C, and high thermostability at 50°C. The obtained aggregates remained stable after 10 cycles of reuse and after maintenance for 15 days at room temperature. Furthermore, the evaluation of the saccharification of pre-treated rice bran with immobilized xylanase resulted in the production of XOS with a low degree of polymerization, formed by 2 to 6 xylose residues, known for their prebiotic potential in the food industry.

Conflict of Interest

The authors declare that they have no conflict of interest in the publication.

Funding

This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Author Contributions

LPF participated in the study design and conducted analysis; RZV participated in the analysis of experimental data and manuscript preparation; MGO participated in the execution of biochemical characterization; LFA participated in the XOS production evaluation; VMG contributed to the study design and analysis of experimental data; and GPMA coordinated study design, analysis, and manuscript preparation.

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Xylooligosaccharides production by an immobilized recombinant fungal xylanase

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Version 1

Abstract

Figures

Introduction

Methods

Cultivation and maintenance

Crude extract production

Activity assay

Protein concentration

Xylanase immobilization

Biochemical characterization

Immobilized xylanase reuse tests

Immobilized xylanase storage test

Enzymatic hydrolysis of NaOH-treated rice bran and evaluation of xylooligosaccharides (XOS) release

Statistical analysis

Results and Discussion

Production and immobilization of SM2 xylanase

Biochemical characterization

Saccharification of rice bran to produce xylooligosaccharides (XOS)

Reuse

Storage assessment

Conclusions

Declarations

Conflict of Interest

Funding

Author Contributions

References

Status:

Version 1