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Introduction
Although there are worldwide researches focused on the production of basidiomycete fungi ligninases, as a biological method to reduce lignin content of biomass (Janusz et al. 2015), there is still a lack of answers to many questions about obtaining them. In particular, it is necessary to study other potential fungi species in the synthesis and ligninases production (Brijwani et al. 2010) as is the case of Trichoderma genus.
The species of Trichoderma are widely distributed in all latitudes, and occur naturally in different environments, especially in those that contain organic matter or decomposing plant wastes. They have the ability to produce several metabolites and adapt to various environmental conditions and substrates, this gives the Trichoderma genus the possibility of being use in the biotechnology industry.
Laccases generally produced from fungi are accompanied by other types of compounds such as isoforms, proteases, cellulases and other compounds derived from crude extract production (Jia et al. 2019). There are several methods that are used for the separation and purification of laccases from crude extracts of fungi, such as chromatography, centrifugation, phase formation, precipitation and filtration. They are used depending on the objective with the purified protein, either identification or improvement of a subsequent process (Camperi et al. 2014 and Borges et al. 2019).
The mutant strain identified in previous studies as Trichoderma viride M5-2 has the capacity to biotransform highly lignocellulosic substrates such as sugarcane bagasse (Valiño et al. 2004) and some species of temporary legumes (Valiño et al. 2015ab), where the production of its cellulase enzymes was quantified. However, the study of ligninolytic enzyme production of this fungus has not been deepened for enzymatic induction studies by biological interactions with other high laccase-producing fungi in wheat bran. These studies are focused on the industrial importance approach for biotechnological applications (Zhao et al. 2018 and Junior et al. 2020).
For these reason, it is necessary to elucidate and define the methodologies, design parameters and operating conditions for the separation, concentration and purification of ligninolytic enzymes synthesized by this strain. The objective of this research was to study the production of lignocellulases enzymes from Trichoderma viride M5-2 in wheat bran and the purification of their laccases.
Materials and Methods
Microorganism. The mutant strain of lignocellulolytic T. viride, identified as M5-2 fungus, belonging to the strains collection from Biotechnology Department of the Institute of Animal Science was used (Sosa et al. 2017).
Culture conditions. The fungus strain was cultured in a dish with Potato Dextrose Agar (PDA) medium. It was incubated for 7 days at a temperature of 30 ºC. After this period it was passing to an enriched medium for fungi according to table 1.
Table 1 Composition of the enriched medium for fungal growth
| Culture medium | Quantity (1L) |
|---|---|
| Glucose | 10.0 g |
| Potassium phosphate | 2.0 g |
| Magnesium sulphate | 0.5 g |
| Malt extract | 3.5 g |
| Yeast extract | 2.0 g |
| Bactopeptone | 0.1g |
| Metallic salts solution | 1.0mL |
| Copper sulphate | 0.5 mg |
| Magnesium sulphate | 0.16 mg |
| Zinc sulphate | 1.14 mg |
| Cobalt chloride | 0.29 mg |
Fermentation process. One aliquot of 5mL was taken from the enriched media and inoculate into a flask containing 3 g of wheat bran (table 2) and 100 mL of citrate buffer (50 mM, pH 5.0). Liquid cultures were incubated in an orbital shaker at 120 rpm for 10 days at 30 °C. Fermentation samples were taken every 24 hours, filtered through Büchner funnnel, and the resulting liquid was centrifuged (10 000 rpm, 3 min at 4°C) and reserved for further analysis.
Table 2 Description for every 40 grams of high-fiber wheat bran
| Content | Grams |
|---|---|
| Proteins | 6.0 0 |
| Fats | 1.50 |
| Available carbohydrates | 18.00 |
| Sugars | 7.00 |
| Dietary fiber | 11.00 |
| Sodium | 0.11 |
Assay of enzymatic cellulase activities (endo β1, 4-glucanase and exo β 1, 4-glucanase). Endo β1,4-glucanase (CMCasa) activity was determined on the carboxymethylcellulose substrate and exo β1,4-glucanase (PFasa) activity on crystalline cellulose. They were calculated and expressed in international units per milliliter (U/ mL). This activity refers to glucose micromoles released per minute of reaction under the conditions of the activity assay (Mandels et al. 1976). The content of reducing sugars released was determined by the 3.5-dinitrosalicylic acid method (Miller, 1959).
Xylanase activity was determined by mixing 0.9 mL of 1% (w/v) birch wood xylan (prepared in 50 mM Sodium-citrate buffer, pH 5.3) with 0.1 mL of suitably diluted enzyme and the mixture was incubated at 50 °C for 5 min (Bailey et al. 1992). The reaction was stopped by addition of 1.5 mL of 3, 5 dinitrosalicylic acid (DNS) and the content was boiled for 5 min (Miller 1959). After cooling, the developed color was read at 540 nm. The amount of liberated reducing sugar was quantified using xylose as standard. One unit of xylanase activity was defined as the amount of enzyme that liberates 1 mol of xylose equivalents per minute under the assay.
The enzymatic assay of laccase and lignin peroxidase activity was carried out according to the methodology proposed by Kumar et al. (2016). Samples were taken from the submerged fermentation of wheat bran as previously described.
Laccase activity was determined with a spectrophotometer, by syringaldazine oxidation under aerobic conditions. The resulting violet color was measured at 530 nm. The analytical conditions were 5 mM of syringaldazine, 50 mM buffer citrate, pH 4.5, 30°C and 1 minute reaction time. A laccase unit (U) is the amount of enzyme that catalyzes the conversion of 1.0 mmol of syringaldazine per minute under these conditions. Lignin peroxidase was determined by the H2O2-dependent oxidative dimerization of 2.4-dichlorophenol at 25°C, where the reaction mixture contained 26 mM of 4-amino-antipyrin, 20 mM of 2.4-dichlorophenol and 3 mM of H2O2 in 20 mM with pH 4.5 of sodium succinate. The change in absorbance was monitored at 510 nm (ε = 1.85x104 M-1cm-1).
Estimation of protein. Protein concentration was determined at different fermentation hours and after each step of the purification process by the Bradford method, with the use of a standard curve of bovine serum albumin (BSA) (AOAC 2005).
Statistical analysis. A completely randomized design was used, in which differences of pH, enzymatic activity, productivity and humidity were measured, with respect to fermentation times. All this determination was made with three repetitions, where each Erlenmeyer constituted an experimental unit. For result processing, a simple classification was carried out in the statistical system INFOSTAT (di Rienzo et al. 2012). The differences between means were established according to Duncan (1955) test.
Purification of laccase of the T viride M5-2 fungus (Gagaoua and Hafid 2016). From laccase production kinetics, establish for the strain, the fermentation time of greater production of this enzyme was used to produce more extract in order to carry out the purification process. According to this, 1 L of liquid medium was prepared, which was inoculated with an equal proportion of 5 mL of inoculum of the fungus per 100 mL of medium. After the fermentation time, the culture was filtered in sterile gauze and centrifuged at 1 400 rpm for 1 min. The supernatant was again subjected to enzymatic laccase analysis and the protein concentration was measured. The enzymatic extract was concentrated by filtration in membrane Pellicon ® XL filter of 30 KDa (Millipore, Germany) and five washings were performed with phosphate buffer (10 mM, pH 7)
Anion exchange chromatography. Anion exchange chromatography was developed according to the methodology proposed by Janson (2011). The sample was applied to a DEAE anion exchange sepharose fast flow column in a fast protein liquid chromatography (FPLC) BioRad, United States. The resin was packed in a 20 cm by 1 cm column to a volume of 15 mL. It was balanced by three volumes of buffer A (sodium acetate 10 mM, pH 5) at a flow rate of 3 mL/min. After sample application, a gradient program was followed by steps from 100 to zero percent of buffer A, with a consequent increase in buffer B or elution buffer (sodium acetate 10 mM, pH 5 with molar sodium chloride 2). Fractions of 3 mL were collected throughout the program running. Each of the collected fractions was dialyzed and concentrated with an Amicon® Ultra fifteen device, Germany and the laccase activity was analyzed with syringaldazine as a substrate. The fractions found as positive were pooled and concentrated through the Amicon® device to calculate the specific activity and purification parameters.
Purification parameters (purification factor and enzyme yield). From the values of enzymatic activity and protein concentration, the purification parameters were calculated as:
Results and Discussion
Cellulolytic and xylanolytic enzymatic activity. Figure 1 shows the determinations of endo and exo β 1,4-glucanase and xylanases cellulolytic activity in solid medium submerged in wheat bran with the pH and temperature conditions established for this fermentation p <0.0001. The maximum production achieved for exo β 1,4-glucanase was 0.222 U/mL at 48h. However, for endo β 1,4-glucanase and xylanases, these conditions favor its production after 72h of fermentation, with 0.214 and 0.31U/mL respectively.
Figure 1 Cellulolytic activity endo β1, 4 glucanase (P <0.0001, SE ±0.0013) and exo β 1.4 glucanase (P <0.0001, SE ±0.0033) and xylanase (P <0.0001, SE ±0.0021) by T. viride M5-2 in submerged medium of wheat bran. The activity maximum values were obtained at a temperature of 30ºC.
Differences between maximum enzyme production and low values found in relation to fermentation time suggest that they may be related to wheat bran chemical composition, that do not favor the action of exoglucananase and the rest. This requires additional experimental justification.
Cellulase production in cultures is associated with growth and is influenced by various parameters including the nature of cellulosic substrate, medium pH and nutrient availability. In addition, a large-scale production of cellulases requires understanding and proper control of the growth and enzyme production capabilities of the producer (Singhania et al. 2017). This is, however, extremely complicated because many factors and their interactions can affect cellulase productivity. Media formulation for fermentation is of significant concern because no general composition can provide the optimum growth and cellulase production. Amorim et al. (2019) proposed liquefied wheat bran as carbon source and inducer in high-solids submerged cultivation for xylanase production. Enzyme synergism results improved reduced sugar yield and it depends on enzyme composition and ultrastructural features of substrate (Meehnian et al. 2017). Structural features of substrate are sometimes altered in the presence of additives (Obenga et al. 2017).
This result differs from the fermentation in sugar cane bagasse in terms of production time of the enzyme, where cellulolytic activity is higher. The major technical limitation in fermentative production of cellulases remains the increased fermentation times with low productivity (Van Dyk and Pletschke 2012 and Singhania et al. 2017). The candidate strain for enzymatic production fermentative processes are those capable of expressing their maximum capacity during the first fermentation hours, since this reduces the working time and optimizes the process as the results achieved in this study (Valiño et al. 2016a b).
Laccase and peroxidase enzymatic activity. Figure 2 shows the results obtained in the enzymatic laccase activity assay. The maximum production of laccases was reached at 48 h of submerged fermentation in wheat bran, with 0.22 U/mL. To be a conidial fungus, it has an important production of enzymes in a short time of incubation.
Figure 2 Laccase activity of T. viride M5-2 in submerged medium of wheat bran, in a fermentation kinetics of 250h (EE ±1, 07; P<0, 0001)
These values are similar to those reported in some basidiomycetes. According to Ozcirak and Ozturk (2017), patterns of ligninolytic enzymes production in Pleurotus (excellent lignin degrader), during 23 days at 25°C in solid state fermentation using pretreated potato peel were 6 708.3 ± 75 U/L for laccase and 2 503.6 ± 50 U/L for manganese peroxidase.
Lignin degradation is catalyzed by an extracellular complex produced by some fungi, when they have been exposed to nutrient limiting conditions in the culture medium (Pollegioni 2015). This complex is composed by three ligninolytic enzymes involved in the degradation of lignin, manganese peroxidase (MnP), lignin peroxidase (LiP) and laccase (Selvam et al. 2003 and Rybczyńska and Korniłłowicz, 2017).
According to Moreno (2013) and Rezaei et al. (2017), delignification process can increase enzyme accessibility by increasing the number of pores and the accessible surface area, thus improving the yields of enzymatic hydrolysis (Oliva et al. 2015).
Laccase activity can be detected in lignin bioassays as the only carbon source, although the analytical tests for lignin determination make them expensive. However, other organic compounds such as syringaldazine, used in this study, are also substrates of others lignin-modifying enzymes, for example the lignin peroxidase, where the activity is easily revealed by a change in the coloring of the culture medium (Mehandia et al. 2020).
The results of the assay to detect lignin peroxidase did not show rapid activity. Notable changes in the coloration of substrate were manifested after 5 min of the enzymatic reaction (figure 3). This indicates that lignin peroxidase is at a lower concentration compared to laccase, probablemente due to fermentation time and conditions for peroxidase activity assay.
Figure 3 Peroxidase activity of T. viride M5-2 in submerged medium of wheat bran in a fermentation kinetics of 230h (EE ±0.0021; P<0, 0001)
Kinetics of peroxidase production by the T. viride M5-2 strain is different from laccase production kinetics, possiblly due to different mechanisms of action of these enzymes, where lignin peroxidase is capable of acting directly on phenolic (Guerberoff and Camusso 2019) and non-phenolic (Kumar et al. 2016) lignin wastes, while laccases can also act on non-phenolic wastes through a lipid peroxidation secondary reaction (Moreno 2013).
Laccase purification. The enzymatic concentrate obtained from the fermentation of wheat bran with T. viride M5-2 strain was worked according to a laccase purification methodology. In this process, it is known that they have an acidic isoelectric point, so it is feasible to purify them at pH above this one by means of ion exchange chromatography.
In this study, a positively charged DEAE sepharose matrix was used, which allowed the enzyme to be negatively charged above its isoelectric point and could adhere to the matrix, and as a consequence could be eluted by pH variation or ionic strength.
During the study, different bands associated with the presence of laccases were observed in the exchange columns, so the activity test of this enzyme was performed on the different collected fractions. According to the dish test, it was determined that the T viride M5-2 fungus was able to oxidize phenolic substrates such as siringaldazyne and has two types of laccases, the first eight fractions collected have a high enzymatic activity and the fractions of the eighteen to the twenty seven have another type of laccase with lower activity.
The fractions with eluted laccase activity, showed different chromatographic performances, which could be closely associated to their structure. The first eight fractions, as well as the fractions from eighteen to twenty-seven, were joined to form two new fractions called Fraction I and Fraction II. These fractions were again concentrated by membrane filtration with nitrogen current.
It was found that Enzymatic Fraction I showed higher laccase activity than the most retained Enzymatic Fraction II, due to the low activity of this fraction. Purification parameters of laccase from T. viride M5-2 were determined with Fraction I as shown in figure 4.
Figure 4 Purification process parameters of the Fraction I laccase of T. viride M5-2. CE: Crude extract, PL: Purified Laccase, Pc: Protein content (EE ± 0.10), Ea: Enzymatic activity (EE ± 5.68), Sa: Specific activity (EE ± 1.05), PF: Purification factor, Y: Yield, The significance was the same for all the parameters evaluated (P <0.0001)
Specific activity of the enzymatic extract in this Fraction was increased by achieving the removal of a large part of the contaminating proteins and reaching a purification factor higher than twelve, which indicates that this Fraction is twelve times purer than the initial preparation. The purification scheme used in this study also allowed not compromising the enzyme yield, obtaining a value of 182 %.
Regarding the overall performance of the process, this study coincides with the results proposed by Gagaoua and Hafid (2016), who obtained yields greater than 100% with low purification factors, which is associated with the presence of laccase as a predominant protein. However, there are crude extracts that have a high protein concentration that are not precisely the protein of interest, where the presence of more pollutants affects the process performance. Liu et al. (2015) reported yields below 70% due to these reasons.
Another aspect that should be highlighted is the use of wheat bran as raw material for obtaining ligninase enzymes and the selection of fermentation system in submerged solid medium that guarantees a faster and simpler obtaining process. It is also necessary to point out the effectiveness of the purification system used in this study in the selective separation of laccase enzymes present in the crudes.
There are several methods that are used for the separation and purification of laccases from raw fungus extracts. These include chromatographic methods, ultra centrifugation, phase formation, precipitation and ultra-filtration. These methods are used depending on the utility pursued with the purified protein, whether it is identification or improvement of a subsequent process (Camperi et al. 2014).
To select the specific purification method for laccases, factors such as: application availability, enzyme activity conservation, purification efficiency and amount of purified protein must be taken into account. With these factors, centrifugation is initially ruled out to separate laccase because this method is ideal for separating enzymes from biomass remnants in the enzyme extract and for analyzing structures and variable separation times. However, the use of this method generates phases where the target protein is, together with other proteins of similar molecular weight, which creates confusion. In addition, structural modifications of protein occur, which affects the performance of the enzymatic activity (Shi, 2016).
It is important to highlight that chromatographic methods are the most used to purified enzymes, but sometimes yields are not so high. This is a problem, especially if it is intended to use the purified enzyme in pretreatment processes of lignocellulosic material. In general, these methods are used in the identification and molecular study of proteins (Camperi et al. 2014). However, results of the present study show a purification factor that allows the enzyme to be concentrated without affecting the yield, which is an aspect that is important to propose this working methodology in the purification of lacases from this species
It is concluded that T. viride strain has the capacity to produce the lignocellulolytic enzyme complex in wheat bran. The separation method used to purify laccase enzymes is effective. It is recommended to add successive steps of purification depending on the degree of purity.
