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Cuban Journal of Agricultural Science

Print version ISSN 0864-0408On-line version ISSN 2079-3480

Cuban J. Agric. Sci. vol.54 no.2 Mayabeque Apr.-June 2020  Epub June 01, 2020



Ligninolytic potential of Curvularia kusanoi L7 laccases for animal production

1Institute of Animal Science (ICA), km 47 ½ Carretera Central San José de Las Lajas, 32700, Mayabeque, Cuba

2Scienze Agrarie, Alimentari e Forestali (SAAF) Department, Università degli Studi di Palermo, Viale Delle Scienze, Ed. 5, 90128 Palermo, Italy


The present research was conducted with the objective of evaluating the ligninolytic potential of Curvularia kusanoi L7 laccases in improving the nutritional quality of fibrous substrates for animal feed. Laccase production was obtained from C. kusanoi L7 under native conditions by solid submerged fermentation of wheat bran and by biological interactions with Trichoderma viride M5-2 and Trichoderma pleuroticola. The enzymes were purified from the culture broths by triphasic partition. The degradative potential of native and induced laccases where evaluated by determining foliar lignololytic capacity, raw wheat (Triticum aestinum) straw delignification and the effect of enzyme pretreatment on fibrous fractionation and in vitro digestibility of sugarcane bagasse. Laccases, both native and induced, presented high foliar degradation and delignification of raw wheat straw. Regarding to the improvement of nutritional quality of sugarcane bagasse, all the enzyme pretreatments managed to decrease the fiber content and increase in vitro digestibility. Compared to untreated bagasse, native laccase pretreatment obtained the highest digestibility values (P<0,05) for dry matter (54.71%), organic matter (63.14%), acid detergent fiber (63.59%) and cellulose (65.46%). It is concluded that C. kusanoi L7 laccases can be used as a novel tool in improving the nutritional quality of fibrous sources for animal feed.

Key words: Enzyme; induction; fiber; pretreatment; in vitro digestibility

The rapidly grow of world population results in the depletion of many resources and aggravates the international food situation. According to FAO (2019), hunger affects over 42.5 millions of people only in the Caribbean and Latin America region. For these reasons, the development of tropical animal production is essential as one of the important ways to reduce the negative impact of the food deficit.

Several studies are conducted with the aim of achieving more sustainable productions, capable of supplying international demand and promoting the use of alternative sources for animal feed that do not compete with human consumption. In this sense, different strategies are valued, where enzymatic pretreatment of fibrous sources stands out (Pinos et al. 2019). However, the structural complexity of lignin, as well as its close association and chemical crosslinking with the carbohydrate fraction of the plant cell wall, make it a biomolecule with a recalcitrant character and difficult degradation.

The enzymes that modify this biopolymer are oxidative, nonspecific and, in addition, they act through non-protein mediators in contrast to hydrolytic cellulase and hemicellulase enzymes (Mehandiaa et al. 2020). One of the main lignolytic enzymes that catalyze the progressive depolymerization of lignin is laccase and within their group, they have the greatest industrial application (Munk et al. 2015).

Compared to the use of lignocellulolytic microorganisms, the enzymatic pretreatment of high fiber substrates has great economic benefits and reduces the time of the degradation process (Zhao et al. 2019). However, there are only few studies on the evaluation of ligninolytic enzymes in improving the nutritional quality of fibrous sources for animal feed, and none of them highlight the importance of laccase for these purposes. That´s why the present research aims to: Evaluate the ligninolytic potential of Curvularia kusanoi L7 laccases for its use in animal feed

Material and Methods

Enzyme crudes obtaining by solid submerged fermentation of wheat bran. Microorganism. Curvularia kusanoi L7 strainwas used, isolated from lemon tree, with number of nucleotide sequences registered in the GenBank, and accession number KY795957.

Procedure: From the pure culture of C. kusanoi L7, 3 cm2 were taken and inoculated in an Erlenmeyer flask containing 3 g of wheat bran and 100 mL of citrate buffer (50 mM, pH 5.0), and they were incubated at 30 °C in an orbital shaker at 120 rpm for 168 h of fermentation. The contents of each Erlenmeyer were filtered through a Buchner funnel, the resulting liquid was centrifuged (4 °C, 10 000 rpm, 3 min) and the supernatant (crude enzyme extract) were stored in Corning tubes at -20 °C for subsequent determinations (Wang et al. 2014).

Laccase induction in C. kusanoi L7 cultures through biological interactions with Trichoderma viride M5-2 and Trichoderma pleuroticola. The induction of laccase enzymes in C. kusanoi L7 cultures was carried out through biological interactions with two strains of Trichoderma (T. viride M5-2 and T. pleuroticola, with nucleotide sequences registered in the GenBank and number of accession KY977981 and MK992922, respectively). Inoculums of these strains (1x107 CFU/g of substrate) were incorporated into the C. kusanoi L7 cultures at 48h of growth. The induction process was carried out in solid submerged fermentation of wheat bran as described in the previous section. For both Trichoderma strains the procedure was similar.

Laccase purification by three phase partition. The enzymatic extracts of C. kusanoi L7 and those resulting from the induction process, were purified using the three-phase partitioning methodology proposed by Alberto et al. (2018). Ter-butanol was added to the crude extracts in a ratio of 1.0: 1.1 (v/v), respectively. This mixture was saturated at 78% with ammonium sulfate. The system formed was homogenized by vortex for 1 minute and then incubated for 1 hour at 38 °C in a temperature controlled incubation bath until the phases were separated. The system was centrifuged (4000 rpm x 10 minutes at 25 °C) in an IEC CL31R thermo-scientific centrifuge. The middle phase was separated from the rest and re-dissolved in phosphate buffer (50 mM, pH 7). Finally, enzyme activity and protein concentration were determined.

Determination of laccase activity. Laccase activity was determined using a UV-Vis split beam spectrophotometer, Rigol Ultra-3400. The reaction mixture formed by 100 µL of syringaldazine (5 mM in ethanol) and 800 µL of citrate buffer (50 mM, pH 4.5) was incubated at 30 °C for 1 minute. An aliquot of 100 µL of the enzyme crude was added to reach the final reaction volume of 1mL and the syringaldazine oxidation reaction was monitored kinetically for 1 minute under aerobic conditions at 530 nm. The amount of enzyme that catalyzes the conversion of 1.0 mmol of syringaldazine per minute was considered as a unit of laccase activity (U) according to Perna et al. (2018).

Determination of protein concentration. Protein concentration was determined by the method proposed by Bradford (1976) using a standard curve of bovine serum albumin (BSA) in the concentration range of 50 - 0.01 mg/mL. The procedure was conducted in the same way for initial crude extracts and those resulting from the purification process.

Evaluation of the ligninolytic capacity of C. kusanoi L7 laccases on raw wheat straw by Attenuated Total Reflection Infrared Spectroscopy with Fourier Transform (ATR-FT-IR). To evaluate the ligninolytic capacity of C. kusanoi L7 laccases on raw wheat straw, the substrate was incubated with the different enzymes preparation (native laccase of C. kusanoi L7, laccase induced by T. viride M5-2 and laccase induced by T. pleuroticola) at a ratio of 1: 1 (w/v) in glass tubes, for 5 days at 40ºC. The ligninolytic capacity of the enzyme treatments was compared with a control treatment where the enzyme was replaced by 1mL of distilled water. Differences in lignin degradation were evidenced by Total Attenuated Reflection Infrared Spectroscopy (ATR-FT-IR) on Pelkin Elmer equipment with ATR diamond base and MCT/A detector. The scans were performed from 4000 to 400 cm-1.

Fibrous fractioning and in vitro digestibility of sugarcane bagasse pretreated with purified laccases from C. kusanoi L7. To evaluate the activity of C. kusanoi L7 laccases on the fibrous components of sugarcane bagasse, a completely randomized design with four treatments was used: control (untreated bagasse) and three enzymatic treatments (bagasse treated with native laccase, bagasse treated with laccase induced by T. viride M5-2 and bagasse treated with laccase induced by T. pleuroticola). The analyses where performed with six repetition for a total of 24 samples.

To obtain each treatment, the different enzymes were incubated with 1g of substrate at a rate of 4 IU/g in an orbital shaker at 40 °C for 72 hours. Fibrous fractioning was performed according to Goering and Van Soest (1970).

The digestibility of sugarcane bagasse pretreated with C. kusanoi L7 laccases was evaluated using the in vitro technique proposed by Marrero et al. (1998) with pig fecal inoculum. A crossbred pig (Yorkshire x Landrace x Duroc) was taken as the feces donor, who received 1.5 kg of corn-soybean based feed and water at will. Feces were taken immediately after defecation. Subsequently, in the residues was determined the content of dry matter (DM) and organic matter (OM) by AOAC (1995), acid detergent fiber (FND) lignin and cellulose by Goering and van Soest (1970). Incubation medium was used as the blank for digestibility calculations.

Statistical analysis. Both experiments were processed according to a simple classification model by the InfoStat statistical package (Di Rienzo et al. 2012). Duncan (1955) test was used when necessary to discriminate differences between the means.

Foliar ligninolytic capacity of laccase C. kusanoi L7 in lemon tree (Citrus x limon)To evaluate the ligninolytic effect of C. kusanoi L7 laccases on the lemon tree leaf surface, 3 young plants were used for each treatment (control with distilled water, native laccase of C. kusanoi L7, laccase induced by T. viride M5-2 and laccase induced by T. pleuroticola). All of these treatments were applied in 5 leaves per each plant, for a total of 15 leaves per treatment. The leaf surface was cleaned with distilled water and 1 mL of the enzyme was sprayed. Foliar degradative capacity was evaluated every 24 hours for a period of 7 days. The experiment was carried out at room temperature at the experimental station of the Agri-Food and Forest Sciences of the University of Palermo, Sicily, Italy.

Results and Discussion

Evaluation of the ligninolytic capacity of C. kusanoi L7 laccases on raw wheat straw by Attenuated Total Reflection Infrared Spectroscopy with Fourier Transform (ATR-FT-IR). The ATR-FT-IR spectra of raw wheat straw and straw treated with C. kusanoi L7 laccases are shown in Figure 1. In accordance with previous studies by Xu et al. (2004 and 2006), the presence of lignin in the sample is detected through characteristic signals in the spectrum.

Figure 1 ATR-FT-IR spectrum of raw wheat straw and treated with native and induced lacases of C. kusanoi L7 in the range 400 to 4000 cm-1. The numbers 1-12 correspond to the characteristic signals of lignin. Blue line represent raw wheat straw, green line represent raw wheat straw degraded by native laccase, purple line represent raw wheat straw degraded by T. viride M5-2 induced laccase and red line represent raw wheat straw degraded by T. pleuroticola induced laccase 

In the present investigation, the following bands associated with lignin were identified:

  1. Stretching vibration of O-H groups (phenolic and aliphatic) corresponding band in the range of 3400 to 3200 cm-1

  2. Stretching vibration of C-H of the CH3 and CH2 groups detected at 2930 cm-1

  3. Small band at 2850 cm-1 attributed to vibration of OCH3 groups.

  4. Band at 1711 cm-1 attributed to the C=O stretching of unconjugated ketones, carbonyl groups, conjugated aldehyde or ester groups and carboxylic acids.

  5. Band at 1610 cm-1 associated with aromatic C=C double bonds

  6. Band at 1510 cm-1 associated with vibrations of C=C bonds of the aromatic and phenolic units of lignin

  7. Band detected at 1420 cm-1 associated with vibrations of lignin phenylpropane aromatic skeleton

  8. Band detected at 1333cm-1 associated with the vibrations of the aliphatic C−H bonds (CH or CH2 groups)

  9. Band detected at 1320 cm-1 associated with symmetrical flexing of C-H aliphatic bonds.

  10. Band detected at 1210 cm-1 associated with the presence of C−O bonds of the guaiacyl ring.

  11. Band detected at 1160 cm-1 associated with the antisymmetric stretch vibration C-O of the secondary alcohols or of the hydroxycinnamic acids ester-bound (such as esterified ρ-coumaryc acid and ferulyc acid) (Sun and Cheng, 2002)

  12. Band detected at 1035 cm-1 associated with the vibrations produced by the O-CH3 bonds of the guaiacyl and syringyl type units

Laccase treatments, both induced and native, show a marked intensities reduction of the lignin associated signals. T. pleuroticola induced laccase treatment achieves the greatest reduction of these signals, although there are only small differences from the rest. In all cases, the greatest reductions were observed in the characteristic triplet (5, 6 and 7), attributed to the vibrations of the lignin aromatic ring, which corresponds to similar studies made by Ibarra et al. (2004)

These results are also agree to those proposed by Mwaikambo and Ansell (2011), who found a marked decrease in the band between 3300 and 3100 cm-1 after treatments of straw fibers with laccase enzyme. It is known that the action mechanism of this enzyme itself implies the oxidative depolymerization of phenolic compounds through the formation of unstable phenoxyl radicals (Ghoul and Chebil, 2012), which is why variations in the intensity of these bands are indicative of the enzyme action on the phenolic units of lignin. According to Mattinen et al. (2005) and Kahar (2013) changes at 1512 cm-1 attributed to lignin aromatic ring vibrations due to aromatic skeletal vibration (C═C), are indicative of changes in lignin surface, by what a lower intensity of this band demonstrates the laccase capacity to oxidize the phenolic units present on the fibers surface (Oliva et al. 2015 and Liu et al. 2014).

In vitro digestibility and fibrous fractioning and of sugarcane bagasse pretreated with purified laccases from C. kusanoi L7.Table 1 summarizes the fibrous fractioning of sugarcane bagasse pretreated with the laccase enzymes of C. kusanoi L7. In all cases, the enzymatic pretreatment process allowed a decrease in both the acid detergent fiber and the levels of lignin and cellulose. The reduction of these indicators is an important aspect for a better utilization of sugarcane bagasse in animal production.

Table 1 Fibrous fractioning of sugarcane bagasse pretreated with laccase enzymes from C. kusanoi L7 

Indicators (%) Sugarcane bagasse SE ± sign.
Bagasse control Treated with native laccase Treated with T. viride induced laccase Treated with T. pleuroticola induced laccase
DM 94.65 94.68 94.79 94.28 ±0.24 P=0.4875
ADF 51.88 a 40.14 b 39.72 c 39.74 c ±0.09 P<0.0001
Lignin 8.93 a 6.12 b 6.12 b 5.35 c ±0.10 P<0.0001
Cellulose 39.49 a 30.06 b 28.07 c 27.57 c ±0.26 P<0.0001

a, b, c Different letters indicate significant differences for P <0.05 (Duncan, 1955). DM (dry matter), ADF (acid detergent fiber)

López et al. (2018) stated that pretreatment methods must be able to improve the biodegradability of the substrate and present low energy consumption, ease waste disposal and low economic cost. That is why pretreatment methods constitute a sustainable and effective alternative for the bioconversion of lignocellulosic biomass.

The modification of the sugarcane bagasse fibers through the action of laccase enzymes, allows obtaining a more accessible and more biodegradable substrate. The in vitro digestibility of pre-treated bagasse is summarized in table 2. As observed, the difference compared to the control, clearly indicate that degradation process was effective in improving the nutritional quality of this fibrous source.

Table 2 In vitro digestibility of sugarcane bagasse pretreated with the laccase enzymes of C. kusanoi L7 

Indicators (%) Sugarcane bagasse SE ± sign
Bagasse control Treated with native laccase Treated with T. viride M5-2 induced laccase Treated with T. pleuroticola induced laccase
DMD 34.88 d 54.71 a 44.59 c 48.60 b ±0.48 P<0.0001
OMD 45.43 d 63.14 a 54.78 c 58.91 b ±0.60 P<0.0001
ADFD 50.53 d 63.59 a 56.88 c 61.16 b ±0.36 P<0.0001
CD 52.45 c 65.46 a 60.88 b 64.48 a ±0.51 P<0.0001

a, b, c, d Different letters indicate significant differences for P <0.05 (Duncan, 1955). DMD (dry matter digestibility), OMD (organic matter digestibility), ADFD (acid detergent fiber digestibility) and CD (cellulose digestibility).

Vargas and Pérez (2018) stated that the utilization of sugarcane bagasse is affected by its low digestibility, which is the fundamental reason for the application of pre-digestion processes that improve its nutritional quality. These types of pretreatments vary from the use of alkalis such as sodium hydroxide to the use of physical methods with high energy consumption that can increase digestibility up to 60%.

Lagos and Castro (2019) stated that sugarcane bagasse, unlike whole cane, has higher fiber content, so its digestibility is lower (around 25%). These authors pointed out that the use of this fibrous source in animal feed requires the incorporation of different methodologies and procedures that improve its bioconversion and extend its use. An example of this statement is the investigations made in Veracruz, Mexico, where a predigested feed was developed from sugarcane bagasse for animal feed. The increase in the fibrous component digestibility was achieved by alkaline pretreatment, obtaining greater digestibility and applicability of this resource in cattle feeding (Llanes 2012).

Other strategies to increase the fibrous digestibility of highly fiber substrate are the use of fibrolytic enzymes (Gado et al. 2009). These types of enzymes are used in the feeding of monogastrics and ruminants species and allow a better utilization of these sources.

In ruminant species, fibrolytic enzymes are used mainly as additives in cattle feed, where they present important results by increasing the digestibility of fiber, improving the efficient use of energy from pastures and reducing the costs of diets (Mendoza 2000). In monogastric species, the addition of fibrolytic enzymes allows altering the structure of the cell wall and improving the utilization of the fibrous fraction of the nutrients. According to Aranda et al. (2004), the fibrolytic pretreatment of alternative food sources allows increase its inclusion levels in the diets of these species. The use of fibrolytic products in animal production has an important impact in the diet optimization and nutrient assimilation, especially because fibrolytic enzymes stimulate the complex fiber degradation mechanism.

Most of the international companies that commercialize fibrolytic enzyme products, presents formulation based on cellulase, hemicellulase and xylanase enzymes, such as Grasszyme®, Alfazyme® and Fibrozyme® (Zilio et al. 2019). However these preparations doesn´t have lignin-modifying enzymes. It is known that ligninolytic enzymes are fundamentals for accomplish the lignin rupture and bring better access to cellulose fibers. According to Lillington et al. (2020) the joint action of cellulolytic and ligninolytic enzymes are essential to accomplish a better degradation of fibrous substrates. For these reasons, the inclusion of laccase like enzymes in fibrolytic preparations might convert these products in much more efficient technologies.

Foliar lignolytic capacity of C. kusanoi L7 laccases on the lemon tree (Citrus aurantifolia). C. kusanoi L7 fungus was isolated from lemon tree, that’s why the evaluation of the laccases lignolytic capacity on it constitute an example of how these enzymes are essential in the colonization and pathogenicity of the microorganism. On the other hand, it is known that enzymatic activity is affected in highly degree by substrate conditions, for that is important to evaluate different reaction environments to be sure of enzyme catalytic capacity. According to this, the evaluation of C. kusanoi L7 laccases on lemon tree, allows to confirm the fibrolytic ability of these enzymes.

The present study showed that C. kusanoi L7 laccases treatments had high differences respect to the untreated control. After spraying the enzyme, the wilting state of the leaves was observed almost immediately. After the first 24 hours (figure 2), great degradation of the leaf surface was found and after 72 hours of application, general tissue necrosis and loss of all the treated leaves were observed. Although the induced laccases differ from the native enzyme, all the treatments generated in the same level the progressive depolymerization of lignin and the consequent degradation of the plant tissue.

Figure 2 Foliar lignolytic capacity of C. kusanoi L7 laccases at 24 h of treatment 

It’s known that Curvularia species, together with other species of the genera Cladosporium, Alternaria, Epicoccum and Nigrospora are known to be considered as primary degrading organisms, which proliferate as the leaves aged. Its spores accumulate on the leaf surface and remain latent until the death of plant tissues. These species are generally colonizers of most senescent leaf tissues of old trees, shrubs, and grasses; action that largely depends on its enzymatic production (Hudson 1968). Valenzuela et al. (2001) also stated that about 80% of the leaf litter degradation is due to the degrading activity of the extracellular enzymes secreted by these fungi. That is why the enzymes isolation from these microorganisms assurance a great degradation capacity on these type of substrates


C. kusanoi L7 laccases, both native and induced, presented high ligninolytic potential. They are able of modifying lignin structure and improve nutritional quality and in vitro digestibility of sugarcane bagasse. These novel results lead futures investigations to in vivo evaluation of fibrous diets treated with laccase for animal feed. Furthermore it is presented the first evaluation of these purified enzymes in this field where the modification of non-conventional feed sources can constituted a sustainable alternative to develop the animal production.


The authors thank the Department of Agrarian, Food and Forest Sciences of the Università degli Studi di Palermo, Sicily, Italy, for the personal and material support. The authors also wish to thank the Microbiology laboratory of the Institute of Animal Science, Cuba, for the maintenance of the strains.


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Received: January 15, 2020; Accepted: March 29, 2020

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