Abstract
Trichoderma strains were extensively studied as biocontrol agents due to their ability of producing hydrolytic enzymes, which are considered key enzymes because they attack the insect exoskeleton allowing the fungi infection. The present work aimed to evaluate the ability of chitosanase production by four Trichoderma strains (T. harzianum, T. koningii, T. viride and T. polysporum) under solid stated fermentation and to evaluate the effect of pH and temperature on enzyme activity. pH strongly affected the enzyme activity from all tested strains. Chitosanase from T. harzianum and T. viride presented optimum activity at pH 5.0 and chitosanase from T. koningii and T. polysporum presented optimum activity at pH 5.5. Temperature in the range of 40–50°C did not affect enzyme activity. T. polysporum was found as the most promising strain to produce chitosanase with maximal enzyme activity of about 1.4 IU/gds, followed by T. viride (~1.2 IU/gds) and T. harzianum (1.06 IU/gds).
Keywords: Trichoderma spp., Chitosanase, Enzyme activity parameters optimization, Response surface methodology
Introduction
Chitin is the second-most abundant polysaccharide with an annual production of 1010–1011 tons, which is only next to that of cellulose. Chitin and chitosan are commercially obtained from shrimp and crab shells from the fishing industry. Chitosan, a d-glucosamine polymer, is a totally or partially deacetylated derivative of chitin. It is usually obtained by the artificial deacetylation of chitin in the presence of alkali [1]. Recent studies on chitin and chitosan have attracted interest for their conversion to oligosaccharides, because these oligosaccharides are not only water-soluble, but also possess versatile functional properties such as antitumor and antimicrobial activities [2, 3].
Chitosanases (E.C. 3.2.1.132) hydrolyze β-1,4-glycosidic linkage of chitosan, a polysaccharide consisting mainly of d-glucosamine with a variable content of N-acetyl-d-glucosamine. Chitooligosaccharides can induce the expression of pathogenesis-related proteins in higher plants. They can even act as immuno-potentiating effectors [4]. The oligosaccharides of chitin/chitosan, prepared by hydrolyzing chitin/chitosan with chitinase/chitosanase, have various potential applications in food, agricultural, and pharmaceutical industry [2].
Trichoderma spp. are among the most frequently isolated soil fungi and present in plant root ecosystems [5]. These fungi are opportunistic, avirulent plant symbionts, and function as parasites and antagonists of many phytopathogenic fungi, protecting plants from disease. So far, Trichoderma spp. is among the most studied BCAs fungal and it is commercially marketed as biopesticides and biofertilizers [6–8]. Its entomopathogenicity is close related to its ability of synthesize hydrolytic enzymes. Despite several studies on Trichoderma chitinase production has been published [5, 9–12], the most studied strains are T. harzianum and T. viride, and to date, in the published studies, no reports on the production of chitosanase by Trichoderma spp. were found.
Before considering a process optimization for large scale enzyme production, to check the strain ability to produce the desired enzyme and a suitable method to assay the enzyme activity are needed. For industrial purposes, a suitable low cost and easy method is desirable. Even belonging to the same species each microbial strain might present different optimum conditions for enzyme activity determination. This work aimed to evaluate the suitability of chitosanases production from Trichoderma spp. and the study of pH and temperature effect on enzyme activity (optimization of the environmental assay parameters for Trichoderma spp. chitosanase activity determination).
Materials and Methods
Microorganisms and Maintenance
The fungal strains (T. harzianum, T. koningii, T. viride and T. polysporum) used in the present work belongs to the collection of Embrapa Semi-Arido (Petrolina—PE, Brazil) and were isolated as a biocontrol agent. Spores suspension was obtained by growing the fungus in wheat bran at 30°C for 7 days and harvesting the spore with 0.01% (v/v) Tween-80 in distilled water. Spore count was adjusted to 1 × 107 spores/ml. Fungus was maintained in spore form supported in wheat bran at 4°C [13].
Determination of Optimum pH for Chitosanase Production
The best pH for chitosanase production was determined by fungal radial growth in chitosan detector agar (CDA). The CDA was prepared by mixing 1.3 g/l of Na2HPO4; 3.0 g/l of KH2PO4; 0.5 g/l of NaCl; 1.0 g/l of NH4Cl; 0.24 g/l of MgSO4; 0.01 g/l of CaCl2; 20 g/l of agar and 10 g of chitosan dissolved in acetic acid 1% (v/v). The pH was adjusted to 4.5; 5.5 or 6.5 with NaOH. The culture medium was sterilized at 121°C for 15 min and used in the Petri dishes. After cooling and the agar solidification, Trichoderma spores were seeded at the center of the Petri-dish and incubated at 30°C for 5 days. The microbial growth was accompanied by the radial mycelium growth in the Petri dish.
Solid-state Fungal Cultivation
The fungal strains were cultivated in solid substrate containing wheat bran (5 g) commercial chitosan (1 g), and 2.5 ml of a saline solution containing NaNO3 (1.0 g/l); (NH4)2HPO4 (1.0 g/l); MgSO4·7H2O (1.0 g/l); NaCl (1.0 g/l) [14]. The pH of the saline solution was adjusted to 5.5 because the best microbial growth in CDA plates was observed at this pH value for all strains. The medium was autoclaved at 121°C/15 min, cooled, inoculated with 1 ml of spore inoculums, prepared as described above, and incubated statically at 30°C for 48 h. Fermentations were carried out in 250 ml Erlenmeyer’s flasks covered with cotton plugs.
Wheat bran was purchased from the local market (Mercado São Sebastião, Fortaleza-CE, Brazil). Commercial chitosan, from shrimp shell with 80% of deacetylation degree, was purchased from a local industry (Polymar, Fortaleza-CE, Brazil).
Enzyme Extraction
The content of each Erlenmeyer flask was mixed with 20 ml of sodium acetate buffer solution (200 mM), with pH values changing according to a Faced Centered Central Composite experimental design (Table 1). The flasks were gentile hand shake at room temperature (30 ± 1°C) for 1 min and the liquid was filtrated in Whatman no 1 filter paper. The filtrate was immediately used to determine the chitosanase activity.
Table 1.
Experimental design and chitosanase Trichoderma spp. activity
| Run | pH | Temperature (°C) | T. harzianum (IU/gds) | T. koningii (IU/gds) | T. viride (IU/gds) | T. polysporum (IU/gds) |
|---|---|---|---|---|---|---|
| 1 | 4.5 | 40 | 0.000 ± 0.016 | 0.036 ± 0.056 | 0.181 ± 0.005 | 0.073 ± 0.045 |
| 2 | 4.5 | 60 | 0.098 ± 0.005 | 0.007 ± 0.007 | 0.180 ± 0.004 | 0.129 ± 0.071 |
| 3 | 5.5 | 40 | 0.092 ± 0.005 | 0.606 ± 0.1,1 | 0.051 ± 0.021 | 1.342 ± 0.563 |
| 4 | 5.5 | 60 | 0.298 ± 0.014 | 0.196 ± 0,019 | 0.053 ± 0.031 | 1.408 ± 0.110 |
| 5 | 4.5 | 50 | 0.001 ± 0.004 | 0.051 ± 0,084 | 0.119 ± 0.004 | 0.105 ± 0.014 |
| 6 | 5.5 | 50 | 0.260 ± 0.016 | 0.531 ± 0,086 | 0.150 ± 0.051 | 1.435 ± 0.061 |
| 7 | 5.0 | 40 | 0.900 ± 0.045 | 0.012 ± 0,055 | 0.618 ± 0.005 | 0.220 ± 0.583 |
| 8 | 5.0 | 60 | 0.890 ± 0.003 | 0.025 ± 0,013 | 1.172 ± 0.004 | 0.268 ± 0.025 |
| 9 | 5.0 | 50 | 1.020 ± 0.051 | 0.197 ± 0,025 | 0.920 ± 0.003 | 0.340 ± 0.098 |
| 10 | 5.0 | 50 | 1.061 ± 0.053 | 0.189 ± 0,069 | 0.941 ± 0.001 | 0.278 ± 0.011 |
| 11 | 5.0 | 50 | 1.0451 ± 0.042 | 0.195 ± 0.058 | 0.930 ± 0.035 | 0.298 ± 0.045 |
Chitosanase Activity Environmental Parameters Optimization
Chitosanase activity was measured in culture filtrates using as substrate a solution containing 0.4% (w/v) of chitosan dissolved in sodium acetate buffer (200 mM), with the same pH used to extract the enzyme (Table 1). The assay was carried out by mixing 100 μl of the crude enzyme with 400 μl of substrate and incubating for 60 min at the desired temperature, which was changed according to the experimental design presented in Table 1. Enzyme activity was determined by measuring the released reducing sugar by DNS method [15] and results were expressed as IU/gds. One IU is the amount of enzyme that releases 1 μmol of reducing sugar per minute at the assay conditions.
Functional relationships (fitted regression models) between the responses and the factors where expressed by a second-degree polynomial function, obtained by multiple regression, as presented in Eq. 1.
 |
1 |
where Yi is the response variable; β0 a constant; βi is the coefficient for the linear effect; βii is the coefficient of the quadratic effect; βij is the coefficient of the interaction effect. Xi and Xj are the independent variables.
Statistical Analysis
The software Statistica v7.0 (Statsoft) was used to built and analyze the experimental designs.
Results and Discussions
Table 1 depicts the experimental design and the enzyme activity obtained for chitosanase produced by the studied Trichoderma spp. In some experimental runs, mainly the ones with low pH values, enzyme activity was null or very low. Pareto charts of the estimated effects of the independent variables on the studied response (enzyme activity) are presented in Fig. 1. A confidence interval of 95% was considered and the pH effect was statistically significant for all chitosanase produced by the tested Trichoderma strains. On chitosanase from T. harzianum and T. viride, pH presented negative effect. The negative pH quadratic effect on T. harzianum was higher than the observed on T. viride. On T. koningii and T. polysporum, pH presented positive effect with the highest effect on chitosanase from T. polysporum. Temperature was significant only on T. koningii, chitosanase activity but its effect was slightly negative. Interaction between pH and temperature was not significant on chitosanase activity from any studied strain.
Fig. 1.
Pareto chart of standardized effects on chitosanase activity. a Chitosanase from T. harzianum, b chitosanase from T. koningii, c chitosanase from T. viride, d chitosanase from T. polysporum
Regression coefficients of the fitted quadratic model (Eq. 1) obtained for chitosanase activity as function of pH and temperature are presented in Table 2. Statistical significance was evaluated by ANOVA and F test (Table 3) considering a confidence interval of 95%. All fitted models were statistically significant since Fcalculated > Flisted [16]. The regression coefficients were also good for all fitted models (R2 > 0.90).
Table 2.
Regression coeficients of RSM model
| Factor | Y1 | Y2 | Y3 | Y4 |
|---|---|---|---|---|
| Mean | −84.51 | 5.33 | −78.46 | 40.64 |
| pH (L) | 33.45 | −4.53 | 31.39 | −17.92 |
| pH (Q) | −3.35 | 0.59 | −3.15 | 1.91 |
| Temperature (L) | 5.20 × 10−2 | 0.21 | 3.48 × 10−2 | 4.57 × 10−2 |
| Temperature (Q) | −7.00 × 10−4 | −1.25 × 10−3 | −3.00 × 10−4 | −5.00 × 10−4 |
| pH × temperature (L) | 5.40 × 10−3 | −1.91 × 10−2 | 1.00 × 10−4 | 5.00 × 10−4 |
Y1 enzyme activity from T. harzianum, Y2 enzyme activity from T. koningii, Y3 enzyme activity from T. viride, Y4 enzyme activity from T. polysporum
Table 3.
ANOVA analysis for the fitted regression models
| Variation source | Quadratic sum | Degrees of freedom | Mean square | F value |
|---|---|---|---|---|
| T. harzianum | ||||
| Regression | 2.090 | 5 | 0.418 | 80.38 |
| Error | 0.026 | 5 | 0.0052 | |
| Total | 2.110 | 10 | ||
| R2 | 0.988 | |||
| Flisted (95%) | F5,5 = 5.05 | |||
| T. koningii | ||||
| Regression | 0.398 | 5 | 0.080 | 14.81 |
| Error | 0.027 | 5 | 0.0054 | |
| Total | 0.425 | 10 | ||
| R2 | 0.936 | |||
| Flisted (95%) | F5,5 = 5.05 | |||
| T. viride | ||||
| Regression | 1.780 | 5 | 0.356 | 16.18 |
| Error | 0.111 | 5 | 0.022 | |
| Total | 1.891 | 10 | ||
| R2 | 0.940 | |||
| Flisted (95%) | F5,5 = 5.05 | |||
| T. polysporum | ||||
| Regression | 3.112 | 5 | 0.622 | 889.14 |
| Error | 0.004 | 5 | 0.007 | |
| Total | 3.116 | 10 | ||
| R2 | 0.999 | |||
| Flisted (95%) | F5,5 = 5.05 | |||
Figure 2 present the response graphs built using the fitted models. As previously mentioned, temperature did not presented significant effect on chitosanase activity from Trichoderma strains under study. A slight effect was observed only on chitosanase activity from T. koningii for temperature in the range of 50°C at low pH values. Maximal activity was observed for pH-values around 5.0 for chitosanase from T. harzianum and T. viride for any considered temperature. For these strains, the maximal enzyme activity was obtained around pH 5.0 and higher or lower pH-values strongly decreased the chitosanase activity.
Fig. 2.
Response surface graph for the chitosanase activity. a Chitosanase from T. harzianum, b chitosanase from T. koningii, c chitosanase from T. viride, d chitosanase from T. polysporum
Chitosanase activity from T. koningiiI was also strongly affected by pH and enzyme activity was enhanced at pH values above 5.0 presenting maximal values around pH 5.5 and temperature in the range of 40–50°C. T. polysporum chitosanase activity was also strongly affected by pH values with a sharply increase for values above 5.2. Again maximal activity was found around pH 5.5 for any temperature.
Although the four tested strains belongs to the same genera and were cultivated in the same substrate and under the same environmental condition, they excreted different amounts of chitosanase, which presented different behavior due to pH changes. Chitosanase produced from T. harzianum and T. viride presented similar enzyme behavior with maximal activity around 1.0 IU/gds. T. koningii and T. polysporum presented maximal enzyme activity at pH 5.5. However T. koningii presented the enzyme activity (~0.6 IU/gds) and T. polysporum presented the highest enzyme activity compared to the other strains (>1.4 IU/gds).
It is difficult to compare the results obtained herein to other published works on chitosanase production under solid-state fermentation by Trichoderma species because the published studies with these fungi have been focused on the production of chitinase and cellulases due their biocontrol ability against insects and other plant pathogens. To our knowledge, this is the first study on Trichoderma spp. chitosanase production because no previous studies on chitosanase production by the strains studied herein were found.
Conclusions
Trichoderma spp. is a suitable microorganism to produce chitosanase enzymes by solid-state fermentation. Temperature effect on enzyme activity was only significant for T. koningii. On the other hand, pH presented a strong and significant effect on enzyme activity for all studied strains. The effect of pH on chitosanase activity depends on the strain under study. Chitosanase activity from the Trichoderma species studied herein can be determined at any temperature in the range of 40 to 50°C. Chitosanase from T. harzianum and T. viride presented optimum activity at pH 5.0 and chitosanase from T. koningii and T. polysporum presented optimum activity at pH 5.5. According to the results presented herein, T. polysporum is the most promising strain to produce chitosanase with maximal enzyme activity of about 1.4 IU/gds, followed by T. viride (~1.2 IU/gds). T. harzianum (1.06 IU/gds). The lowest enzyme activity was obtained from T. koningii (~0.6 IU/gds).
Acknowledgments
The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Aparo à Pesquisa do Estado de São Paulo (FAPESP) for the research scholarship and financial support and to Embrapa Semi-Árido (CPATSA-Brazil) for the microbial strain.
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