Production of β-(1,3)-glucanases by Trichoderma harzianum Rifai: Optimization and Application to Produce Gluco-oligosaccharides from Paramylon and Pustulan

1Departamento de Química e Ciências Ambientais, Universidade Estadual Paulista, IBILCE, São José do Rio Preto, São Paulo, Brazil 2Biorefining Research Institute, Lakehead University, Thunder Bay, ON, Canada P7B 5E1 3Departamento de Bioquímica e Biotecnologia, CCE, Universidade Estadual de Londrina, UEL, Londrina, Paraná, Brazil 4Departamento de Física, Química e Biologia, Universidade Estadual Paulista, FCT, Presidente Prudente, São Paulo, Brazil


Introduction
Many fungal β-D-glucans have been described that possess biological activities including immunomodulation [1]. Modification of polysaccharides has contributed to the development of new industrial applications for these biopolymers in a variety of commercial sectors such as pharmaceuticals, cosmetics and foods [2,3]. Modification through hydrolysis produces gluco-oligosaccharides which have demonstrated health benefits that influence the immune system, and are recognized as having biological response modifying activities [4,5].
In this work, we report on the production of β-(1→3)-glucanases by Trichoderma harzianum Rifai PAMB-86 cultivated in a fermenter on (1→3)(1→6)-β-D-glucan (botryosphaeran) as sole carbon source, and their optimization by the response surface method (RSM). The β-(1→3)-glucanases produced were partially fractionated and employed to enzymatically hydrolyze paramylon and pustulan. Studies involving specific β-glucanases in the hydrolysis of algal and lichen β-glucans are important to understand the nature of the hydrolysis reactions involved, and to develop new strategies to obtain glucooligosaccharides on a large scale for biotechnological applications.

Microorganism and culture conditions
Trichoderma harzianum Rifai (isolate PAMB-86) was obtained from decaying peroba rosa wood (Aspidosperma spp.) and maintained on xylose-agar at 4°C [11] incorporating Vogel minimal salts medium (VMSM) [19]. T. harzianum Rifai PAMB-86 conidia were used to inoculate (1×10 8 spores/flask) three 125 ml Erlenmeyer flasks containing 25 ml liquid medium comprising VMSM and glucose (10 g/l) during 72 h, 180 rpm at 28°C. The mycelium resulting was used as inoculum in fermenter-based experiments. The fungal isolate was cultivated in a laboratory fermenter of 2.5 l capacity equipped with a pH electrode, and facilities for control of agitation and aeration (Technal, Brazil), and was operated in a batch continuous mode. The fermenter was equipped with four removable standard baffles, and a top driven agitator shaft mounted with three standard blades (Rushton impellers). Fermentation was carried out using 600 ml medium comprising VMSM and botryosphaeran (1.5 g/l) as sole carbon source at 28°C and 120 rpm. Aeration, initial pH and time of growth for the production of β-(1→3)-glucanases by T. harzianum Rifai PAMB-86, were used as variables in the statistical experimental design outlined below. Extracellular fluid (ECF) was recovered following centrifugation (7000xg/10 min), exhaustively dialyzed against deionised water at 4°C, and used as the source of enzyme.

Analytical procedures
Hydrolysis products arising from various glucans were measured by the reducing sugars method [20]. Glucose from gluco-oligosaccharides was measured by the glucose oxidase method using a kit (Glicose Enz Color reagent kit; Bio Diagnóstica, Curitiba, Brazil).

HPAEC/PAD analysis of enzymatic hydrolysates
Following enzymatic hydrolysis, undigested polymeric material remaining in the hydrolysates was removed by precipitation with 3 volumes of ethanol [10]. The supernatant was recovered by centrifugation (7000x g/10min), and the ethanol evaporated under vacuum. The resulting syrup was re-solubilized in water, and used for determination of reducing sugars, and aliquots of 0.025 ml taken for sugar analysis by High Performance Anionic Exchange Chromatography with Pulsed Amperometric Detection (HPAEC/PAD) on a Dionex Chromatograph DX 500. Mono-and oligo-saccharides were separated on a CarboPac PA100 (Dionex Chromatography) column (4×250 mm) equipped with a PA-100 guard column at a flow rate of 1.0 ml/ min. The column was equilibrated in 0.1 M NaOH (97%) and 0.5 M sodium acetate (3%). After 15 min, a linear 0-0.25 M concentration gradient of sodium acetate was applied over a 70 min interval while the concentration of NaOH remained at 0.1 M. Sugar quantification was carried out from peak area measurements using response factors obtained from authentic standard sugars, and identified by their retention times (T R ± SD min): glucose (2.93 ± 0.06 min), gentiobiose (4.57 ± 0.21 min), laminaribiose (7.12 ± 0.80 min) and laminaritriose (17.34 ± 0.20 min). Peaks exiting the chromatography column after 20 min were considered oligosaccharides of DP 4 and greater than 4.

Factorial design and analysis
Fermentation conditions to optimise β-(1→3)-glucanase production by T. harzianum Rifai PAMB-86 were studied as variables in a factorial design and analysis by the response-surface method ( Table 1). The independent variables were x 1 = aeration (vvm), x 2 = initial pH, and x 3 = time of growth. The level of these variables and the variation levels for experimental studies on β-(1→3)-glucanase production (Y 1 = U/ ml) are shown in Table 1. Analysis of variance (ANOVA) and multiple regression analysis were performed using STATISTICA Version 6.

Fractionation of β-glucanolytic complex from T. harzianum Rifai PAMB-86
Extracellular fluid (600 ml) containing the enzyme was lyophilized following centrifugation to remove the mycelium. Lyophilized ECF was dissolved in 20 mM sodium acetate buffer (pH 5.0) and applied to a column of Sephadex G-100 (2.5×90 cm, Pharmacia Biotech, Sweden), and eluted with 20 mM sodium acetate buffer (pH 5.0) at a flow rate of 15 ml/h. Fractions of 2.5 ml were collected and analyzed for β-(1→3)glucanase, β-(1→6)-glucanase and β-glucosidase activities. Two fractions were isolated (designated F-I and F-II), and used as the source of enzyme to hydrolyse paramylon and pustulan. All fractionation steps were performed at 4°C.

Production of β-(1→3)-glucanases by Trichoderma harzianum Rifai PAMB-86
Production of microbial β-(1→3)-glucanases are strongly  influenced by the level of β-D-glucan present as inducer, and the types of glucosidic linkages present in the β-glucans used as carbon sources [21][22][23][24]. In a previous study, production of β-(1→3)-glucanases by T. harzianum Rifai grown on botryosphaeran (a (1→3)(1→6)-β-D-glucan from B. rhodina MAMB-05 [23] was optimized in shake-flasks. The results showed that production of β-(1→3)-glucanases was dependent upon the concentration of botryosphaeran and the time of growth, and highest enzyme titres obtained were 1.2 U/ml. A statistical mixturedesign indicated a synergistic effect of botryosphaeran on β-(1→3)glucanases production by T. harzianum Rifai in combination to glucose and lactose as a mixture of carbon sources [24]. It was impractical to increase the botryosphaeran concentration in the nutrient medium in attempts to enhance enzyme titres as this exopolysaccharide is sparingly soluble in water (limit of 3 g/l), and aqueous solutions formed are rather viscous and difficult to manage. Instead, fermentation variables such as initial pH and aeration rate were evaluated in a 2.5 l fermenter (600 ml) to improve β-(1→3)-glucanase titres by T. harzianum Rifai PAMB-86 when cultivated on botryosphaeran. Enzyme production was optimised by the response surface method.
Through multiple regression analysis of the experimental data, a second-order polynomial equation was obtained for β-(1→3)-glucanase production (Equation 1).
Linear-effect terms of the variables x 1 and x 2, and squared-effect terms of the variables x 1 and x 2 were discarded as being non-significant. According to the results, an intercept was significant indicating that the central-points (aeration, 1.5 vvm; initial pH, 5.5; and 5 d growth) were correctly chosen. The variable more important for β-(1→3)-glucanase production by T. harzianum Rifai PAMB-86 was the initial pH followed by the time of growth. The analysis of variance (ANOVA) showed the lack-of-fit (p >0.05) was not significant, indicating that the model was predictive ( Table 2). The experimental and predicted β-(1→3)glucanase activities were in agreement ( Table 1). The R-squared value implied 97% of the variability in the observed response values could be explained by the model, or by experimental factors and their interactions. The pure error was low, indicating good reproducibility of the experimental data.
Statistical analysis by RSM has also been successfully employed by Donzelli et al. [25] to optimise the production of β-(1→3)glucanases by Trichoderma atroviride in shake-flask cultivation using different (1→3)-β-D-glucans as sole carbon sources. According to our experimental data, maximum β-(1→3)-glucanase production (3.7 U/ ml) by T. harzianum Rifai PAMB-86 occurred within 5 d of growth, an initial pH of 5.5 and aeration of 1.5 vvm. This is a 3-fold improvement over shake flask-grown cultures at the same concentration of botryosphaeran. The 3-dimensional surface and contour plot showing the effect of β-(1→3)-glucanase production as a function of initial pH and time of growth is shown in Figure 1.
There were no significant observed alterations (p <0.05) between the initial and final pH in the experimental runs. This behavior was also reported by Théodore & Panda [22] with cultures of a T. harzianum strain using glucose as sole carbon source, and an initial pH of 4.7 was chosen as the best pH for optimal β-(1→3)-glucanase production. Donzelli et al. [25] evaluated growth and β-(1→3)-glucanase production by T. atroviride grown on scleroglucan (a (1→3)(1→6)-β-D-glucan from Sclerotium glucanicum) and glucose as carbon sources, and verified a relationship between β-(1→3)-glucanase activities and initial pH. An increase in initial pH from 5.5 to 6.5 increased enzyme production 16fold depending on other factors that were applied.

Enzymatic hydrolysis of paramylon and pustulan
ECF containing β-(1→3)-glucanase activity was concentrated by lyophyllization and the re-solubilised enzyme preparation submitted to gel filtration chromatography. Fractionation revealed the presence of two enzyme fractions (F-I and F-II) hydrolyzing laminarin (a low branched (1→3)-β-D-glucan with (1→6)-β-D-glucosidic linkages, ~5%). F-I contained mainly β-(1→3)-glucanase activity (0.44 U/ml) as well as β-glucosidase (0.12 U/ml) and β-(1→6)-glucanase activities (0.24 U/ml), while F-II contained lower β-(1→3)-glucanase activity (0.30 U/ml), but no β-(1→6)-glucanase activity. Enzymes F-I and F-II exhibited broad substrate specificity (Table 3; relative activity assigned 100% to laminarin). The enzyme fractions showed no activity towards carboxymethylcellulose and dextran. F-II was not able to hydrolyse lichen pustulan and the oligosaccharides cellotriose and cellotetraose. Both fractions preferred laminaribiose and laminaritriose as substrates to laminarin, while F-I showed highest activity towards cellobiose and gentiobiose.   The β-glucanase complex from T. harzianum Rifai PAMB-86 was fractionated in attempts to separate the enzyme components so as to minimize attack on the oligosaccharides liberated during the early stages of hydrolysis of β-D-glucans by enzyme. Earlier studies using a crude enzyme preparation had demonstrated that gluco-oligosaccharides of DP >3 were largely absent from hydrolysates of laminarin and botryosphaeran [10]. To resolve and avoid this situation, we examined each enzyme fraction on its ability to hydrolyze paramylon and pustulan for the production of fragments of DP >3. Enzyme F-I degraded both β-D-glucans differently as judged by the array of hydrolysis products determined by HPAEC/PAD analysis (Table 4), while F-II hydrolysed paramylon in a similar manner to F-I. Glucose was released in relatively large amounts early during hydrolysis by both enzymes attacking paramylon, and the amounts of reducing sugars liberated increased with time. By 30 min ~15-20% of paramylon had been degraded by both enzymes. Under similar conditions, hydrolysis of pustulan by F-I showed that glucose too was rapidly released, and the reducing sugar content in the hydrolysates increased with time, and after 30 min, ~2% of the pustulan was degraded. Enzymatic hydrolysates of paramylon by F-I and F-II contained laminaribiose and laminaritriose, but gentiobiose was absent, confirming that paramylon from Euglena gracilis did not carry glucose branches linked through β-(1→6) bonds. Gluco-oligosaccharides of DP ≥4 produced from paramylon decreased with time for both enzymes. F-I hydrolysates of pustulan contained gentiobiose and gluco-oligosaccharides of DP ≥4 that increased with time of hydrolysis (Table 4). F-I thus appears to be an excellent enzyme preparation for the production of gluco-oligosaccharides of higher DP's from pustulan. The absence of laminaribiose and laminaritriose in F-I hydrolysates of pustulan confirmed that the pustulan of the lichen, Actinogyra muehlenbergii, did not carry any β-(1→3)-linked glucose residues.

Degrees of freedom Mean square F-test p
Curdlan, a bacterial linear (1→3)-β-D-glucan, was reported to be hydrolyzed by a crude β-(1→3)-glucanase preparation from T. harzianum, and under the conditions, 80% of this polysaccharide was hydrolyzed [13]. β-Glucanases from Streptomyces sp. were also reported to degrade curdlan to mono-, di-and various lower oligosaccharides [15]. The crude β-glucanolytic enzyme preparation from T. harzianum Rifai revealed that these enzymes were also able to produce a broad range of products from botryosphaeran in short incubation times [10] with only tetrasaccharides as the highest DP liberated. Grandpierre et al. [26] observed that the rapid release of glucose could inhibit enzyme action on curdlan.
Analysis of the products obtained after incubation of the β-Dglucans, paramylon and pustulan, with β-glucanases from T. harzianum Rifai PAMB-86 revealed more information about the specificity of the enzymes produced by this fungus, and this information can be useful in further studies to optimize gluco-oligosaccharide production.
In conclusion, the β-glucanases from T. harzianum Rifai PAMB-86 showed maximal enzyme titres at 5 days fermentation, initial pH 5.5 and aeration of 1.5 vvm. This enzyme complex was demonstrated to be useful in the production of gluco-oligosaccharides from algal paramylon and lichen pustulan, which may find applications as prebiotics (nutraceuticals) and modifiers of immunological activities. Enzymatic hydrolysis of paramylon and pustulan (1 g/l; solubilised by autoclaving) was conducted in 20 ml solution (25 mM sodium acetate buffer, pH 4.5) using 1 unit of each enzyme fraction (F-I, F-II; activity measured against laminarin) at 40°C. 1 G 1 , glucose; G 2G , gentiobiose; G 2L , laminaribiose; G 3L , laminaritriose; DP, degree of polymerization; 2 ND, not detectable