Commercial cellulases from Trichoderma longibrachiatum enable a large-scale production of chito-oligosaccharides

Introduction

Chitosan (CTS) is the second most abundant polysaccharide next to cellulose and found in crustaceans, insects and fungi. Consisting of glucosamine (GlcN) and to a minor percentage of N-acetyl glucosamine (GlcNAc), CTS is one of few native cationic polysaccharides known to date [1]. Among bioactive polysaccharides, CTS is of particular interest due to the variety of biological activities that were observed for this biopolymer, including antimicrobial activity and antitumor activity [2, 3]. The range of CTS applications is broad, comprising the food industry, agriculture and biomedical applications [4]. CTS is soluble in dilute acid solutions, however, shows poor solubility under physiological conditions, which is the major limitation of further utilization for various applications [5, 6]. The increase in solubility can be achieved by decreasing the molecular weight of CTS, which results in low molecular weight chitosan (LMWC) and chito-oligosaccharides (COSs). These hydrolysis products are soluble in water and physiological media, which facilitates their characterization and usage. The increased solubility results in higher bioavailability and consequently leads to the enhancement of the biological activities, with preferred application of COSs when compared to the native polymer [7, 8]. Chemical and enzymatic hydrolysis of CTS is used to date, but these methods thereby greatly differ regarding their hydrolysis products. Chemical CTS hydrolysis is performed using harsh chemicals resulting in heterogeneous pools of COSs with oligomers of inconsistent degree of polymerization (DP), degree of N-acetylation (DA) and pattern of N-acetylation (P_A) [9, 10]. Better controlled and environmentally friendlier hydrolysis is possible using chitosan hydrolyzing enzymes, which specifically cleave the polymer chain on the preferred cleavage sites and thus release a defined mix of COSs. Certain heterogeneity remains, which requires thorough analysis of the enzymatic hydrolysis products to refer distinct biological properties to structural properties of the COSs [11]. To date, only few studies on COS production from CTS include comprehensive product analysis. The use of chitosan specific enzymes like chitosanases is still costly and further impeded by the limited availability of these enzymes. Thus, great potential thereby relies on the use of readily available, commercial enzymes with chitosan hydrolyzing activity. A variety of glycoside hydrolases (GHs) is described to possess chitosan hydrolyzing activity and recent studies revealed the potential of commercial cellobiohydrolases (CBHs) to produce COSs. Classified as exoglucanases, CBHs commonly cleave cellobiose from cellulose, however, endo-activity was observed for the hydrolysis of solubilized chitosan [12, 13]. It was further shown that CBHs of different origin produce different COSs in term of DP and DA, whereby CBHs from Trichoderma longibrachiatum and from Hypocrea jecorina were successfully proven for chitosan hydrolysis.

Within this study, a commercial cellulase preparation from Trichoderma longibrachiatum was used for the large-scale production of COSs and comprehensive product analysis unveiled their structural properties. The investigation of the enzyme preparation regarding its chitosan hydrolyzing activity and the impact of different process parameters on the COS composition strengthened the potential of this low cost enzyme source for the production of well-defined COSs for various applications.

Materials and methods

Results and discussion

Enzymatic production of COSs lead to more homogeneous reaction products which are easier to purify when compared to chemical hydrolysis. However, the use of pure chitosanases for this purpose is still expensive and thus not economically feasible. Cellulases are a well-studied enzyme class and used for many industrial processes [17]. Preparations containing different individual enzymes such as endoglucanases (EG) and CBH are to date commercially available and affordable, which also renders them of particular interest for COS production. The cellulase preparation used in this study is derived from Trichoderma longibrachiatum (TrlCel) and was to the best of our knowledge so far not considered for COS production. Previous studies on glycoside hydrolases of this fungus revealed their chitosan hydrolyzing activity, in particular of the respective cellobiohydrolases [12].

In a first step, the pH and temperature optimum of TrlCel was determined assessing the concentration of released sugars. A pH optimum of pH 6 was obtained (Fig. 1a), which was in accordance to the pH optima described for various cellulases for cellulose hydrolysis [18]. Likewise, pH optima of cellobiohydrolases reported for chitosan hydrolysis lie in this pH range [12]. The temperature optimum of the TrlCel was expectably in the range of 60°C (Fig. 1b), which was in agreement with temperature optimua of fungal cellulases [19]. Although the assessment of the chitosan hydrolyzing activity based on the general determination of released sugars is valid, more detailed information about the impact of the hydrolysis conditions on the reactions products is of high interest. COSs are a diverse substance class, which are characterized by multiple parameters like the molecular weight and the DA. It is obvious that the reaction conditions can influence the structural properties of the reaction products. In the case of glycoside hydrolases (GHs) and their catalytic acid/base residues, the pH is a parameter, which can alter the catalytic mode of action of the enzyme [20]. The ionization behavior of the catalytic residues and other residues of the active side has a high impact on the substrate binding and cleavage sites. LCMS analysis of the reaction products obtained after chitosan hydrolysis at the optimal pH and temperature revealed COSs without N-acetyl groups in the range of DP 2–6. The impact of the pH on the COS composition was further studied in the pH range from 4 to 6. Expectably higher yields were observed for all oligomers at pH 5.5 and 6.0 (temperature optimum), however, an inverse picture was observed in the case of the mono-acetylated trimer, which was mainly formed at pH 4.0 and 4.5 (Fig. 1c). Since this was the only found COS bearing an N-acetyl group, it is assumed that a specific ionization of the residues in the active side plays a role in substrate binding and subsequent cleavage of the polymer chain. As in chemical catalysis, enzymatic reactions also rely on the chemistry of the chosen solvent, a fact that holds true for reactions in aqueous and organic solvents [21]. Studies on the impact of the pH on chitosan hydrolysis of various GHs could be a promising route to direct the physicochemical properties of COSs by choosing the proper solvent.

Fig. 1:

Determination of the effect of the pH and temperature on the hydrolysis of chitosan by TrlCel. pH optimum (a), temperature optimum (b) and the impact of the pH value during enzymatic hydrolysis on the COS composition (c).

The COS mixture obtained from enzymatic hydrolysis of chitosan hydrolysis at optimal conditions was analyzed by NMR (Fig. 2). The data was analyzed consulting previously published spectra analysis of COS mixtures obtained from chitosan hydrolysis by cellobiohydrolases [12, 22]. The COSs were partially desalted prior to NMR analysis, however, a high acetate signal could still be observed. The overall DA of the COS mixture was determined by referring the H-2 protons of the glucosamine (GlcN) moieties at 2.60–262 ppm to the acetyl signals of occurring N-acetyl glucosamines (GlcNAc) at 2.08–2.09 ppm. A GlcN:GlcNAc ratio of 5:1 was calculated, which confirmed the low content of N-acetyl groups in the produced COSs that was also described by the LCMS data. The ¹H NMR spectrum (Fig. 2) contained proton peaks in the anomeric region at 4.34–4.50 ppm, which refer to the reducing and non-reducing GlcN units in the COSs. Only very minor ¹H/¹³C signals of GlcNAc were found at 5.18/91.1 ppm. The low signal of α-GlcNAc suggests that fractions GlcNAc residues also occur at the reducing end. HSQC and HMBC experiments further confirmed that the reducing and non-reducing end were mainly composed of GlcN residues, which assumed that the preferred cleavage site of TrlCel is GlcN-GlcN. This cleavage pattern was previously observed for a cellobiohydrolase from Trichoderma longibrachiatum [12], which led to the assumption that this enzyme occurs in the cellulase preparation used within this study.

Fig. 2:

600 MHz 1H NMR spectrum of the COSs obtained after chitosan hydrolysis by TrlCel.

Native PAGE analyisis was performed to assess the protein composition of the crude cellulase mix. Two main bands were identified at 70 kDa and around 90 kDa, respectively. The bands were resected from the gel and incubated with chitosan to assess the chitosan hydrolyzing activity of the respective proteins. Only the enzyme in band 2 at 70 kDa hydrolyzed chitosan (Fig. 3) and was thus identified as the chitosan responsive enzyme in the applied TrlCel mix. The band was analyzed via MS/MS after digestion with trypsin and the obtained results from the ion search were compared to the UniProt database to determine the responsible enzyme. Due to the lack of entries in the sequence database of Trichoderma longibrachiatum, the database of Trichoderma reesei was considered because of the high homology. An exoglucanase 2 from Hypocrea jecorina (the anamorph of T. reesei) was identified for the chitosan hydrolyzing band 2 with a log(e) score of –441 and 38 found ion fragments. The protein refers to a cellobiohydrolase II, which was previously identified to have chitosan hydrolyzing activity [12, 13].

Fig. 3:

Native PAGE analysis of the cellulose enzyme preparation hydrolyzing chitosan (M: page ruler pre-stained protein ladder; lane 1: enzyme mix) and the reducing sugar concentration obtained after incubation of chitosan with the resected gel band. Only the incubation of chitosan with band 2 led to chitosan hydrolysis.

After thorough analysis of the COS production by the commercial TrlCel mix, the process was scaled up to a 20 g batch and the most crucial parameters were monitored over 24 h. The progress in COS production was monitored as function of the increasing concentration of reducing sugars (Fig. 4a). A decreased production over time was observed after 6 h of incubation and the curve further flattened until 24 h. Previous studies did not show a significant increase of the reducing sugar concentration after 24 h (data not shown). Another important parameter is the change in viscosity over time, which gives information about the mode of action of the enzyme on the respective polymer. Cellobiohydrolases are commonly exo-acting on cellulose, however show endo-activity in case of chitosan hydrolysis [12, 13]. Applying the TrlCel mix, an immediate decrease of viscosity was observed, which confirmed the endo-acting activity of TrlCel (Fig. 4b). The different mode of action on chitosan compared to cellulose can be explained by the solubility of chitosan, which facilitates an endo-mode of action.

Fig. 4:

Process characterization of the large-scale production of COSs by TrlCel. Progression of the chitosan hydrolysis illustrated in terms of the reducing sugar concentration (a), the decrease in viscosity over time (b) and the enzyme activity of TrlCel along the process.

When applying enzymes without prior immobilization on carriers, the stability of the enzyme over time is a crucial parameter, which can greatly alter the production process. The described COS production was carried out at 50°C, a temperature that effects the activity of many non-stabilized enzymes after long exposition. The activity of TrlCel was monitored and a continuous decrease of the enzyme activity was observed, which finally resulted in around 80% activity loss after 24 h. The loss of activity could be retarded by the immobilization of the respective enzyme on a stabilizing carrier [23]. Less enzyme would thus be necessary to achieve similar yields and the same enzyme could be used for several cycles. Whether this is practicable or not for readily available enzyme mixes like the used TrlCel remains part of discussion. The COSs were separated from the reaction broth like mentioned in the method section and freeze dried. A crude yield could be identified to be 60% of COS referred to the initial chitosan amount applied in the hydrolysis.

Conclusion

This study demonstrated the potential of a commercial cellulase preparation from Trichoderma longibrachiatum for the large-scale production of COSs. The optimum hydrolysis parameters were determined and the oligomeric products produced by the TrlCel mix were thoroughly analyzed according to their DP and DA. Only COSs without N-acetyl groups were detected and the pH was assumed to be crucial factor for the binding and cleavage specificity due to traces of mono-acetylated COSs that were mainly found at lower pH values. The chitosan hydrolyzing enzyme in the cellulase preparation was identified to be a cellobiohydrolase, an enzyme class where chitosanase activity was only described recently. In a final experiment, the suitability of this low-cost source for chitosan hydrolysis was successfully proven for large-scale production of COSs. The combination of the use of a low cost enzyme source for the efficient production of COSs and the detailed analysis of the oligomeric products is important to improve the access to this bioactive substance class. Based on large amounts of COSs, separation protocols can be developed to obtain pure COSs from the produced mixtures Large amounts of COSs with defined structural properties could be produced, which facilitates the study of the many interesting bioactivities that are described to date.