**2.2 Hydrolytic potential of** *P. verruculosum* **cellulase complex in comparison with industrial analogs**

One of the important criteria for comparing the effectiveness of enzyme preparations intended for the biodegradation of cellulose-containing raw materials is their specific activity on a number of substrates—soluble and insoluble polysaccharides (FP, MCC, CMC, xylan), synthetic substrates (pNPG, pNPX), and oligosaccharides (cellobiose). These activities on the example of the enzyme preparations obtained with the help of fungi of the genus *Trichoderma* (all the preparations Accelerase and Cellic CTec) and *Penicillium* as producers are shown in **Table 2**.

The presented data make it possible to characterize the individual components of the enzyme complex and their balance in the composition of the preparation, which mainly determines the effectiveness of their action in the hydrolysis of a substrate of complex composition, which is plant biomass.

Activity on filter paper characterizes the overall activity of enzyme preparations in relation to insoluble cellulose [59]. On average, it was 0.6–1.2 U/mg of protein, while the commercial preparation Accelerase1000 was characterized by the increased activity of 1.7 U/mg, and Accelerase XY had extremely low activity on filter paper.

MCC activity illustrates the ability of enzyme preparations to hydrolyze highly ordered crystalline zones of cellulose (crystallites).

Preparations of Cellic CTec-1 and Cellic CTec-2 had the same MCC activity (0.3 U/ mg protein); preparations of Accelerase 1500 and DUET, as well as *P. verruculosum*

*Cellulases from Mycelial fungi* Penicillium verruculosum *as a Real Alternative to* Trichoderma*… DOI: http://dx.doi.org/10.5772/intechopen.111851*


#### **Table 2.**

*Specific activities (units/mg of protein) of enzyme preparations obtained with the help of fungi of the genus*  Trichoderma *and* Penicillium *as producers.*

B151, were characterized by activity of 0.7–0.8 U/mg of protein; while Accelerase XY corresponded to a low level of MCC activity (0.1 U/mg of protein), and Accelerase 1000 had the highest level (1.2 U/mg of protein) compared to other drugs.

The activity of preparations on CMC demonstrates their ability to hydrolyze less ordered, amorphous zones of cellulose [60, 61]. The hydrolysis of these regions of the substrate is carried out mainly by endoglucanases, which hydrolyze internal β-1,4 glucosidic bonds remote from the ends of the cellulose polymer chain, with the formation of fragments of the polymer substrate chain and cellooligosaccharides, which is accompanied by a significant decrease in the degree of substrate polymerization and, as a result, a decrease in its viscosity (which is especially important in the first stages of the bioconversion process). Reducing the viscosity of the reaction mixture is a very significant factor in the industrial implementation of enzymatic hydrolysis processes since increased viscosity can lead to a significant decrease in the efficiency of some production stages (for example, due to a decrease in heat transfer rates, mixing, mass transfer, etc.). The considered commercial enzyme preparations, on average, were characterized by CMC activity of 8–12 U/mg of protein, while Accelerase XY corresponded with a low level of activity (0.8 U/mg of protein), and the *P. verruculosum* B151 preparation had the highest value (18, 3 U/mg protein) compared to other preparations.

In general, the specific activities of the *P. verruculosum* B151 preparation in relation to various substrates, which characterize the effectiveness of individual components of multienzyme complexes, are comparable with similar activities of commercial *Trichoderma* preparations. However, the specific activities given in **Table 2** were determined from the initial rates of hydrolysis of the corresponding substrates, and their comparison is not sufficient to compare the hydrolytic activity of enzyme preparations, since during long-term hydrolysis of biomass, the processes of enzyme inactivation, their inhibition by lignin (for due to unproductive adsorption of enzymes on it), as well as inhibition of enzymes by the reaction products.

For a more detailed consideration, **Table 3** shows the data characterizing the results of testing enzyme preparations during long-term (exhaustive) hydrolysis of biomass of various types [62]. Pretreated corn stalks, bagasse, and coniferous and deciduous wood were used as substrates. The selected substrates differ in composition, including the content of cellulose and hemicelluloses (xylans), as well as the content of lignin. The content of xylans decreased in the series: corn stalks, bagasse, aspen wood, and pine wood. In addition, MCC was used, which made it possible to evaluate the effectiveness of cellulase enzyme preparations in relation to a lignin-free substrate [63].

The hydrolytic efficiency of enzyme preparations based on fungi of the genus *Trichoderma* and *Penicillium* was evaluated by the formation of reducing sugars (RS) at various dosages of enzyme preparations (**Table 3**).

The enzyme preparations Accelerase 1000, Accelerate 1500, and Accelerate DUET had approximately the same efficiency of hydrolysis (RS yield) of pretreated corn stalks and bagasse, shredded pine, and aspen wood. The effectiveness of these preparations can be taken as an "average level".

Accelerase XY, which is essentially a xylanase preparation, turned out to be the least effective among those studied for the hydrolysis of pretreated corn stalks, shredded pine and aspen wood, and MCC but proved to be competitive in the hydrolysis of pretreated bagasse.

Cellic CTec-1 and Cellic CTec-2 preparations had approximately equal activity values for FP and MCC, which turned out to be less than the corresponding values for Accelerase 1000, Accelerase 1500, and Accelerase DUET preparations (**Table 2**). At the same time, Cellic CTec-2 was characterized by increased activities in relation to CMC, pNPG, and cellobiose, compared to Cellic CTec-1, as well as Accelerase 1000,


#### **Table 3.**

*Yields of RS as a result of 48-hour hydrolysis of biomass with the enzyme preparations obtained using fungi of the genus Trichoderma and Penicillium. The dosage of enzyme preparations is 2, 5, and 10 mg of the preparation protein per 1 g of dry matter of the cellulose-containing substrate, 50°C, pH 5.0. The substrate concentration was 100 mg/ml on a dry matter basis.*

*Cellulases from Mycelial fungi* Penicillium verruculosum *as a Real Alternative to* Trichoderma*… DOI: http://dx.doi.org/10.5772/intechopen.111851*

Accelerase 1500, and Accelerase DUET. In addition, Cellic Ctec-2 also had high xylanase activity. Such an advantage of Cellic CTec-2 in relation to Cellic CTec-1 and other studied commercial preparations, revealed in a comparison of their activities in terms of initial hydrolysis rates, was also observed during long-term biomass hydrolysis. Cellic CTec-2 has proven to be the most effective for hydrolyzing pretreated corn stalks and bagasse, ground pine wood, and MCC. The overall effectiveness of the Cellic CTec-1 preparation in long-term biomass hydrolysis was lower than the efficiencies of the Accelerase1000, Accelerase1500, and Accelerase DUET preparations, despite comparable values of the main corresponding activities (**Table 2**). Thus, among the studied commercial preparations, Cellic CTec-2 was the most effective for the hydrolysis of the considered biomass samples.

According to the data given in **Table 2**, the *P. verruculosum* B151 enzyme preparation had increased activities for FP, MCC, CMC, and xylan compared to the commercial Cellic CTec-2 preparation. At the same time, the BGL activity of the *P. verruculosum* B151 preparation was lower; therefore, the combined preparation *P. verruculosum* B151 + *P. verruculosum* F10 in a ratio of 9:1 for the protein was used in the hydrolysis experiments. The combination of enzyme preparations B151 + F10 exceeded the effectiveness of the preparation Cellic CTec-2 with prolonged hydrolysis of crushed aspen wood, both preparations hydrolyzed pine wood in the same way; with the hydrolysis of pretreated corn stalks and bagasse, the effectiveness was slightly lower than the result of Cellic CTec-2, but nevertheless higher than the effectiveness of the other investigated commercial preparations.

As can be seen from **Table 3**, the increase in the depth of hydrolysis of pretreated corn stalks (48 hours of hydrolysis) with an increase in the dosage of the most effective enzyme preparations (Cellic CTec-2 and B151 + F10) turns out to be very insignificant, which indicates the achievement of the maximum degree of enzymatic conversion of this substrate and its high reactivity (and high efficiency of the pretreatment processing of this type of biomass). Pretreated bagasse and crushed aspen wood were characterized by a greater increase in the yield of RS than crushed pine wood with an increase in the dosage of enzyme preparations, which is explained by the increased content of lignin in pine wood compared to other substrates under consideration. Also in pine, unlike bagasse, there are pitch and hardly hydrolyzable galactomannan. For the hydrolysis of bagasse, which contains readily available polysaccharides (arabinoxylan and xylan), the presence of xylanase, which is present in penicillin preparations, is important.

The maximum values of the RS increase also allow us to characterize and compare the effectiveness of the studied enzyme preparations conveniently analyzed by the results of saccharification of the MCC model substrate. It was shown that the degree of hydrolysis of MCC by all enzyme preparations except Accelerase XY turned out to be significantly higher than the maximum degree of hydrolysis of various types of biomass provided by them. At the same time, the maximum increase in the RS yield per unit mass of the consumed enzyme preparation corresponded to the Cellic CTec-2 and *P. verruculosum* B151 + F10 enzyme preparations, which demonstrated the highest efficiency in the hydrolysis of various types of biomass.

The effectiveness of the action of enzyme complex that performs the bioconversion of plant biomass is its most important characteristic, which determines the feasibility of its use in biotechnology and the resulting economic benefit. This characteristic, in turn, generalizes such important properties of the preparation as specific activities for a number of substrates and the limiting degrees of conversion of plant materials during long-term hydrolysis.

A comparative study of commercial enzyme preparations based on *Trichoderma* and an enzyme preparation based on *P. verruculosum* indicates that it is not inferior to commercial *Trichoderma* biocatalysts or surpasses them in hydrolytic activity in the processes of saccharification of various types of plant materials. This allows us to state that biocatalysts obtained on the basis of *P. verruculosum* producer strains can be a competitive alternative when scaling up biotechnological processes of bioconversion of renewable plant materials.
