**2. Enzymatic complex for deep destruction of cellulose-containing biomass**

Enzymatic hydrolysis allows the destruction of plant raw materials to monosaccharides without significant energy costs and anthropogenic impact on the environment. Since plant biomass is a complex substrate that includes polysaccharides of various compositions, a complex of enzymes of various specificities is necessary for its deep destruction, which carries out cooperative hydrolysis of its constituent

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

components. In industrial biotechnology, various microscopic fungi are widely used as producers of such enzyme complexes. They are the main source of commercial cellulase preparations produced on an industrial scale in different countries of the world. For a long time, it was believed that cellulolytic fungi belonging to the genus *Trichoderma* are the undisputed leaders in the secretion of the most active cellulases [15–19]. Commercial cellulase preparations based on mutant strains of *Trichoderma reesei* (synonym of *Hypocrea jecorina*) are most common on the enzyme market. Therefore, it is not surprising that most of the research and developments on the bioconversion of lignocellulose raw materials (primarily for the production of second-generation bioethanol) were based on the use of *T. reesei* cellulases. However, in recent years, more and more publications demonstrating that there are alternatives to *T. reesei*-based enzyme preparations have appeared [16–18, 20–22]. In particular, it is related to cellulases produced by fungi from the genera *Penicillium* and *Myceliophthora*. Modern developments aimed at obtaining cellulose preparations of a new generation are characterized by using methods of protein and genetic engineering; recombinant (modified) enzymes with improved properties from various microorganisms are included in the composition of these preparations.

The leading role of mutant or recombinant strains of fungi of the genus *Trichoderma* (*T. reesei, T. viride, T. longibrachiatum*) is explained, firstly, by their high secretory ability, and, secondly, by the variety of enzymes produced with different substrate specificity [23–26]. Therefore, it is not surprising that biocatalysts (enzyme preparations) of cellulases based on *Trichoderma* fungi are produced in many countries by leading manufacturers of industrial enzymes, in particular, Novozymes (Denmark), Genencor and Danisco Division (USA), Iogen (Canada), PrimAlko (Finland), Röhm Gmbh (Germany), EnMex (Mexico), Meiji Seika Kaisha Ltd. and Shin Nihon Chemical Co. (Japan), etc. At the same time, the search for new producers of enzymes suitable for the hydrolysis of plant raw materials, as well as an increase in the overall activity and balance in the component composition of already known enzyme complexes, is still an actual task of modern biotechnology.

Strains of fungi of the genera *Penicillium*, *Acremonium*, *Chrysosporium*, *Chaetomium*, and *Humicola* can become a worthy alternative to strains of the genus *Trichoderma* since the enzyme complexes produced by them are not inferior in hydrolytic ability and sometimes are superior to those obtained with the help of known strains of *Trichoderma* [27–31].

Promising producers of highly active cellulase complexes are some species of fungi from the genus *Penicillium*. Cellulases from *Penicillium*, as a rule, surpass *T. reesei* enzymes in the rate of hydrolysis and glucose yield from various cellulose-containing substrates at the same dosage of protein concentration or cellulase activity (**Table 1**). Almost all the authors note a relatively high level of beta-glucosidase activity in enzyme preparations based on penicillin. This property is one of the main advantages of fungi of the genus *Penicillium* over *Trichoderma*. As a consequence, the gain of penicillin enzymes in glucose output sometimes reaches a 5-fold size (**Table 1**) [17, 18, 32–34]. Only after the addition of an excess of exogenous BGL, *T. reesei* preparations are able to provide comparable glucose yields. However, in some cases, even with the addition of BGL cellulase from *T. reesei* could not compete with cellulase complexes from *Penicillium* [17, 18, 20]. For example, with the same cellulase activity on filter paper in the reaction mixture (10 U/g of substrate), the preparation of *T. reesei* RUT-C30, enhanced with the addition of aspergilli BGL (6 U/g), was inferior in depth of hydrolysis of microcrystalline cellulose (MCC) after 48 hours to the preparation of *P. pulvirrolum* F-2220 (the difference


*\*The ratio of product concentrations (usually glucose) obtained by hydrolysis of cellulose-containing substrates under the action of enzyme preparations based on fungi Penicillium and T. reesei.*

#### **Table 1.**

*Comparison of the hydrolytic ability of cellulase complexes from fungi of the genus Penicillium and T. reesei.*

was 25%), whereas in the hydrolysis of pretreated spruce wood and wheat straw, both preparations showed comparable results [20].

Sequencing and annotation of the genomes of *P. decumbens* 114-2, *P. funiculosum* NCIM 1228, and *Piptochaetium verruculosum* TS63-9 have shown that these species of fungi differ in a richer set of enzymes that catalyze the cleavage of components of lignocellulose materials compared with *T. reesei* [35–37]. It especially concerns cellulases with a cellulose-binding module (CSM) and hemicellulases. Analysis of *P. decumbens* 114-2 secretome obtained by cultivation on a medium with wheat bran showed that it was richer in carbohydrates than that obtained on a medium with glucose [35]. In the secretome of *P. funiculosum* NCIM 1228, 113 enzymes acting on carbohydrates were detected by quantitative mass spectrometry, of which 92 were glycoside hydrolases [37]. Apparently, the genomes and secretomes, which are characterized by a high content of glycoside hydrolases and their accompanying enzymes, are also characteristics of other species of fungi of the genus *Penicillium*. Another reason for the high efficiency of cellulase complexes based on *Penicillium* fungi is the high specific activity of their key components, namely CBH I and CBH II, compared to the corresponding *T. reesei* enzymes. The difference can reach 2–2.5 times. In particular, this was demonstrated in the example of CBH *P. funiculosum*, *P. pulvirrolum*, *P. verruculosum*, and *P. canescens* [18–20, 38, 39]. One of the reasons for such high specific activity in the case of CBH I and CBH II of *P. verruculosum* is the optimal distribution of N-bound glycans on the surface of the catalytic domain of these enzymes [40, 41].

One of the properties that negatively affect the activity of cellulases during the hydrolysis of lignocellulose substrates is their unproductive adsorption on lignin [42–48]. During the hydrolysis of MCC, enzymes from *P. verruculosum* are significantly less inhibited by two types of lignin artificially added to the reaction system *Cellulases from Mycelial fungi* Penicillium verruculosum *as a Real Alternative to* Trichoderma*… DOI: http://dx.doi.org/10.5772/intechopen.111851*

than cellulases from five different *T. reesei* preparations. At the same time, the lignin regenerated from the solution in the organosolve caused a stronger inhibition (by 11–84%) than the residual lignin obtained by exhaustive enzymatic hydrolysis of pretreated wood (8–58% inhibition). Similar results demonstrating a lower negative effect of lignin on *P. verruculosum* cellulase compared to *T. reesei* enzymes were obtained in the work [34].

In the example of enzymes from *P. oxalicum*, it was shown that the degree of unproductive adsorption on lignin decreases in the raw of CBH – xylanase – EG – BGL, and mainly electrostatic interactions are responsible for the binding of xylanase and EG to lignin, whereas, in the case of CBH and BGL, hydrophobic interactions have a decisive influence. CBM in CBH plays a noticeable role in binding to the lignin [48]. The changes in the properties of lignin, caused by the pretreatment of lignocellulose raw materials, had a noticeable effect on the adsorption of *P. oxalicum* enzymes, as in the work [46]. Thus, as a result of pretreatment with hot water, lignin from corn waste got an increased ability to bind proteins [48].

Despite the high hydrolytic potential, commercial cellulase preparations from penicillins have not yet become widespread, although they are produced on an industrial scale by the company Adisseo in France (Rovabio ® Excel based on *P. funiculosum*) [36] and in China (enzyme preparations based on *P. decumbens*) [35]. Perhaps this is due to the fact that the existing strains of *Penicillium* are still inferior to the best industrial strains of *T. reesei* in extracellular protein secretion [17, 18]. One of the reasons may be the insufficiently aggressive marketing policy of the producers of these enzymes.
