**2.1 Recombinant strains of** *Penicillium verruculosum***—Highly effective producers of cellulases**

It is possible to increase the effectiveness of complex cellulase preparations both by improving the properties of individual enzymes and by optimizing the component composition of preparations. Protein engineering allows to change in the properties of already known individual enzymes, significantly increasing their activity and operational stability [49–51]. Since the composition and structure of various types of cellulose-containing raw materials can vary quite a lot depending on the source and type of pretreatment, the component composition of the enzyme preparations used for their hydrolysis should correspond to the composition of the raw materials.

The selection of the composition optimal for processing a particular type of raw material preparation is carried out by comparing the hydrolytic ability of different mixtures of individual enzymes [31, 52]. Genetic engineering of cellulase producers with a subsequent screening of the obtained mutants allows the creation of new highly effective strains of microorganisms secreting a complex of enzymes of optimal composition. At the same time, classical nondirectional mutagenesis also remains an effective tool for obtaining such producers.

The wild strain of *P. verruculosum* WA30 was isolated about 40 years ago. He was identified as a producer of a cellulase complex with high activity in relation to MCC. The productivity of the wild strain was low and was about 7 g/l of secreted protein in the culture fluid [53]. On the basis of the wild strain WA30 by the method of mutagenesis using nitrosoguanidine and ultraviolet irradiation with subsequent selection *P. verruculosum* strains were obtained. The protein concentration in the culture fluid reached 30–40 g/l [54, 55]. The synthesis of extracellular cellulolytic enzymes of *P. verruculosum* is induced by cellulose and gentiobiose, and in most cases, it is subject to catabolic repression by glucose. The peculiarity of the obtained *P. verruculosum* mutants with a high level of extracellular cellulase secretion is that, during the mutagenesis, catabolic repression was reduced. Changes in pH during fermentation can influence the composition of the secreted cellulase complex. Some side activities, such as xylanase and amylase, become higher at higher pH values.

The enzyme complex secreted by *P. verruculosum* contains more than 20 enzymes differing in biochemical and catalytic properties. The main cellulases secreted by the control highly productive strains B1-221-151 (contracted B151) and B1-537 are CBH I (Cel7A), CBH II (Cel6A), EG I (Cel7B), EG IIa (Cel5A), EG IIb (Cel5B), EG III (Cel12A), and BGL (Cel3A). The content of CBH I is about 35%, CBH II is 21–33%, the content of these EGases varies from 2 to 5%, and the percentage of BGL is 3–4% [38, 56, 57]. In addition to cellulases, the enzyme complex also includes other carbohydrases—xyloglucanases, xylanases, α-galactosidase, and glucoamylase (a total of 18–30% of the total protein). The molecular weights of enzymes vary from 19 to 120 kDa and isoelectric points from 2.0 to 5.8. For most of these cellulases, the pHoptima of activity is in the pH range of 4.0–5.0 [38]. As a result of the limited proteolysis of the linker *P. verruculosum* cellulases, having CBM (CBH I, CBH II, EG I), as a rule, is represented by high-molecular (full-size) and low-molecular forms, the latter being the catalytic domain of a specific enzyme without CBM [38].

As mentioned above, CBH I and CBH II of *P. verruculosum*, when acting on crystalline cellulose, are significantly superior in hydrolytic ability to both CBH from *T. reesei* (of which CBH I is actually the "gold standard" in the study of properties of the cellulases [58]); at the same time, even the low molecular weight form of CBH II from *P. verruculosum* is comparable in effectiveness to the full-size CBH I from *T. reesei* [18]. This, together with a higher level of BGL activity, leads to a higher potential of *P. verruculosum*-based enzyme preparations compared to traditional commercial *T. reesei* preparations [17, 18, 34].
