Comparison of the hydrolytic ability of cellulase complexes from fungi of the genus
Abstract
Abstract The possibility of using the recipient strain Penicillium verruculosum B1-537 (ΔniaD) as a producer of laboratory and industrial enzymes was considered. The advantage of this strain is its ability to secrete a basic cellulase complex consisting of cellobiohydrolases, endoglucanases, and β-glucosidase, which exceeds in its hydrolytic ability the enzyme complex of Hypocrea (Trichoderma) strains. Using the expression system, the basic complex of cellulases of the recipient strain Piptochaetium verruculosum B1-537 (ΔniaD) was supplemented with new (booster) enzymes that are necessary to increase its hydrolytic activity. Enzyme preparations adapted to the processing of various types of renewable plant biomass were obtained.
Keywords
- Penicillium verruculosum
- Trichoderma
- cellulase complex
- cellulose bioconversion
- renewable plant biomass
- industrial enzymes
1. Introduction
Currently, the attention of scientists is focused on the depletion of reserves of fossil energy sources. Rapid population growth provides high energy needs for fossil resources such as coal, natural gas, and crude oil [1]. The burning of fossil fuels poses a threat to the environment due to the emission of greenhouse gases, which, in turn, lead to global climate change. As for renewable energy sources, fuels and products derived from cellulosic waste do not harm the environment and can be considered as an alternative to fossil energy sources. Plant waste not only supports economic development but also creates an ecologically friendly environment for the production of energy and biochemicals [2, 3, 4, 5, 6, 7, 8, 9, 10].
Biomass cellulose is a plant polysaccharide and is an almost inexhaustible source of renewable raw materials that can be converted enzymatically into glucose. In turn, glucose is a raw material for microbiological processes of obtaining liquid and gaseous fuels (ethanol, butanol, etc.), organic and amino acids, feed protein, and many other useful products of microbiological synthesis [11]. Coniferous and deciduous wood and its waste are of particular interest [12].
An important stage of bioconversion of cellulose-containing biomass, which prevents its commercial use, is the enzymatic conversion of cellulose into glucose. Natural wood and other lignocellulose materials are resistant to enzymes due to the crystalline structure of cellulose and the presence of lignin and hemicellulose-protecting cellulose fibers [12]. The reactivity of natural crystalline cellulose (for example, cotton) or lignocellulose (wood of various species, grass straw) during the enzymatic conversion is low, which is accompanied by extremely low yields of glucose and other sugars. Effective enzymatic hydrolysis of cellulose-containing biomass requires its pretreatment to increase reactivity by destroying the crystal structure of cellulose and completely or partially removing the lignin. As methods of pretreatment, gamma irradiation, mechanical grinding in a ball mill, treatment with mineral acids (sulfuric, phosphoric), cadoxene, and alkali delignification are used [12].
The influence of such factors on the efficiency of enzymatic conversion as changes in the degree of crystallinity of cellulose (determined by X-ray diffraction), the availability of cellulose surface to enzyme molecules (measured by such methods as protein adsorption, as well as by thermal desorption of nitrogen-hydrogen mixture), the size of cellulose particles (by dispersion analysis using optical microscopy), and the degree of polymerization of cellulose (determined by the viscosity of cellulose solutions in cadoxene) was investigated. Based on the results of these studies, the influence of these parameters on the efficiency of enzymatic cellulose conversion was quantified—it linearly depends on a decrease in the degree of crystallinity and an increase in the surface area available to the enzyme molecules, but it does not depend much on the geometric size of cellulose particles and its degree of polymerization [13].
Deep cleavage of crystalline cellulose to soluble sugars (glucose) is carried out by a complex of cellulolytic enzymes, including endoglucanases (EG), cellobiohydrolase (CBH), and β-glucosidase (BGL). The activity of the components of this complex and their interaction determine the action of the enzymes on cellulose-containing substrates [14].
Many research groups are looking for new effective producers of cellulases and hemicellulases. Research is being conducted to improve existing strains of microorganisms in order to increase the production of various cellulases and reduce their cost. In this chapter, we compare the hydrolytic potential of multi-enzyme cellulose complexes produced by different strains of fungi and evaluate the role of different enzymes in the hydrolysis of pretreated cellulose-containing substrates.
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 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
The leading role of mutant or recombinant strains of fungi of the genus
Strains of fungi of the genera
Promising producers of highly active cellulase complexes are some species of fungi from the genus
Cellulase producer | Substrate | Difference, times* |
---|---|---|
Abies alba pretreated by steam explosion or organosolve | 1.6−3.6 [17, 18] | |
Poplar wood pretreated by organosolve | 1.4−2.0 [17, 18] | |
Red maple wood pretreated by organosolve | 1.5−2.1 [17, 18] | |
Spruce wood pretreated by steam explosion | 2.1 [18] | |
Bleached eucalyptus kraft pulp | 1.1 [17, 18] | |
Crushed corn cobs | 1.1−1.5 [18] | |
Stipa tenacissima | 1.8 [18] | |
Corn cobs alkali pretreated | 2.0−2.2 [32] | |
Microcrystalline cellulose | 3.6 [33] | |
Spruce wood pretreated by steam explosion | 3.1 [17, 18] | |
Microcrystalline cellulose | 1.3−1.7 [18] | |
Cellulose from eucalyptus | 1.3−3.9 [17, 18] | |
Coniferous wood pretreated by organosolve | 1.3−5.1 [17, 18] | |
Wheat straw alkali pretreated | 1.6 [34] |
Sequencing and annotation of the genomes of
One of the properties that negatively affect the activity of cellulases during the hydrolysis of lignocellulose substrates is their unproductive adsorption on lignin [42, 43, 44, 45, 46, 47, 48]. During the hydrolysis of MCC, enzymes from
In the example of enzymes from
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
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, 50, 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
The enzyme complex secreted by
As mentioned above, CBH I and CBH II of
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
Preparation | FP | MCC | CMC | pNPG | Cellobiose | Xylan | pNPX |
---|---|---|---|---|---|---|---|
50°C, pH 5.0 | 40°C, pH 5.0 | 50°C, pH 5.0 | 40°C, pH 5.0 | 40°C, pH 5.0 | 50°C, pH 5.0 | 40°C, pH 5.0 | |
Accelerase 1000 | 1.7 | 1.2 | 12.3 | 3.6 | 2.7 | 2.4 | <0.01 |
Accelerase 1500 | 1.2 | 0.8 | 10.5 | 3.8 | 2.3 | 1.7 | <0.01 |
Accelerase XY | <0.1 | 0.1 | 0.8 | 0.6 | 0.4 | 92.5 | 0.3 |
Accelerase DUET | 1.1 | 0.8 | 7.9 | 3.1 | 2.2 | 14.0 | 1.3 |
Cellic CTec-1 | 0.7 | 0.3 | 10.9 | 2.8 | 2.4 | 1.5 | <0.01 |
Cellic CTec-2 | 0.6 | 0.3 | 15.6 | 5.5 | 4.0 | 28.2 | <0.01 |
0.9 | 0.7 | 18.3 | 1.7 | 0.7 | 30.3 | <0.01 | |
0.2 | 1.1 | 5.9 | 45.5 | 60.2 | 0.8 | <0.01 |
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
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
In general, the specific activities of the
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].
Preparation | RS yield (mg/ml) | ||||
---|---|---|---|---|---|
Pretreated with steam explosion | Grind wood | MCC | |||
Corn stalks | Bagasse | Pine | Aspen | ||
Accelerase 1000 | 37.8-38.0-38.5 | 19.6-25.8-40.0 | 22.4-25.3-30.6 | 21.5-34.3-41.3 | 26.0-52.2-76.8 |
Accelerase 1500 | 33.9-36.5-38.9 | 17.6-24.4-34.6 | 19.9-24.5-29.1 | 19.3-35.2-40.9 | 27.0-50.6-77.3 |
Accelerase XY | 18.2-18.3-18.3 | 18.7-19.5-32.8 | 3.6-4.9-8.6 | 11.8-13.9-17.3 | 2.9-4.8-5.8 |
Accelerase DUET | 34.3-34.8-36.9 | 20.3-31.7-43.0 | 20.4-24.5-29.5 | 25.9–41.5-49.6 | 25.3-53.5-75.6 |
Cellic CTec-1 | 23.3-26.3-26.3 | 12.0-21.8-32.0 | 12.1-14.7-22.6 | 9.2-18.9-23.0 | 16.2-30.2-60.8 |
Cellic CTec-2 | 48.8–53.2-54.4 | 28.5-37.0-50.5 | 24.5-29.2-31.6 | 27.6–40.7-48.4 | 35.5-73.7-84.4 |
48.1-51.0-52.2 | 32.2-34.5-48.6 | 27.3-29.0-32.4 | 29.8-44.9-52.3 | 32.5-58.0-75.1 |
The hydrolytic efficiency of enzyme preparations based on fungi of the genus
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, 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
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
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
2.3 Methods for increasing the efficiency of bioconversion of plant materials under the action of an enzyme complex produced by P. verruculosum
An auxotrophic mutant B1-537 (ΔniaD) was obtained for genetic engineering manipulations based on the highly productive strain
The expression of the gene of the target enzyme can be carried out under the control of various “promoter-terminator” systems selected by the researcher. This may be a proprietary system inherent in the target protein gene, or a promoter and terminator of another gene. To express various genes in the strain B1-537 (ΔniaD), a promoter and a transcription terminator of the inducible CBH I gene
Cloned genes | Expressed enzymes | Content in the preparation, %* |
---|---|---|
Xylanase A | 18 (0) [55] | |
Mannanase B | 45–70 (0) [55] | |
EG I | 3 (0) [55] | |
eglIV T. reesei | LPMO (EG IV) | 15 (0) [55] |
CBH II | 2 (0) [55] | |
Xylanase III | 17 (0) [55] | |
bgl1 A. niger | BGL I | 21 (0) [56] |
CBH I | 66 (20) [65] | |
CBH I + EG II | 56 + 19 (20 + 3) [65] | |
CBH I + EG II + BGL I | 37 + 34 + 12 (20 + 3 + 0) [65] | |
LPMO (EG IV) | 24 (0) [65] | |
LPMO (EG IV) + BGL I | 1 + 18 (0 + 0) [57] | |
CBH I + EG II + BGL I + LPMO (EG IV) | 24 + 35 + 10 + 1 (20 + 3 + 0 + 0) [57] |
One of the approaches to obtain recombinant strains is so-called “fusion construct”, consisting, for example, of sequentially connected genes encoding CBH I, EG II
Another example, as described in the previous chapter, is the composite preparation of
To obtain the strains with a moderate level of expression of target recombinant enzymes alternative expression systems can be used. This type includes, for example, a constitutive expression system based on the histone gene promoter hist4.1 [64]. Its use makes it possible to increase the production of the target protein without significantly changing the composition of the main secreted enzyme complex. Using an expression system based on a histone promoter, the B1_PrHist enzyme preparation was obtained with heterologous
Thus, both of the genetic engineering approaches discussed above, in which the target protein is expressed either under the control of a strong inducible (
Cellulases are an example of enzymes that are extremely demanded in practice and are used in Europe, the USA, and a number of other countries to implement the ideas of bioeconomics, implying the replacement of non-renewable fossil raw materials and chemical processes of its processing with alternative biotechnological processes with integrated use of renewable raw materials and various wastes. In particular, with the use of cellulases in a number of countries (USA, Italy, Brazil), the concept of biofactory (biorefinery) has been practically implemented, consisting in the realization of large-scale processes of enzymatic conversion of renewable lignocellulose plant biomass (agricultural waste) into sugars, followed by the production of alcohols (second-generation biofuels) from them [67]. Other biotechnological processes based on the use of lignocellulose raw materials have been tested and are close to practical implementation in order to obtain solvents, organic and amino acids, biologically active substances, monomers for the synthesis of various polymers, etc. In addition, cellulases are traditionally used in the textile industry, pulp and paper industry, and various branches of the food industry, such as brewing, alcohol, and bakery, as well as feed additives in poultry and animal husbandry [68].
3. Conclusions
Cellulolytic enzymes are found in various living systems, but the main destroyers of cellulose are microorganisms, primarily microscopic aerobic fungi, as well as various types of aerobic and anaerobic bacteria. Fungi from the genus
Using the methods of mutagenesis, selection, and genetic engineering, highly productive
Abbreviations
endoglucanase | |
cellobiohydrolase | |
β-glucosidase | |
lytic polysaccharide monooxygenase | |
cellulose binding domain | |
microcrystalline cellulose | |
carboxymethyl cellulose | |
filter paper | |
reducing sugars | |
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