Application of the “fusion” approach for the production of highly efficient biocatalysts based on recombinant strains of the fungus Penicillium verruculosum for the conversion of cellulose-containing biomass

Hydrolysis of cellulose-containing biomass mediated by biocatalysts (enzyme preparations, EP) is one of the most advanced and environmentally friendly methods of obtaining a range of useful substances. A new approach to creating recombinant EPs with predefined properties, which consists in applying fusion constructs for the cloning of genes encoding target enzymes, was used in the present study. A number of EPs with different properties was derived from a strain of the fungus Penicillium verruculosum using fusion constructs; these preparations are of interest primarily as additives enhancing the hydrolytic capacity of the basic cellulolytic complex from P. verruculosum. Use of the new EPs in combination with the basic EP from P. verruculosum resulted in an increase of the biocatalytic (hydrolytic) efficiency of the latter towards cellulose-containing raw materials of plant origin. Addition of 20% of the new EP to the basic EP without changing the total EP dose in the reaction mixture resulted in a significant (up to 70%) increase of the efficiency of hydrolysis of cellulose-containing substrates (ground aspen wood and shredded deresined pine wood).     

Using an Inducible Promoter of the Glucoamylase Gene to Construct New Multienzyme Complexes from <Emphasis Type="Italic">Penicillium verruculosum</Emphasis>

New recombinant strains of Penicillium verruculosum are created using a new plasmid construct based on an inducible promoter of glucoamylase gene (gla1) that secretes heterologous xylanase E (XylE) from P. canescens. New biocatalysts are produced that contain cellulolytic enzyme preparations (EPs) enriched with XylE. The amount of XylE in the recombinant EPs varies in the optimum range of 11–24% of the total protein while generally preserving the P. verruculosum cellulose complex. The hydrolytic activity of the new EPs with respect to polymeric plant-derived substrates exceeds that of EPs produced using other expression systems and commercial preparations. The new EP glaX-17 surpasses in particular a control EP based on the recipient strain by 13% in the efficiency of hydrolyzing aspen wood and is 20% more efficient than the commercial EP Accelerase Duet. The new EP glaX-17 displays 25% greater efficiency (35–43%) during the hydrolysis of wheat bran than the commercial EP Accelease Duet. The effectiveness is demonstrated of using the new gla1 promoter for the production of EPs (biocatalysts) while preserving the balanced cellulose complex of the strain and optimum yield of heterologous XylE required for the deep hydrolysis of xylan-containing plant biomass.     

Comparing the efficiency of plant material bioconversion processes using biocatalysts based on Trichoderma and Penicillium verruculosum enzyme preparations

A comparative study of the effectiveness of six commercial biocatalysts based on enzyme preparations derived from a fungus of the genus Trichoderma as a producer (Cellic Ctec-1, Cellic Ctec-2, Accelerase 1000, Accelerase 1500, Accelerase XY, Accelerase DUET), and of laboratory biocatalysts based on enzyme preparations derived from the fungus Penicillium verruculosum for the hydrolysis of the four types of plant cellulose material (steam explosion pretreated corn stalks and bagasse, ground pine and aspen), and of microcrystalline cellulose is performed. Biocatalysts activities toward various substrates, and the dependence of the depth of exhaustive hydrolysis of plant material on the dosage of these biocatalysts are determined. It is shown that biocatalysts based on the strains of P. verruculosum are competitive with widely used commercial biocatalysts based on the Trichoderma strain if we scale the biotechnological processes of bioconversion of renewable plant raw materials.     

Improving the efficiency of the bioconversion of plant raw materials with mutant cellulases of <Emphasis Type="Italic">Penicillium verruculosum</Emphasis>

Plant biomass is the main type of organic material on Earth. The efficiency of biocatalytic conversion of plant raw materials determines the cost of their biotechnological processing to produce commercially valuable products such as organic alcohols and acids, carbohydrates, and hydrocarbons. New recombinant Penicillium canescens strains that produce not only their own enzyme complex but also heterologous cellulases (i.e., mutant and wild-type cellobiohydrolase I (CBH I) and endoglucanase II (EG II) of P. verruculosum) are constructed. Enzymatic agents (EAs) prepared on the basis of recombinant strains of P. canescens are found to be more active in the hydrolysis of crushed aspen wood. Yields of glucose and reducing sugars are observed 24–72 h after hydrolysis with EAs prepared in recombinant strains to be from 48 to 52 and 60 to 64%, respectively, higher than those for hydrolysis with EAs prepared in the initial recipient strain. The presence of N45A and N194A site-specific mutations introduced to reduce surface glycosilation thus results in a substantial increase in the yields of desired CBH I and EG II.     

Biocatalytic conversion of kraft pulp using cellulase complex of <Emphasis Type="Italic">Penicillium verruculosum</Emphasis>

The efficiency of biocatalysts based on the cellulase complex from the Penicillium verruculosum fungus in the hydrolysis of kraft pulp from soft and hardwood is investigated. The activities of biocatalysts with respect to unbleached and bleached cellulose samples and the dependence of the degree of cellulose conversion on the content of noncellulose components are determined. It is shown that wet kraft pulp exhibits high reactivity in enzymatic hydrolysis with cellulase complex from P. verruculosum and is undoubtedly of interest as a substrate for scaling up biotechnological processes of the bioconversion of renewable plant-derived materials.     

Properties of Chimeric Polysaccharide Monooxygenase with an Attached Cellulose Binding Module and Its Use in the Hydrolysis of Cellulose-Containing Materials in the Composition of Cellulase Complexes

The use of recently discovered polysaccharide monooxygenases (PMO) in the composition of cellulase complexes greatly enhances their saccharification ability. Genetic engineering is used in this work to produce a chimeric enzyme based on the Thielavia terrestris PMO with cellulose binding module (CBM) from the Penicillium verruculosum cellobiohydrolase I attached to the PMO С-terminus via a peptide linker. Chimeric PMO exhibits higher (by 24%) activity toward amorphous cellulose and wider substrate specificity than the initial PMO. As a result of the CBM attachment, chimeric PMO acquires the ability to cleave xylan and carboxymethyl cellulose in addition to cellulose and β-glucan, and its activity toward xyloglucan increases by one order of magnitude. Replacing 10% of the highly active cellulase preparation hBGL2 produced by P. verruculosum with the chimeric PMO while retaining the overall dose of the enzymes with regard to their protein concentration increases the yield of sugars during the hydrolysis of microcrystalline cellulose and powdered aspen wood by 24 and 47%, respectively. In addition, the maximum yield of sugars during wood hydrolysis is achieved in 24 h of reaction time, in contrast to hydrolysis with the indicated preparation without the added PMO, which requires 48 h.     

Optimizing the composition of cellulase enzyme complex from <Emphasis Type="Italic">Penicillium verruculosum</Emphasis>: Enhancing hydrolytic capabilities via genetic engineering

Modern technologies for the enzyme hydrolysis of cellulose-containing raw materials allow the production of sugars from which alcohols (biofuel), organic and amino acids, biopolymers, feed additives, and other value-added products can be obtained via microbiological conversion. Three types of cellulolytic enzymes are required for the bioconversion of cellulose containing materials: endoglucanase, cellobiohydrolase, and ß-glucosidase. The prospects for improving the hydrolytic capabilities of the enzyme complex secreted from Penicillium verruculosum are investigated in this work by means of genetic engineering to add different combinations and ratios of homologous and heterologous cellulases: endoglucanase IV (EGIV) of Trichoderma reesei, endoglucanase II (EGII), and cellobiohydrolase I (CBHI) of P. verruculosum, along with ß-glucosidase (ß-GLU) of Aspergillus niger. The optimum ratio of components is determined and the catalytic activity of enzymatic complexes is increased by as much as 100%.     

Preparation and properties of new biocatalysts for the degradation of nonstarch plant polysaccharides

Recombinant strains of Penicillium verruculosum are developed that produce the homologous endoglucanase 2 (Eg2) and the P. canescens heterologous xylanase E (XylE). The recombinant strains are used to obtain new biocatalysts, i.e., enzyme preparations (EPs) that are substantially enriched with Eg2 and XylE. These preparations are highly active with respect to nonstarch plant polysaccharides (NPSes): cellulose, β-glucan, and xylan. The qualitative and quantitative compositions of the new EPs are studied by protein chromatography. It was shown that the EPs contained (in terms of total protein content) ~16–17% Eg2, 48–63% XylE, and 17–30% cellobiohydrolases, while the EP obtained using the recipient strain contained 1.4% Eg2, ~60% cellobiohydrolase and no XylE. The optimum pH values for cellulase (with respect to carboxymethylcellulose, CMC) and the xylanase activity of the EPs are 4.0 and 5.5, respectively. The EPs exhibit the abovementioned activities within a wide range of pH (3 to 7). The EPs exhibit CMC-ase and xylanase activities in the temperature range of 20–80°С with maxima at 60 and 70°C, respectively. The xylanase activity of the new EPs is virtually uninhibited by protein inhibitors of rye.     

Comparative analysis of the effect of pretreating aspen wood with aqueous and aqueous-organic solutions of sulfuric and nitric acid on its reactivity during enzymatic hydrolysis

The effect of aspen wood pretreatment methods with the use of both aqueous solutions of sulfuric and nitric acids and aqueous-organic solutions (ethanol, butanol) of sulfuric acid (organosolv) on the limiting degree of conversion of this type of raw material into simple sugars during enzymatic hydrolysis are compared. The effects of temperature, acid concentration, composition of organic phase (for sulfuric acid), and pressure (for nitric acid) on the effectiveness of pretreatment were analyzed. It is shown that the use of organosolv with 0.5% sulfuric acid allows us to increase the reactivity of ground wood by 300–400%, compared to the initial raw material. Pretreatment with a 4.8% aqueous solution of nitric acid (125°C, 1.8 MPa, 10 min) is shown to be most effective, as it increases the reactivity of the ground aspen wood by more than 500%.     

Preparing catalyst based on recombinant cellulolytic enzyme specimen <Emphasis Type="Italic">Penicillium verruculosum</Emphasis> and its use in the paper industry

The effective application of a biocatalyst based on recombinant enzyme preparations obtained using Penicillium verruculosum is studied for the modification of marketable bleached sulfate hardwood cellulose. The effect of treatment with cellulase complex on the structure, morphology, and surface state of cellulose fibers is evaluated. After biocatalytic exposure, cellulose is shown to have an improved capacity for beating and the formation of bonds in paper sheets, along with greater strength than the initial cellulose. Application of the enzymatic modification of cellulose in the paper industry during biotechnological processes allows a range of problems in the efficient use of energy and resources to be solved.     

Emerging role of nanobiocatalysts in hydrolysis of lignocellulosic biomass leading to sustainable bioethanol production

Catalytic conversion (hydrolysis) of carbohydrate polymers present in the lignocellulosic biomass into fermentable sugars is a key step in the production of bioethanol. Although, acid and enzymatic catalysts are conventionally used for the catalysis of various lignocellulosic biomass, recently application of immobilized enzymes (biocatalysts) have been considered as the most promising approach. Immobilization of different biocatalysts such as cellulase, β-glucosidase, cellobiose, xylanase, laccase, etc. on support materials including nanomaterials to form nanobiocatalyst increases catalytic efficacy and stability of enzymes. Moreover, immobilization of biocatalysts on magnetic nanoparticles (magnetic nanobiocatalysts) facilitates easy recovery and reuse of biocatalysts. Therefore, utilization of nanobiocatalysts for catalysis of lignocellulosic biomass is helpful for the development of cost-effective and ecofriendly approach. In this review, we have discussed various conventional methods of hydrolysis and their limitations. Special emphasis has been made on nanobiocatalysts used for hydrolysis of lignocellulosic biomass. Moreover, the other most important aspects, like nanofiltration of biomass, conversion of lignocellulose to nanocellulose, and toxicological issues associated with application of nanomaterials are also discussed.     

Trichoderma reesei endoglucanase IV: A new component of biocatalysts based on the cellulase complex of the fungus Penicillium verruculosum for hydrolysis of cellulose-containing biomass

The problem of raising the efficiency of enzyme preparations catalyzing cellulose conversion is among the present-day technological challenges. Here, we report the enhancement of the hydrolytic capacity of cellulase preparations by introducing nonhydrolytic enzymes (polysaccharide monooxygenases) into the cellulolytic complex. An enzyme preparation with an increased hydrolytic capacity has been obtained from the recombinant strain of the fungus Penicillium verruculosum that carries the Trichoderma reesei endoglucanase IV gene. This method allows the efficiency of the cellulase complex to be increased by 20%.     

The effect of moisture on the dynamical mechanical properties of bacterial cellulose/glucuronoxylan nanocomposites

The plant cell wall possesses unique material properties due to its hierarchical organisation. In order to biomimic a native structure like a plant cell wall, a model system consisting of microfibrillar cellulose, produced by the gram-negative bacteria Acetobacter xylinum, and a glucuronoxylan matrix derived from aspen holocellulose was constructed. The glucuronoxylan was extracted from delignified aspen (Populus tremula) wood chips using DMSO to preserve its native chemical composition. Dynamic mechanical analysis (DMA) measurements performed with moisture scans showed a moisture-induced softening of delignified aspen wood fibres due to the plasticization of glucuronoxylan. A similar result was observed for the model system. However, the softening behaviour of the delignified aspen fibre and the model system was not identical, most probably due to differences in spatial organisation of the components. Dynamic FTIR-studies indicated that interactions between the cellulose and the glucuronoxylan exist in the aspen holocellulose while the components in the nanocomposite appear to be more isolated.     

Enzymatic production of Xylooligosaccharide from selected agricultural wastes

Four different agricultural wastes, namely tobacco stalk (TS), cotton stalk (CS), sunflower stalk (SS) and wheat straw (WS) were tested for the production of Xylooligosaccharide (XO). XO production was performed by enzymatic hydrolysis of xylans which were obtained by alkali extraction from the agricultural wastes. Depending on the source, it was found that these four agricultural wastes contained different amount of xylan, cellulose and lignin and the xylan obtained from these source contained different amount of sugar and uronic acid. The highest amount of arabinose was in xylan from WS while the other xylans mainly had xylose and small amount of glucose. Different xylanase preparations were evaluated for production XO from these xylan sources. Aspergillus niger xylanase produced lower amount of XO from wheat straw xylan (WSX) than cotton stalk xylan (CSX), sun flower xylan (SSX) and tobacco stalk xylan (TSX) while Trichoderma longibrachiatum xylanase hydrolyzed highly branched WSX better. The HPLC analysis of the hydrolysis products indicated that depending on structure and composition of xylan, A. niger xylanase produced less amount of xylose than T. longibrachiatum xylanase, and the hyrolysis product of A. niger xylanase contained different amount of oligosaccharides (X2 > X3 > X4 > X5 > X6, >X6). Regardless of the structural differences of the xylan types presented in this paper, all xylans generated XO with different degree of polymerization (DP), but the DP of XO depended on the enzyme specificity and the structure of substrate.     

Second-generation ethanol production from steam exploded barley straw by Kluyveromyces marxianus CECT 10875

Barley straw is nowadays being considered a potential lignocellulosic raw material for fuel-ethanol production as an alternative to starch- or sugar-containing feedstock. In this work, several configuration strategies for ethanol production from steam-exploded barley straw by Kluyveromyces marxianus CECT 10875 have been studied with the aim of obtaining higher ethanol concentrations.Different substrate loading (2-15%, w/v) were studied during enzymatic hydrolysis. The xylanase contribution on glucose production and glucan conversion at different substrate loading was also investigated. In addition, three different process configurations, separate hydrolysis and fermentation, simultaneous saccharification and fermentation and presaccharification and simultaneous saccharification, were compared at different water insoluble solids concentration (5%, 10% and 15%). The influence of xylanase addition on the ethanol yield was studied as well.Results show that endo-xylanases improved glucan conversion and ethanol yield compared with a standard enzymatic mixture, markedly at low substrate concentration. The positive effect of added xylanase was most evident at early stages of enzymatic hydrolysis. Regarding process configurations for the period of 72 h, SSF with endo-xylanases provided the best ethanol yield, nearly 70%, for 10% WIS. Nonetheless, the higher ethanol concentration, 29.4 g/l, was obtained at 15% WIS.     

Improvement of enzymatic xylooligosaccharides production by the co-utilization of xylans from different origins

This study aimed to improve XOs production by enzymatic hydrolysis of xylans from various lignocellulosic waste biomasses namely corn cob, cotton and sunflower stalks, rice hull, wheat straw by using two commercial xylanase preparations, Shearzyme 500L and Veron 191. Shearzyme 500L showed better xylan hydrolysis capacity with high amount of xylose liberation. Xylobiose was the main hydrolysis product in each case. Even though the enzymatic hydrolyses using Shearzyme 500L resulted higher reducing sugar production compared to those of Veron 191, the hydrolysis of complex xylan structures was improved and the production of undesirable xylose was lowered by the co-utilization of xylanase preparations. By the co-utilization of xylanase preparations, the reducing sugar production from wheat straw, corn cob and sunflower stalk originated xylans was increased by 36%, 33% and 13%, respectively, compared to the expected reducing sugar yields. The highest reducing sugar production was obtained from complex corn cob xylan. The depolymerization of cotton and sunflower stalk xylan was poorest even though they have simple structures. Poor utilization of these xylans might be related to their high residual lignin content which might hinder the accessibility of xylan by the xylanases. However, the utilization of sunflower and cotton stalk xylan was improved when they were hydrolyzed within a xylan mixture containing equal amounts of each of five different xylans. In short, XOs production efficiency from agricultural waste materials was improved by the co-utilization of suitable xylanase and/or xylan mixtures considering the heterogeneous structures of xylan and different substrate specificities of xylanases.     

Quantitative Phosphorus-31 NMR Analysis of Six Soluble Lignins

By using a set of lignin samples, which have been subjected to thorough analyses by the international wood chemistry community, the recently developed quantitative method of ³¹P NMR spectroscopy was comprehensively examined. The values of total phenolic hydroxyl groups and those of total hydroxyl groups were found to favourably compare with those obtained by other laboratories, applying independent methods of analysis. Furthermore, the application of quantitative ³¹P NMR spectroscopy offered additional detailed structural information for the examined lignins which was in accord with literature accounts for similar wood species and lignin preparations. More specifically, the steam explosion lignins from aspen and yellow poplar woods and that produced by ball milling/enzyme hydrolysis of cottonwood were found to contain relatively high amounts of β-O-4 structures. In contrast, the kraft, organosolv, and the acid hydrolysis processes were found to induce significant chain scission on the resulting lignins. Ball milled cottonwood lignin contained the highest frequency of β-O-4 bonds and the lowest amount of free phenolic hydroxyls. The erythro form of β-O-4 structures were invariably predominant in the lignins from aspen, yellow poplar and cottonwood, in accord with the conclusions of previous reports on hardwood lignins. Thus, the application of quantitative ³¹P NMR spectroscopy offered the detailed chemical composition of the examined lignins.     

Flakeboards Made From Aspen and Southern Pine Wood Flakes Reacted with Gaseous Ketene

Abstract

Southern pine and aspen wood flakes were chemically modified by reaction at ca. 55°C with ketene in the absence of solvent. Reactions were relatively slow, with weight gains of up to 17% and 20%, respectively, obtained. Acetyl content correlated with weight gain only up to the 12% level. Water and solvent extraction of ketene-modified southern pine and aspen flakes showed very little loss in acetyl.

Flakeboards made from southern pine and aspen flakes treated with ketene showed a greatly reduced rate and extent of swelling resulting from liquid water sorption as compared to control boards. Similar results were obtained in swelling tests done in water vapor. Ketene modification had a much greater effect on improving dimensional stability properties of aspen flakeboards than on southern pine flakeboards.     

毕赤酵母产重组木聚糖酶发酵条件的优化及其酶学性质

目的优化毕赤酵母工程菌GS115/xyn11A产重组木聚糖酶的发酵条件,并检测其酶学性质。方法采用单因素试验和L(934)正交试验考察摇瓶发酵条件下培养基起始pH值、诱导剂甲醇添加量、诱导温度及诱导时间对产酶活性的影响;并分析重组木聚糖酶的酶学性质。结果影响重组毕赤酵母产酶的因素重要性依次为:培养基起始pH值>诱导时间>诱导温度>甲醇添加量,重组酵母产酶最佳条件为:起始pH值7.5,甲醇添加量1.5%,32℃诱导96 h,在此条件下进行诱导表达重组木聚糖酶的酶活性可达228.35 IU/ml;酶的最适反应温度为50℃,最适反应pH值为5.5,在低于40℃和pH 4.5～7.5的范围内较稳定。结论优化了毕赤酵母产重组木聚糖酶的发酵条件,为木聚糖酶的工业化生产及应用提供了依据。     

Optimization of pelletisation and combustion in a boiler of 17.5 kWth for vine shoots and industrial cork residue

Wood pellets have become an important renewable energy fuel. Nowadays the main raw materials used for their production are wood wastes from wood industries. However, these wood wastes have other uses in Spain and it is necessary to look for other possible raw materials. In this work, vine shoots and industrial cork residue were studied as raw materials. The results showed that pelletisation of vine shoots presented a high energy demand. This energy requirement was reduced with the addition of industrial cork residue. Moreover, industrial cork residue decreased the ash content of pellets and increased their heating value, although it decreased their physical properties at the same time. Regarding combustion, the addition of industrial cork residue decreased the accumulation of ash in the pellet burner and its sintering tendency. The major conclusion of the work is that the most appropriate blend to improve pelletisation and combustion processes is 30% wt. of vine shoots and 70% wt. of industrial cork residue.