Trichoderma Xylanases,Their Properties and Application

Abstract

Trichoderma spp. are known to produce enzymes with high xylanolytic activity. Different xylanases and various components of their xylanolytic system have been identified and purified. Some of the xylanases have been characterized extensively with respect to their physicochemical, hydrolytic, and molecular properties. Cellulase-free xylanase preparations have been tested successfully in industrial applications such as the prebleaching of kraft pulps in the pulp and paper industry. Future work on understanding the functional significance of xylanase multiplicity, the mechanisms of xylanase prebleaching, and the structural conformation of xylanases could lead to improved or alternative applications of Trichoderma xylanases.     

Origin of fungal biomass degrading enzymes: Evolution,diversity and function of enzymes of early lineage fungi

The aim of this study was to elucidate the evolution of enzyme secretome of early lineage fungi to contribute to resolving the basal part of Fungal Kingdom and pave the way for industrial evaluation of their unique enzymes. By combining results of advanced sequence analysis with secretome mass spectrometry and phylogenetic trees, we provide evidence for that plant cell wall degrading enzymes of higher fungi share a common ancestor with enzymes from aerobic ancient fungi. Sequence analysis (HotPep, confirmed by dbCAN-HMM models) enabled prediction of enzyme function directly from sequence. For the first time, oxidative enzymes are described here in early lineage fungi (Chytridiomycota & Cryptomycota), which supports the conceptually new understanding that fungal LPMOs were also present in the early evolution of the Fungal Kingdom. Phylogenetic analysis of fungal AA9 proteins suggests an LPMO-common-ancestor with Ascomycetes and Basidiomycetes and describes a new clade of AA9s. We identified two very strong biomass degraders, Rhizophlyctis rosea (soil-inhabiting) and Neocallimastix californiae (rumen), with a rich spectrum of cellulolytic, xylanolytic and pectinolytic enzymes, characteristically including several different enzymes with the same function. Their secretome composition suggests horizontal gene transfer was involved in transition to terrestrial and rumen habitats. Methods developed for recombinant production and protein characterization of enzymes from zoosporic fungi pave the way for biotechnological exploitation of unique enzymes from early lineage fungi with potential to contribute to improved biomass conversion. The phyla of ancient fungi through evolution have developed to be very different and together they constitute a rich enzyme discovery pool.     

Tamarind kernel powder co-induces xylanase and cellulase production during submerged fermentation of <Emphasis Type="Italic">Termitomyces clypeatus</Emphasis>

Tamarind kernel powder (TKP), a soluble agro-residue, was used to examine the production of both cellulolytic and xylanolytic enzymes in a submerged culture of Termitomyces clypeatus, an edible mushroom. Soluble TKP containing xyloglucan as the major polysaccharide induced all cellulolytic and xylanolytic enzymes, and enzyme production increased up to 3% (w/v) TKP with culture filtrate consisting of xylanase and CMCase at a ratio of 4: 1 app. Strong catabolic repression of enzyme production was also observed with the soluble substrate, although fed-batch addition of soluble substrate at late growth phase modified the enzyme kinetics by improving the yield by 30%. The results indicate that inducers were possibly released from TKP by cellulose and xylan fractions of the lignocellulosic polymer. Therefore, the present study reports the successful economic utilization of TKP, an abundantly available soluble agro-residue, for the production of both cellulolytic and xylanolytic enzymes in a single fermentation method.     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.     

A novel family of hemicellulolytic α-glucuronidase

Investigation of the xylanolytic enzyme system of the xylose-fermenting yeast Pichia stipitis resulted in the discovery of an extracellular α-glucuronidase efficiently debranching hardwood glucuronoxylan. This activity is not exhibited by more extensively investigated α-glucuronidases of glycoside hydrolase (GH) family 67, operating on substrates in which the uronic acid is linked to the non-reducing xylopyranosyl residues of main chain fragments. The N-terminus of the purified enzyme corresponded exactly to the P. stipitis gene ABN67901 coding for a protein of unknown function. BLAST search revealed the presence of similar genes in genomes of other microorganisms. These results lead to the emergence of a new family of α-glucuronidases.     

Characterisation of a Talaromyces emersonii thermostable enzyme cocktail with applications in wheat dough rheology

In this paper, we report new sequence data for secreted thermostable fungal enzymes from the un-sequenced xylanolytic filamentous fungus Talaromyces emersonii and reveal novel insights on the potential role of enzymes relevant as wheat dough improvers. The presence of known and de novo enzyme sequences were confirmed through NanoLC-ESI-MS/MS and resultant peptide sequences were identified using SWISS PROT databases. The de novo protein sequences were assigned identity based on homology to known fungal proteins. Other proteins were assigned function based on the limited T. emersonii genome coverage. This approach allowed the identification of enzymes with relevance as wheat dough improvers. Rheological examination of wheat dough and wheat flour components treated with the thermostable fungal enzyme cocktail revealed structural alterations that can be extrapolated to the baking process.Thermoactive amylolytic, xylanolytic, glucanolytic, proteolytic and lipolytic enzyme activities were observed. Previously characterized T. emersonii enzymes present included; β-glucosidase, xylan-1,4-β-xyloxidase, acetylxylan esterase, acid trehalase, avenacinase, cellobiohydrolase and endo-glucanase. De novo sequence analysis confirmed peptides as being; α-glucosidase, endo-1,4-β-xylanase, endo-arabinase, endo-glucanase, exo-β-1,3-glucanase, glucanase/cellulase, endopeptidase and lipase/acylhydrolase. Rheology tests using wheat dough and fractioned wheat flour components in conjunction with T. emersonii enzymes show the role of these novel biocatalysts in altering properties of wheat substrates. Enzyme treated wheat flour fractions showed the effects of particular enzymes on appropriate substrates. This proteomic approach combined with rheological characterization is the first such report to the authors’ knowledge.     

Microbial xylanases and their industrial applications: a review

Despite an increased knowledge of microbial xylanolytic systems in the past few years, further studies are required to achieve a complete understanding of the mechanism of xylan degradation by microorganisms and their enzymes. The enzyme system used by microbes for the metabolism of xylan is the most important tool for investigating the use of the second most abundant polysaccharide (xylan) in nature. Recent studies on microbial xylanolytic systems have generally focussed on induction of enzyme production under different conditions, purification, characterization, molecular cloning and expression, and use of enzyme predominantly for pulp bleaching. Rationale approaches to achieve these goals require a detailed knowledge of the regulatory mechanism governing enzyme production. This review will focus on complex xylan structure and the microbial enzyme complex involved in its complete breakdown, studies on xylanase regulation and production and their potential industrial applications, with special reference to biobleaching.     

Biomass hydrolyzing enzymes from plant pathogen Xanthomonas axonopodis pv. punicae: optimizing production and characterization

Xanthomonas axonopodis pv. punicae strain—a potent plant pathogen that causes blight disease in pomegranate—was screened for cellulolytic and xylanolytic enzyme production. This strain produced endo-β-1,4-glucanase, filter paper lyase activity (FPA), β-glucosidase and xylanase activities. Enzyme production was optimized with respect to major nutrient sources like carbon and nitrogen. Carboxy methyl cellulose (CMC) was a better inducer for FPA, CMCase and xylanase production, while starch was found to be best for cellobiase. Soybean meal/yeast extract at 0.5 % were better nitrogen sources for both cellulolytic and xylanolytic enzyme production while cellobiase and xylanase production was higher with peptone. Surfactants had no significant effect on levels of extracellular cellulases and xylanases. A temperature of 28 °C and pH 6–8 were optimum for production of enzyme activities. Growth under optimized conditions resulted in increases in different enzyme activities of around 1.72- to 5-fold. Physico-chemical characterization of enzymes showed that they were active over broad range of pH 4–8 with an optimum at 8. Cellulolytic enzymes showed a temperature optimum at around 55 °C while xylanase had highest activity at 45 °C. Heat treatment of enzyme extract at 75 °C for 1 h showed that xylanase activity was more stable than cellulolytic activities. Xanthomonas enzyme extracts were able to act on biologically pretreated paddy straw to release reducing sugars, and the amount of reducing sugars increased with incubation time. Thus, the enzymes produced by X. axonopodis pv. punicae are more versatile and resilient with respect to their activity at different pH and temperature. These enzymes can be overproduced and find application in different industries including food, pulp and paper and biorefineries for conversion of lignocellulosic biomass.     

Effect of hydrolytic and oxidative enzymes produced by solid-state fermentation on greige linen fabric

Solid state fermentation (SSF) was applied for production of fungal enzyme preparations from Phanerochaete chrysosporium, Aspergillus oryzae, Aspergillus giganteus and Trichoderma virens using cotton seed-coat fragment waste as a carbon source and enzyme inducer. Lignin-holocellulose matrix of cotton seed coat fragment proved to be effective in inducing production of ligninolytic, cellulolytic and xylanolytic enzymes in solid-state fermentation. The effect of the enzymes produced by SSF on greige linen fabric is discussed and evaluated. In the first experiment the hydrolytic and accompanying oxidative enzymes in the buffer extract of the whole SSF cultures were used for fabric treatment. In the second trial, the enzymes produced in situ (whole SSF material—mixture of fungal biomass, residual substrate and enzymes) were used for the treatment. Weight loss, reducing sugar liberation and removal of colouring materials were measured. The results showed that at equal enzyme charges the intact SSF materials were more efficient than the enzyme extracts. Of the six strains evaluated, Ph. chrysosporium VKM F-1767 was the most effective in removing colouring matters from greige linen fabric.     

Characterization of a recombinant α-glucuronidase from Aspergillus fumigatus

The degradation of xylan requires the action of glycanases and esterases which hydrolyse, in a synergistic fashion, the main chain and the different substituents which decorate its structure. Among the xylanolytic enzymes acting on side-chains are the α-glucuronidases (AguA) (E.C. 3.2.1.139) which release methyl glucuronic acid residues. These are the least studies among the xylanolytic enzymes. In this work, the gene and cDNA of an α-glucuronidase from a newly isolated strain of Aspergillus fumigatus have been sequenced, and the gene has been expressed in Pichia pastoris. The gene is 2523 bp long, has no introns and codes for a protein of 840 amino acid residues including a putative signal peptide of 19 residues. The mature protein has a calculated molecular weight of 91 725 and shows 99 % identity with a putative α-glucuronidase from A. fumigatus A1163. The recombinant enzyme was expressed with a histidine tag and was purified to near homogeneity with a nickel nitriloacetic acid (Ni-NTA) column. The purified enzyme has a molecular weight near 100 000. It is inactive using birchwood glucuronoxylan as substrate. Activity is observed in the presence of xylooligosaccharides generated from this substrate by a family 10 endoxylanase and when a mixture of aldouronic acids are used as substrates. If, instead, family 11 endoxylanase is used to generate oligosaccharides, no activity is detected, indicating a different specificity in the cleavage of xylan by family 10 and 11 endoxylanases. Enzyme activity is optimal at 37 °C and pH 4.5–5. The enzyme binds cellulose, thus it likely possesses a carbohydrate binding module. Based on its properties and sequence similarities the catalytic module of the newly described α-glucuronidase can be classified in family 67 of the glycosyl hydrolases. The recombinant enzyme may be useful for biotechnological applications of α-glucuronidases.     

Production of acetyl esterase during wood biodegradation byCoriolus versicolor

A role of acetyl esterase in wood biodegradation byCoriolus versicolor was examined by the assay of enzyme production and the chemical analysis of decayed wood meal of Japanese beech (Fagus crenata). Enzyme assay demonstrated that the degradation proceeded in two stages and acetyl esterase production was correlated with the cellulolytic and xylanolytic enzyme production in the second stage, not with the production of phenol-oxidizing enzymes. From the results of chemical analysis, acetyl and xylose contents in wood meal were observed to decrease simultaneously in the second stage. In contrast, rapid decrease of lignin was recognized during the initial three wk of incubation, and it was closely related with the production of phenol-oxidizing enzymes in the first stage. These results show that acetyl esterase ofC. versicolor participates in the degradation of acetylxylan and acts with the cellulolytic and xylanolytic systems, not with the ligninolytic system.     

Influence of vanillin on the production of cellulolytic and xylanolytic enzymes from a wood-rotting fungus,Coriolus versicolor

To examine the influence of a phenolic compound on the production of cellulolytic and xylanolytic enzymes of a woodrotting fungusCoriolus versicolor, a two-dimensional map of enzyme activity was constructed with various concentrations of cellobiose and vanillin. The productions of CMCase, xylanase, β-glucosidase, and β-xylosidase increased with higher cellobiose concentration and were markedly enhanced by addition of vanillin. Higher ratio of vanillin/cellobiose activated the production of these enzymes. Only acetyl esterase, which is not actively produced at the ligninolytic stage ofC. versicolor, was inhibited by the monolignol vanillin. As the presence of vanillin is considered to approximate conditions of wood decay more closely than its absence, the present result demonstrates that addition of vanillin, a phenolic compound, enhanced the production of cellulolytic and xylanolytic enzymes for wood cell wall degradation.     

Effect of silver doping on antidiabetic and antioxidant potential of ZnO nanorods

BackgroundIncreasing resistance to available drugs and their associated side-effects have drawn wide attention towards designing alternative therapeutic strategies for control of hyperglycemia and oxidative stress. The roles of the sizes and shapes of the nanomaterials used in the treatment and management of Type 2 Diabetes Mellitus (T2DM) in preventing chronic hyperglycaemia and oxidative stress are investigated. We report specifically on the effects of doping silver (Ag) into the ZnO nanorods (ZnO:Ag NR’s) as a rational drug designing strategy.MethodsInhibition of porcine pancreatic α-amylase, murine pancreatic amylase, α-glucosidase, murine intestinal glucosidase and amyloglucosidase are checked for evaluation of antidiabetic potential. In addition, the radical scavenging activities of ZnO:Ag NR’s against nitric oxide, DDPH and superoxide radicals are evaluated.ResultsQuantitative radical scavenging and metabolic enzyme inhibition activities of ZnO:Ag NR’s at a concentration of 100 μg/mL were found to depend on the amount of Ag doped in up to a threshold level (3–4 %). Circular dichroism analysis revealed that the interaction of the NR’s with the enzymes altered their secondary conformation. This alteration is the underlying mechanism for the potent enzyme inhibition.ConclusionsEnhanced inhibition of enzymes and scavenging of free radicals primarily responsible for reactive oxygen species (ROS) mediated damage, provide a strong scientific rationale for considering ZnO:Ag NR’s as a candidate nanomedicine for controlling postprandial hyperglycaemia and the associated oxidative stress.     

Recent advances in the improvement of enzyme thermostability by structure modification

Abstract

Thermostability is considered to be an important parameter to measure the feasibility of enzymes for industrial applications. Generally, higher thermostability makes an enzyme more competitive and desirable in industry. However, most natural enzymes show poor thermostability, which restricts their application. Protein structure modification is a desirable method to improve enzyme properties. In recent years, tremendous progress has been achieved in protein thermostability engineering. In this review, we provide a systemic overview on the approaches of protein structure modification for the improvement of enzyme thermostability during the last decade. Structure modification approaches, including the introduction of non-covalent interactions and covalent bonds, increase of proline and/or decrease in glycine, reinforcement of subunit–subunit interactions, introduction of glycosylation sites, truncation and cyclization have been highlighted.     

Production of cellulolytic enzymes containing cinnamic acid esterase from Schizophyllum commune

To develop enzyme preparations capable of digesting plant biomass, we examined the production of cinnamic acid esterase as well as cellulolytic and xylanolytic enzymes in cultures of Schizophyllum commune. The cinnamic acid esterase was produced in the cultures containing solid cellulosic substrates, with production being enhanced by delignifying the wood powder. This indicates that these esterases are produced by cellulose, despite their substrates being phenolic compounds. Cellulolytic and xylanolytic enzymes, with the exception of α-arabinofuranosidase, were also produced in cultures containing cellulosic substances. These results show that enzyme preparation can have high activity of cinnamic acid esterase and cellulolytic and xylanolytic enzymes when S. commune is incubated in the presence of cellulose. These enzyme preparations will be useful for digesting plant biomass and for releasing cinnamic acid derivatives from plant cell walls.     

Secretome analysis of thermophilic mould Myceliophthora thermophila cultivated on rice straw and hydrolysis of lignocellulosic biomass for bioethanol production

Abstract

Myceliophthora thermophila encodes for large number of carbohydrate-active enzymes (CAZymes) involved in lignocellulosic biomass degradation. The mould was grown on rice straw in solid state fermentation at pH 5.0 and 45?°C that produced high levels of cellulolytic and xylanolytic enzymes i.e. 2218.12, 515.23, 478.23, 13.34?U/g DMR for xylanase, CMCase, FPase and β-glucosidase, respectively. The secretome analysis of M. thermophila BJAMDU5 by mass spectroscopy, described 124 different proteins with majority of CAZymes consisting of glycosyl hydrolases (GH), lytic polysaccharide mono-oxygenases (LPMO), carbohydrate esterases (CE) and polysaccharide lyases (PL). Furthermore, the enzyme cocktail of the mould was evaluated for hydrolysis of steam treated rice straw that produced 184.59?mg/g substrate reducing sugars after 24?h, which was used for production of bioethanol by using fast fermenting yeast Saccharomyces cerevisiae resulting in high production of bioethanol.     

碱性蛋白酶SubC在枯草芽孢杆菌中的高效异源表达

【背景】碱性蛋白酶是工业用酶中占比最大的酶类,广泛应用于清洁、食品、医疗等行业。近期研究发现碱性蛋白酶在生产生物活性肽方面有巨大潜力,这将进一步拓宽其在保健食品领域中的应用。【目的】利用枯草芽孢杆菌异源表达地衣芽孢杆菌来源的碱性蛋白酶SubC。【方法】通过筛选3种枯草芽孢杆菌宿主菌株(Bacillus subtilis 1A751、MA07、MA08)和6种信号肽(AmyE、AprE、NprE、Pel、YddT、YoqM),同时优化诱导剂浓度、发酵培养基和发酵时长,最终得到最优重组菌株MA08-AmyE-subCopt。【结果】重组菌株MA08-AmyE-subCopt的胞外酶活力为3.33×103 AU/mL,胞外蛋白分泌量为胞内可溶蛋白表达量的4倍,与携带野生型信号肽的对照组菌株WT相比,酶活提高了73.4%。【结论】异源碱性蛋白酶SubC在枯草芽孢杆菌中成功表达,为碱性蛋白酶SubC的表达和在保健食品领域的工业化应用提供了理论基础。