Lignocellulosic Enzymes Research Articles

BIOCONVERSION OF LIGNOCELLULOSIC MATERIALS WITH THE CONTRIBUTION OF A FERULOYL ESTERASE HYDROLASE FROM Alternaria tenuissima TO RELEASE CARBOHYDRATES

Feruloyl esterase catalyze the hydrolysis of a wide variety of glycosidic linkages between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. Thereby, they play a role in the initial degradation of lignocelluloses, which may be advantageous for the efficient degradation of the plant cell-wall complex that contains both diverse sugar residues and esterified structures. The contribution of the enzyme AltFAE was purified from Alternaria tenuissima to the conversion of lignocellulosic materials without any chemical pretreatment to release the carbohydrates, was studied. It was determined the activity of feruloyl esterase (AltFAE) production by A. tenuissima in liquid cultures with potential inducing agents sawdust, rice straw, tomato, potato, soybean, and corn starch. At the applied concentrations of soybean (2%), the highest levels ~ 689 U L-1 of enzyme activities were obtained. The disintegrating effect of enzymatic lignocellulose treatment can be significantly improved by using different kinds of hydrolytic and oxidizing enzymes. Synergistic conversion of cane bagasse also resulted in a release of 18.4 mg g-1 total carbohydrates (galactose, glucose, mannose, and xylose) per gram substrate using an “enzyme cocktail” [Cell/Xyl 50 U gds-1, AltFAE 10 U gds-1, and Lac 15 U gds-1] after incubating for 48 hrs at 45oC. The information presented in this paper is helpful to better understand the bioconversion of lignocellulosic materials with the contribution of a feruloyl esterase hydrolase from A. tenuissima to release carbohydrates.

Lignocellulosic Ezymes of Basidial Fungi-Isolated from Different Ecological Niches of Georgia

Great interest in basidiomycetes for targeted technological treatment of agro-industrial plant substrates are conditioned by their ability to produce lignocellulosic enzymes. Consisting of 29 soil-climatic zones, Georgia (South Caucasus area) represents a unique place for the isolation of microorganisms. In order to study the biochemistry and physiology of wood-degrading basidiomycetes, samples of wood-degrading basidiomycetes were collected from different taxonomic niches of Georgia. 45 strains were obtained as pure cultures and 32 ones – identified. Producers of lignocellulosic enzymes were revealed among test fungi under solid-state and submerged cultivation conditions. Pleurous ostreatus GV10, Pleurotus drynus IN 11 and Fomes fomentarius GK33 were found to be the best producers of cellulosic enzymes using orange and mandarin peels, wheat and Potato straw, wheat bran as substrates, and Ganoderma lucidum GB03 – the best producer of laccase during cultivation on orange and mandarin peels waste. The influence of lignocellulose on the accumulation of the enzymes laccase, xylanase and Filter paper assay was studied.

Lignocellulosic Ezymes of Basidial Fungi-Isolated from Different Ecological Niches of Georgia

Great interest in basidiomycetes for targeted technological treatment of agro-industrial plant substrates are conditioned by their ability to produce lignocellulosic enzymes. Consisting of 29 soil-climatic zones, Georgia (South Caucasus area) represents a unique place for the isolation of microorganisms. In order to study the biochemistry and physiology of wood-degrading basidiomycetes, samples of wood-degrading basidiomycetes were collected from different taxonomic niches of Georgia. 45 strains were obtained as pure cultures and 32 ones – identified. Producers of lignocellulosic enzymes were revealed among test fungi under solid-state and submerged cultivation conditions. Pleurous ostreatus GV10, Pleurotus drynus IN 11 and Fomes fomentarius GK33 were found to be the best producers of cellulosic enzymes using orange and mandarin peels, wheat and Potato straw, wheat bran as substrates, and Ganoderma lucidum GB03 – the best producer of laccase during cultivation on orange and mandarin peels waste. The influence of lignocellulose on the accumulation of the enzymes laccase, xylanase and Filter paper assay was studied.

Rumen and fecal microbial profiles in cattle fed high lignin diets using metagenome analysis

Coconut coir (a lignin-rich, organic material) is widely used for its commercial and economic benefits. In this study, crossbred (exotic) and Kankrej (indigenous) breeds of cattle were fed diets containing 7 or 14% coconut coir. Metagenomic analyses (16S rRNA gene amplicon and shotgun sequencing) were used to characterize the microbial community in the rumen and fecal samples along with their functional capabilities. Both amplicon and shotgun analyses revealed the predominance of bacterial phyla, Bacteroidetes, Firmicutes, Actinobacteria and Fibrobacter in ruminal liquid, ruminal solid and fecal samples. 16S rRNA gene amplicon sequencing revealed a total of 18 different bacterial taxa were found to be enriched exclusively in the animals fed with 14% coir. The shotgun analysis revealed abundance of bacterial genera, Fibrobacter, Clostridium, Prevotella, Butyrivibrio, and Ruminococcus in both liquid and solid fractions of ruminal contents, while in the fecal sample, Bacteroides, Alistipes, Plaudibacter, Parabacteroides, Porphyromonas, and Victivallis and archaeal genus, Methanocorpusculum were abundant. The functional analysis based on dbCAN database suggested that among the Glycoside hydrolase family, genes that encode oligosaccharide degrading enzymes, GH3, GH13 (p-value < 0.05), and GH43 were abundant in the feces. In ruminal solid, cellulase encoding the GH5 family was abundant. Also, lignocellulosic binding modules encoded by the CBM family, including cellulose (CBM3) and hemicellulose binding modules (CBM32 and CBM67) were abundant. Thus, the study indicated the enrichment of lignocellulosic enzymes in ruminal contents in response to feeding the coconut coir, which could be mined for potential biofuel production and other biotechnological applications.

BIOCONVERSION OF LIGNOCELLULOSIC MATERIALS BY FUNGAL “ENZYME COCKTAIL” WITH THE CONTRIBUTION OF A GLYCOSIDE HYDROLASE FROM XYLARIA POLYMORPHA TO RELEASE CARBOHYDRATES AND BIOMETHANOL

Abstract. The multifunctional GH78 glycoside hydrolase from the soft rot ascomycete Xylaria polymorpha (XpoGH78) catalyzed the conversion of different lignocellulosic materials to release carbohydrates and biomethanol. The disintegrating effect of enzymatic lignocellulose treatment can be significantly improved by using different kinds of hydrolases and a phenol oxidase. Thus, the conversion of rape straw meal by XpoGH78 could be optimized in the presence of accessory enzymes i.e. cellulases, xylanases and/or laccase. Synergistic conversion of rape straw also resulted in a release of 17.3 mg of total carbohydrates (e.g. arabinose, galactose, glucose, mannose, xylose) per gram substrate after incubating for 72 hrs. In addition, the treatment of rape straw with XpoGH78 led to a marginal biomethanol release of approx. 17 µg g-1 and improved to 270 µg g-1 by the cooperation with above accessory enzymes.

Bioconversion of Lignocellulosic Materials with the Contribution of a Multifunctional GH78 Glycoside Hydrolase from Xylaria polymorpha to Release Aromatic Fragments and Carbohydrates.

A bifunctional glycoside hydrolase GH78 from the ascomycete Xylaria polymorpha (XpoGH78) possesses catalytic versatility towards both glycosides and esters, which may be advantageous for the efficient degradation of the plant cell-wall complex that contains both diverse sugar residues and esterified structures. The contribution of XpoGH78 to the conversion of lignocellulosic materials without any chemical pretreatment to release the water-soluble aromatic fragments, carbohydrates, and methanol was studied. The disintegrating effect of enzymatic lignocellulose treatment can be significantly improved by using different kinds of hydrolases and phenoloxidases. The considerable changes in low (3 kDa), medium (30 kDa), and high (> 200 kDa) aromatic fragments were observed after the treatment with XpoGH78 alone or with this potent cocktail. Synergistic conversion of rape straw also resulted in a release of 17.3 mg of total carbohydrates (e.g., arabinose, galactose, glucose, mannose, xylose) per gram of substrate after incubating for 72 h. Moreover, the treatment of rape straw with XpoGH78 led to a marginal methanol release of approximately 17 μg/g and improved to 270 μg/g by cooperation with the above accessory enzymes. In the case of beech wood conversion, the combined catalysis by XpoGH78 and laccase caused an effect comparable with that of fungal strain X. polymorpha in woody cultures concerning the liberation of aromatic lignocellulose fragments.

Biodegradation of Olive Mill Effluent by White-Rot Fungi

The liquid fraction from the two-phase extraction process in the olive industry (alperujo), is a waste that contains lignocellulosic organic matter and phenolic compounds, difficult to treat by conventional biological methods. Lignocellulosic enzymes from white-rot fungi can be an interesting solution to break down these recalcitrant compounds and advance the treatment of that waste. In the present work the ability of Phanerochaete chrysosporium to degrade the abovementioned liquid waste (AL) was studied. Experiments were carried out at 26 °C within the optimal pH range 4–6 for 10 days and with and without the addition of glucose, measuring the evolution of COD, BOD5, biodegradability index, reducing sugars, total phenolic compounds, and colour. The results obtained in this study revealed the interest of Phanerochaete chrysosporium for an economical and eco-friendly treatment of alperujo, achieving COD and colour removals around 60%, and 32% of total phenolic compounds degradation, regardless of glucose addition.

Bioethanol production from grass pea and wild oat hydrolysates using S.cerevisiae ETP53, K. marxianus ETP87, and P. fermentans ETP22

Conversion of lignocellulosic biomass into bioethanol is essential to reduce dependency on fossil fuels. After the grass pea and wild oat straws were characterized, they were hydrolyzed by live fungi (in situ), crude fungal enzymes, and chemical methods (sodium hydroxide and sulphuric acids at 0.5, 1, 2, and 3%). The fungi used for in situ degradation and crude lignocellulosic enzymes were Aspergillus niger JMC22344, Trichoderma reesei JMC22, Pleurotus ostreatus M2191, and Pleurotus sajor-caju M2145. Furfural content of hydrolysates was analyzed by GC–MS. The potential of activated charcoal, overliming, and sequential activated charcoal-overliming potential to reduce furfural in the acid hydrolysates were evaluated. The hydrolysates were fermented by S. cerevisiae ETP53, K. marxianus ETP87, and P. fermentans ETP22. Sulphuric acid hydrolyzed common wild oat (8.91 g/L) yielded higher sugars than NaOH (3.88 g/L) and NaOH treated wild oat (8.14 g/L) was superior in terms of sugars released than grass pea (4.21 g/L). P. ostreatus M2191 liberated the highest sugar (18.63 g/L) extracts than T. reesei JMC22676, A. niger ETP22344 and P. sajor-caju M2145. The 23–77% of the furfural in acid hydrolysates was reduced by overliming, activated charcoal, and the sequential activated charcoal-overliming. Yeasts produced optimal ethanol from straws hydrolyzed by crude enzymes from A. niger JMC22344. Generally, the ethanol titer was directly proportional to the amount of sugars released.

Concurrent production of glycyrrhetic acid 3-O-mono-\u03b2-d-glucuronide and lignocellulolytic enzymes by solid-state fermentation of a plant endophytic Chaetomium globosum

Glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG) as an important derivative of glycyrrhizin (GL) shows stronger biological activities and higher sweetness than GL. The biotransformation process is considered as an efficient strategy for GAMG production, due to its mild reaction, high production efficiency and environmentally friendly status. In this study, licorice straw was used for the first time as a medium for GAMG and lignocellulosic enzyme production via solid-state fermentation (SSF) of endophytic fungus Chaetomium globosum DX-THS3. The fermentation conditions including particle size, temperature, seed age, inoculum size, and moisture of substrate were optimized. Furthermore, additional nitrogen sources and carbon sources were screened for GAMG production by C. globosum DX-THS3 of SSF. Under optimal fermentation conditions, the percent conversion of glycyrrhizin reached 90% in 15 days, whereas the control needed 35 days to achieve the same result. The productivity of optimization (P = 2.1 mg/g/day) was 2.33-fold that of non-optimization (P = 0.9 mg/g/day). Meanwhile, high activities of filter paper enzyme (FPase) (245.80 U/g), carboxymethyl cellulase (CMCase) (33.67 U/g), xylanase (83.44 U/g), and β-glucuronidase activity (271.42 U/g) were obtained faster than those in the control during SSF. Our study provides a novel and efficient strategy for GAMG production and indicates C. globosum DX-THS3 as a potential producer of lignocellulolytic enzymes.

Yeasts in the attine ant-fungus mutualism: Diversity, functional roles, and putative biotechnological applications.

Insects interact with a wide variety of yeasts, often providing a suitable substrate for their growth. Some yeast-insect interactions are tractable models for understanding the relationships between the symbionts. Attine ants are prominent insects in the Neotropics and have performed an ancient fungiculture of mutualistic basidiomycete fungi for more than 55-65 million years. Yeasts gain access to this sophisticated mutualism, prompting diversity, ecological, and biotechnological studies in this environment. We review half a century research in this field, surveying for recurrent yeast taxa and their putative ecological roles in this environment. We found that previous studies mainly covered the yeast diversity from a small fraction of attine ants, being Saccharomycetales, Tremellales, and Trichosporonales as the most frequent yeast or yeast-like orders found. Apiotrichum, Aureobasidium, Candida, Cutaneotrichosporon, Debaryomyces, Meyerozyma, Papiliotrema, Rhodotorula, Trichomonascus, and Trichosporon are the most frequent recovered genera. On the other hand, studies of yeasts' ecological roles on attine ant-fungus mutualism only tapped the tip of the iceberg. Previous established hypotheses in the literature cover the production of lignocellulosic enzymes, chemical detoxification, and fungus garden protection. Some of these roles have parallels in biotechnological processes. In conclusion, the attine ant environment has a hidden potential for studying yeast biodiversity, ecology, and biotechnology, which has been particularly unexplored considering the vast diversity of fungus-growing ants.

Degradation of lignocellulosic matrix of oilseed cakes by solid-state fermentation: fungi screening for enzymes production and antioxidants release.

Vegetable oils are yearly produced in large amounts generating solid by-products, the oilseed cake (OC). OCs are lignocellulosic materials that have been used for animal feed with some limitations due to high fibre content from the plant cell walls. Biotechnological processes can help to overcome these limitations and contribute to up-grading such by-products, enhancing their nutritional value as feed ingredients. All fungal species were able to decrease neutral detergent fibre and acid detergent fibre in all by-products. Additionally, relevant enzymes were produced by the three fungi studied resulting in an improved antioxidant capacity of all fermented OCs. Aspergillus niger led to the highest activity of cellulase (109 U g-1 ), xylanase (692 U g-1 ) and protease (157 U g-1 ) per dry OC matter and to the recovery of an extract rich in antioxidants, with the highest scavenging potential of free radicals and superoxide anion, iron chelation ability and reducing power. Rhyzopus oryzae produced the highest activity of β-glucosidase (503 U g-1 ) and led to the highest liberation of total phenolic content (TPC). Principal components analysis showed that extracts with high antioxidant potential were obtained in solid-state fermentation (SSF) with high enzymatic activity. A positive correlation was established between the action of β-glucosidase and TPC. Within the same bioprocess it was possible to improve the nutritional value of OCs and to obtain relevant bioactive compounds such as lignocellulosic enzymes and phenolic compounds with antioxidant potential, resulting in a significant improvement of already valuable by-products with commercial interest for animal feed. © 2021 Society of Chemical Industry.

Enzymatic effects of Pleurotus ostreatus spent substrate on whole-plant corn silage and performance of lactating goats

Pleurotus ostreatus (oyster mushroom) synthesizes enzymes that degrade lignin, cellulose, and hemicellulose. The objectives of this study were to evaluate the effect of Pleurotus ostreatus spent substrate (POSS) on whole-plant corn silage (WPCS) chemical composition, antioxidant capacity, lignin monomers, and in vitro digestibility, as well as the performance of lactating goats fed corn silage treated with different levels of POSS. In experiment 1, 4 levels of lignocellulolytic enzymes were tested in a complete randomized design: 0, 10, 20, and 30 mg of lignocellulosic enzymes per kilogram of fresh matter, 4 replicates per treatment (vacuum-sealed bags). The bags were opened 60 d after ensiling. In experiment 2, corn silage treated with 3 enzyme levels (0, 10, or 30 mg/kg of fresh matter) was fed to lactating goats as part of the total mixed ration. Nine lactating Saanen goats (62.68 ± 7.62 kg BW; 44 ± 8 d in milk; 2.91 ± 0.81 kg of milk/day, mean ± SD) were assigned to three 3 × 3 Latin squares. Data were analyzed using the GLIMMIX procedure of SAS (version 9.4, SAS Institute Inc.), and means were compared by linear and quadratic orthogonal contrast. In experiment 1, neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, and cellulose quadratically decreased in the WPCS treated with POSS. At the nadir point, POSS decreased NDF by 14.1%, ADF by 19.5%, lignin by 9.07%, and cellulose by 22.1% compared with the untreated silage. Therefore, POSS led to a quadratic increase in in vitro dry matter digestibility of WPCS (+8.88% at the vertex) compared with the untreated silage. In experiment 2, POSS quadratically increased the in vivo total-tract ADF digestibility. Also, the concentration of polyphenols in the milk of goats linearly increased with the addition of POSS, and no differences were observed among treatments for milk yield and composition. In summary, adding 10 mg of lignocellulolytic enzymes from POSS per kilogram of fresh matter of whole-plant corn at ensiling had a more evident reduction in lignin and cellulose concentration, leading to greater in vitro digestibility, as well as greater in vivo ADF digestibility; however, milk yield was not different among treatments.

A genomic perspective on the potential of termite-associated Cellulosimicrobium cellulans MP1 as producer of plant biomass-acting enzymes and exopolysaccharides.

BackgroundLignocellulose is a renewable and enormous biomass resource, which can be degraded efficiently by a range of cocktails of carbohydrate-active enzymes secreted by termite gut symbiotic bacteria. There is an urgent need to find enzymes with novel characteristics for improving the conversion processes in the production of lignocellulosic-based products. Although various studies dedicated to the genus Cellulosimicrobium as gut symbiont, genetic potential related to plant biomass-acting enzymes and exopolysaccharides production has been fully untapped to date.MethodsThe cellulolytic bacterial strain MP1 was isolated from termite guts and identified to the species level by phenotypic, phylogenetic, and genomic analysis. To further explore genes related to cellulose and hemicellulose degradation, the draft genome of strain MP1 was obtained by using whole-genome sequencing, assembly, and annotation through the Illumina platform. Lignocellulose degrading enzymes and levan production in the liquid medium were also examined to shed light on bacterial activities.ResultsAmong 65 isolates obtained, the strain MP1 was the most efficient cellulase producer with cellulase activity of 0.65 ± 0.02 IU/ml. The whole genome analysis depicted that strain MP1 consists of a circular chromosome that contained 4,580,223 bp with an average GC content of 73.9%. The genome comprises 23 contigs including 67 rRNA genes, three tRNA genes, a single tmRNA gene, and 4,046 protein-coding sequences. In support of the phenotypic identification, the 16S rRNA gene sequence, average nucleotide identity, and whole-genome-based taxonomic analysis demonstrated that the strain MP1 belongs to the species Cellulosimicrobium cellulans. A total of 30 genes related to the degradation of cellulases and hemicellulases were identified in the C. cellulans MP1 genome. Of note, the presence of sacC1-levB-sacC2-ls operon responsible for levan and levan-type fructooligosaccharides biosynthesis was detected in strain MP1 genome, but not with closely related C. cellulans strains, proving this strain to be a potential candidate for further studies. Endoglucanases, exoglucanases, and xylanase were achieved by using cheaply available agro-residues such as rice bran and sugar cane bagasse. The maximum levan production by C. cellulans MP1 was 14.8 ± 1.2 g/l after 20 h of cultivation in media containing 200 g/l sucrose. To the best of our knowledge, the present study is the first genome-based analysis of a Cellulosimicrobium species which focuses on lignocellulosic enzymes and levan biosynthesis, illustrating that the C. cellulans MP1 has a great potential to be an efficient platform for basic research and industrial exploitation.

Saccharification of agave bagasse with Cellulase 50 XL is an effective alternative to highly specialized lignocellulosic enzymes for continuous hydrogen production

Agave bagasse is the lignocellulosic residue of the tequila industry and a potential feedstock for hydrogen production (H2) via dark fermentation after proper saccharification. Highly specialized enzymes for hydrolysis of lignocellulosic biomass are costly, hindering their application in continuous processes. This work investigated the use of a generic cellulase (Cellulase 50 XL), to prepare the hydrolysates from agave bagasse, either alone or combined with a highly specialized hemicellulase (Viscozyme L). The combination of Cellulase 50 XL and Viscozyme L increased the release of both total carbohydrates and chemical oxygen demand (COD) in 28.5% and 22.2%, respectively compared with Cellulase 50 XL. Conversely, the maximum volumetric H2 production rate (VHPR, 9.9 L H2/L-d) reached with the Cellulase 50 XL hydrolysates in continuous stirred tank reactors at an organic loading rate of 100 g COD/L-d, is the highest VHPR reported so far with enzymatic agave bagasse hydrolysates obtained with non-specialized enzyme. The estimated cost of H2 production using Cellulase 50 XL at lab-scale was 0.7 USD/L H2. From a systematic comparison of H2 productivities and lab-scale costs with similar studies it was concluded that: i) the composition, and not only the concentration of total carbohydrates plays a critical role in the performance of H2 production, ii) Cellulase 50 XL was one of the best cost-effective alternatives despite being designed for food sectors and not for the hydrolysis of lignocellulosic biomass. Overall, considering H2 productivities and cost of H2 production, Cellulase 50 XL hydrolysates resulted an appropriate option for cost-effective continuous H2 production.

Gut bacterial communities in the freshwater snail Planorbella trivolvis and their modification by a non-herbivorous diet.

The freshwater pulmonate snail Planorbella trivolvis is a common species in various bodies of water but is not native to China. Planorbella trivolvis usually live on diets with high fiber content, such as water grasses, algae and fallen leaves. These snails can attach to the wall of a water tank or to water grass and can be transported overseas to China through the ornamental fish trade. There are few studies investigating the intestinal microbiota of freshwater snails. In this study, using culture-independent molecular analysis, we assessed for the first time the complexity of bacterial communities in the intestines of reared snails. The intestinal microbiota in the snails fed different diets, that is, herbivorous feed (HV) with high cellulose and non-herbivorous feed (NHV) with low cellulose, were analyzed by Illumina sequencing. The results showed that the NHV-based diet significantly increased the body mass, shell diameter and specific growth rate of the snails after 60 days of rearing (P < 0.05). Histological experiments showed that the fat droplets in the epithelium columnar cells of the intestines of the NHV snails increased, and the cilia on these cells fell off. The sequencing results identified 486 and 195 OTUs in HV and NHV, respectively. Lots of bacteria were not reported previously in snails. The intestinal microbiota diversity index (Shannon, Simpson, Ace and Chao) in the NHV snails was significantly lower than that in the HV snails. The gut microbiota in the HV snails were predominantly Proteobacteria (52.97%) and Bacteroidetes (28.75%), while the gut microbiota in NHV snails were predominantly Proteobacteria (95.23%). At the genus level, Cloacibacterium (24.60%), Pseudomonas (4.47%), OM6ON (6.12%), and Rhodobacter (5.79%) were observed to be abundant in HV snails. However, Aeromonas (85.4%) was determined to be predominant in NHV snails. Functional prediction of the gut microbiome in snails by PICRUSt demonstrated a significant difference between the two groups, and the HV snails exhibited higher lignocellulose enzyme activity than did the NHV snails. This study represents a first step in characterizing the gut microbiota of the freshwater snail. Most of these microbes can process plant biomass and digest cellulose and lignocellulose, and the enzymes of these bacteria may have potential biotechnological applications in a variety of industrial processes.

A novel bifunctional glucanase exhibiting high production of glucose and cellobiose from rumen bacterium

Herbivores gastrointestinal microbiota is of tremendous interest for mining novel lignocellulosic enzymes for bioprocessing. We previously reported a set of potential carbohydrate-active enzymes from the metatranscriptome of the Hu sheep rumen microbiome. In this study, we isolated and heterologously expressed two novel glucanase genes, Cel5A-h38 and Cel5A-h49, finding that both recombinant enzymes showed the optimum temperatures of 50 °C. Substrate-specificity determination revealed that Cel5A-h38 was exclusively active in the presence of mixed-linked glucans, such as barley β-glucan and Icelandic moss lichenan, whereas Cel5A-h49 (EC 3.2.1.4) exhibited a wider substrate spectrum. Surprisingly, Cel5A-h38 initially released only cellotriose from lichenan and further converted it into an equivalent amount of glucose and cellobiose, suggesting a dual-function as both endo-β-1,3-1,4-glucanase (EC 3.2.1.73) and exo-cellobiohydrolase (EC 3.2.1.91). Additionally, we performed enzymatic hydrolysis of sheepgrass (Leymus chinensis) and rice (Orysa sativa) straw using Cel5A-h38, revealing liberation of 1.91 ± 0.30 mmol/mL and 2.03 ± 0.09 mmol/mL reducing sugars, respectively, including high concentrations of glucose and cellobiose. These results provided new insights into glucanase activity and lay a foundation for bioconversion of lignocellulosic biomass.

Zinc- and cadmium-tolerant endophytic bacteria from Murdannia spectabilis (Kurz) Faden. studied for plant growth-promoting properties, in vitro inoculation, and antagonism.

This research aims to isolate and identify Zn- and Cd-tolerant endophytic bacteria from Murdannia spectabilis, identify their properties with and without Zn and Cd stress, and to investigate the effect of bacterial inoculation in an in vitro system. Twenty-four isolates could survive on trypticase soya agar (TSA) supplemented with Zn (250-500mg L-1) and/or Cd (20-50mg L-1) that belonged to the genera Bacillus, Pantoea, Microbacterium, Curtobacterium, Chryseobacterium, Cupriavidus, Siphonobacter, and Pseudomonas. Each strain had different indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase and siderophore production, nitrogen fixation, phosphate solubilization, and lignocellulosic enzyme characteristics. Cupriavidus plantarum MDR5 and Chryseobacterium sp. MDR7 were selected for inoculation into plantlets that were already occupied by Curtobacterium sp. TMIL due to them have a high tolerance for Zn and Cd while showing no pathogenicity. As determined via an in vitro system, Cupriavidus plantarum MDR5 remained in the plants to a greater extent than Chryseobacterium sp. MDR7, while Curtobacterium sp. TMIL was the dominant species. The Zn plus Cd treatment supported the persistence of Cupriavidus plantarum MDR5. Dual and mixed cultivation showed no antagonistic effects between the endophytes. Although the plant growth and Zn/Cd accumulation were not significantly affected by the Zn-/Cd-tolerant endophytes, the inoculation did not weaken the plants. Therefore, Cupriavidus plantarum MDR5 could be applied in a bioaugmentation process.

A fast and easy strategy for lytic polysaccharide monooxygenase-cleavable His6-Tag cloning, expression, and purification

Lytic polysaccharide monooxygenases (LPMOs) are industrially important enzymes able to enhance the enzymatic lignocellulose saccharification in synergism with classical glycoside hydrolases. Fungal LPMOs have been classified as AA9, AA11, and AA13-16 families showing a diverse specificity for substrates such as soluble and insoluble beta-glucans, chitin, starch, and xylan, besides cellulose. These enzymes are still not fully characterized, and for example this is testify by their mechanism of oxidation regularly reviewed multiple times in the last decade. Noteworthy is that despite the extremely large abundance in the entire Tree of Life, our structural and functional knowledge is based on a restricted pool of LPMO, and probably one of the main reason reside in the challenging posed by their heterologous expression. Notably, the lack of a simple cloning protocol that could be universally applied to LPMO, hinders the conversion of the ever-increasing available genomic information to actual new enzymes. Here, we provide an easy and fast protocol for cloning, expression, and purification of active LPMOs in the following architecture: natural signal peptide, LPMO enzyme, TEV protease site, and His6-Tag. For this purpose, a commercial methanol inducible expression vector was initially modified to allow the LPMO expression containing the above characteristics. Gibson assembly, a one-step isothermal DNA assembly, was adopted for the direct assembly of intron-less or intron-containing genes and the modified expression vector. Moreover, His6-tagged LPMO constructs can be submitted to TEV proteolysis for removal of the questionable C-terminal His6-Tag, obtaining a close-to-native form of LPMO. We successfully applied this method to clone, express, and purify six LPMOs from AA9 family with different regioselectivities. The proposed protocol, provided as step-by-step, could be virtually applied in many laboratories thanks to the choice of popular and commons materials.

The functions of glutathione peroxidase in ROS homeostasis and fruiting body development in Hypsizygus marmoreus.

Glutathione peroxidase (GPX) is one of the most important antioxidant enzymes for maintaining reactive oxygen species (ROS) homeostasis. Although studies on fungi have suggested many important physiological functions of GPX, few studies have examined the role of this enzyme in Basidiomycetes, particularly its functions in fruiting body developmental processes. In the present study, GPX-silenced (GPxi) strains were obtained by using RNA interference. The GPxi strains of Hypsizygus marmoreus showed defects in mycelial growth and fruiting body development. In addition, the results indicated essential roles of GPX in controlling ROS homeostasis by regulating intracellular H2O2 levels, maintaining GSH/GSSG balance, and promoting antioxidant enzyme activity. Furthermore, lignocellulose enzyme activity levels were reduced and the mitochondrial phenotype and mitochondrial complex activity levels were changed in the H. marmoreus GPxi strains, possibly in response to impediments to mycelial growth and fruiting body development. These findings indicate that ROS homeostasis has a complex influence on growth, fruiting body development, GSH/GSSG balance, and carbon metabolism in H. marmoreus.Key points• ROS balance, energy metabolism, fruiting development.

Evaluating lignocellulosic enzyme activity of termite mound soil from different locations of Tamil Nadu

Termite soils provide a very distinct ecological environment which harbors and promotes very specialized cellulolytic and hemicellulolytic microorganisms. This specialized flora enables the termites to feed on hardly degradable polymers such as lignocellulose. The cellulase enzymes synthesized by the microorganisms have a crucial role in recycling C and nutrients and maintaining soil fertility in nature. Hence in the present study, termite mound soils were collected from different locations of Tamil Nadu and their lignocellulosic enzyme activity viz., Cellulase, Saccharase and β- glucosidase were assessed to know fertility status of the particular habitats.