Β-glucosidase Production Research Articles

Reappraisal of different agro-industrial waste for the optimization of cellulase production from Aspergillus niger ITV02 in a liquid medium using a Box-Benkhen design.

High-value metabolites, such as enzymes and biofuels, can be produced from various agro-industrial waste containing high percentages of cellulose and hemicellulose. Aspergillus niger ITV02 demonstrates high potential in cellulases production, the key enzyme for converting lignocellulosic materials into fermentable sugars to produce second-generation bioethanol (bioethanol 2G). This study evaluated five lignocellulosic residues of agricultural importance: sugarcane bagasse (SCB), sorghum bagasse (SB), corn stubble (CS), barley straw (BS) and rice husk (RH) as substrates for cellulase production. The temperature, pH and stirring conditions were optimized using a Box-Behnken design to identify the most suitable conditions for cellulase production while minimizing nitrogen concentrations. The results indicate that the best way of propagation A. niger ITV02 is through the use of spores as an inoculant, in conjunction with the use of materials with a high cellulose/lignin ratio, such as CS and SB for the generation of cellulases. These conditions promote the expression of cellulases towards β-glucosidase production, unlike materials with lower cellulose/lignin ratios like BS and RH, which exhibited lower cellulase activity. The optimal conditions for cellulase production by A. niger ITV02 were determined to be 33 °C, pH 5.3, and 200rpm, resulting in a 1.7-fold increase in Exoglucanase (FPase activity) (from 0.127 to 0.215 U/mL). These findings demonstrate the potential to enhance FPase activity by utilizing substrates with high cellulose/lignin content and implementing optimal operational conditions without the need to raise the nitrogen content of the basal medium, thus mitigating the economic impact of cellulase production.

Combinational manipulation of transcription factors, CreA and ClbR, is a viable strategy to improve cellulolytic enzyme production in Aspergillus aculeatus

The production of cellulolytic enzymes in response to inducible carbon sources is mainly regulated at the transcriptional level in filamentous fungi. We have identified a cellobiose-response regulator (ClbR) controlling the expression of cellulolytic enzyme-encoding genes in Aspergillus aculeatus. However, the engineering potential of combining the deletion of transcriptional repressors with the overexpression of transcriptional activators to enhance enzyme production has not been analyzed. Here, we investigated the effect of the deletion of the transcriptional repressor creA and the overexpression of the transcriptional activator clbR in enzyme production in A. aculeatus. Here, we verified that a combination of creA deletion and clbR overexpression (Δc&OE) improved cellulase, β-1,4-xylanase, and β-glucosidase production. Cellulase and β-1,4-xylanase production increased 3.4- and 8.0-fold in Δc&OE compared with the host strain (MR12) at 96-h incubation, respectively. β-Glucosidase production in ΔcreA and Δc&OE increased approximately 5.0-fold compared with that in MR12 at 240 h-incubation. Transcriptional analysis revealed that the increase in enzyme production was due to increased expression of cellobiohydrolase, endo-β-1,4-glucanase, β-1,4-xylanase, and β-glucosidase 1 (bgl1). Interestingly, bgl1 expression in ΔcreA increased in a dose-dependent manner in response to glucose. Thus, combinational manipulation of transcription factors improved cellulase, xylanase, and β-glucosidase production in A. aculeatus.

Hyper β-glucosidase producer Beauveria bassiana SAN01—optimization of fermentation conditions and evaluation of saccharification potential

AbstractThe hyper-production of β-glucosidase by a local strain of Beauveria bassiana under submerged conditions is reported in this study. The initial screening of seven agricultural residues showed that the haulm of Bambara—an underutilized African legume—supported the highest β-glucosidase production; hence, statistical optimization of enzyme production was done using this biomass as the sole carbon source. Plackett–Burman design identified the concentrations of Bambara haulm, KCl, and NaCl as well as agitation speed and incubation time as the most significant factors affecting enzyme production. Subsequently, the central composite design predicted the optimal conditions (Bambara 57 g/L, KCl 302 mg/L, NaCl 154 mg/L, agitation speed 150 rpm, and incubation 223 h) for B. bassiana β-glucosidase production, which were further validated. The generated quadratic model was deemed significant judging from its F-value (201.63), adequate precision ratio (45.74), as well as the R2 (0.9988), adjusted R2 (0.9938), and predicted R2 (0.9195) values. The optimization resulted in a ~5.36-fold increase in enzyme levels from the unoptimized production of ~133 to 711 U/mL. The enzyme was also demonstrated to efficiently hydrolyze cellobiose, converting more than 90% of the substrate to glucose. These results further establish the resourcefulness of the B. bassiana strain for the production of β-glucosidase enzyme, having immense potential, especially in the food and energy industries.

Leaching Approach for β-Glucosidase Extraction from Fermented Rice Husk in Solid State Cultivation by Aspergillus protuberus.

Environmental problems are caused by the disposal of agrowastes in developing countries. It is imperative to convert such wastes into useful products, which require enzymes such as β-glucosidase. β-Glucosidase has variety of applications in biotechnology including food, textile, detergents, pulp and paper, pharmaceutical and biofuel industries. β-Glucosidase production was performed using the locally isolated Aspergillus protuberus using best growth circumstances on rice husk in solid-state fermentation (SSF). Leaching of β-glucosidase from fermented rice husk with number of solvents to evaluate their extraction efficacy. Among the different solvents examined, acetate buffer (0.02M, pH 5.0) proved to be the best solvent. The subsequent parameters were optimized with acetate buffer. Two washes with acetate buffer each by shaking (30min) in a ratio of 1g of rice husk: 5ml of acetate buffer together attained maximum recovery of β-glucosidase with 41.95 U/g of rice husk.

A Thermotolerant Yeast Cyberlindnera rhodanensis DK Isolated from Laphet-so Capable of Extracellular Thermostable β-Glucosidase Production.

This study aims to utilize the microbial resources found within Laphet-so, a traditional fermented tea in Myanmar. A total of 18 isolates of thermotolerant yeasts were obtained from eight samples of Laphet-so collected from southern Shan state, Myanmar. All isolates demonstrated the tannin tolerance, and six isolates were resistant to 5% (w/v) tannin concentration. All 18 isolates were capable of carboxy-methyl cellulose (CMC) degrading, but only the isolate DK showed ethanol production at 45 °C noticed by gas formation. This ethanol producing yeast was identified to be Cyberlindnera rhodanensis based on the sequence analysis of the D1/D2 domain on rRNA gene. C. rhodanensis DK produced 1.70 ± 0.01 U of thermostable extracellular β-glucosidase when cultured at 37 °C for 24 h using 0.5% (w/v) CMC as a carbon source. The best two carbon sources for extracellular β-glucosidase production were found to be either xylose or xylan, with β-glucosidase activity of 3.07-3.08 U/mL when the yeast was cultivated in the yeast malt extract (YM) broth containing either 1% (w/v) xylose or xylan as a sole carbon source at 37 °C for 48 h. The optimal medium compositions for enzyme production predicted by Plackett-Burman design and central composite design (CCD) was composed of yeast extract 5.83 g/L, peptone 10.81 g/L and xylose 20.20 g/L, resulting in a production of 7.96 U/mL, while the medium composed (g/L) of yeast extract 5.79, peptone 13.68 and xylan 20.16 gave 9.45 ± 0.03 U/mL for 48 h cultivation at 37 °C. Crude β-glucosidase exhibited a remarkable stability of 100%, 88% and 75% stable for 3 h at 35, 45 and 55 °C, respectively.

Recovery and characterization of β-glucosidase-producing non-Saccharomyces yeasts from the fermentation broth of Vitis labruscana Baily × Vitis vinifera L. for investigation of their fermentation characteristics.

The present study focuses on investigating 60 strains of yeast isolated from the natural fermentation broth of Vitis labruscana Baily × Vitis vinifera L. These strains underwent screening using lysine culture medium and esculin culture medium, resulting in the identification of 27 local non-Saccharomyces yeast strains exhibiting high β-glucosidase production. Subsequent analysis of their fermentation characteristics led to the selection of four superior strains (Z-6, Z-11, Z-25, and Z-58) with excellent β-glucosidase production and fermentation performance. Notably, these selected strains displayed a dark coloration on esculin medium and exhibited robust gas production during Duchenne tubules' fermentation test. Furthermore, all four non-Saccharomyces yeast strains demonstrated normal growth under specific conditions including SO2 mass concentration ranging from 0.1 to 0.3g/L, temperature between 25 and 30°C, glucose mass concentration ranging from 200 to 400g/L, and ethanol concentration at approximately 4%. Molecular biology identification confirmed that all selected strains belonged to Pichia kudriavzevii species which holds great potential for wine production.

A comparative study on flaxseed lignan biotransformation through resting cell catalysis and microbial fermentation by β-glucosidase production Lactiplantibacillus plantarum.

Flax lignan has attracted much attention because of its potential bioactivities. However, the bioavailability of secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, depends on the bioconversion by the colon bacteria. Lactic acid bacteria (LAB) with β-glucosidase activity has found wide application in preparing bioactive aglycone. LAB strains with good β-glucosidase activity were isolated from fermented tofu. Their bioconversion of flax lignan extract was investigated by resting cell catalysis and microbial fermentation, and the metabolism of SDG by Lactiplantibacillus plantarum C5 following fermentation was characterized by widely targeted metabolomics. Five L. plantarum strains producing β-glucosidase with broad substrate specificity were isolated and identified, and they all can transform SDG into secoisolariciresinol (SECO). L. plantarum C5 resting cell reached a maximum SDG conversion of 49.19 ± 3.75%, and SECO generation of 21.49 ± 1.32% (0.215 ± 0.013 mm) at an SDG substrate concentration of 1 mM and 0.477 ± 0.003 mm SECO was produced at 4 mm within 24 h. Although sixteen flax lignan metabolites were identified following the fermentation of SDG extract by L. plantarum C5, among them, four were produced following the fermentation: SECO, demethyl-SECO, demethyl-dehydroxy-SECO and isolariciresinol. Moreover, seven lignans increased significantly. Fermentation significantly increased the profile and level of flax lignan metabolites, and the resting cell catalysis benefits from higher bioconversion efficiency and more straightforward product separation. Resting cell catalysis and microbial fermentation of flax lignan extract by the isolated β-glucosidase production L. plantarum could be potentially applied in preparing flax lignan ingredients and fermented flaxseed. © 2024 Society of Chemical Industry.

Application of Immobilized β-Glucosidase from Candida boidinii in the Hydrolysis of Delignified Sugarcane Bagasse.

Candida boidinii is a methylotrophic yeast with wide geographical distribution. In the present study, the microorganism was isolated from the Bahian semiarid and the enzymatic extract containing β-glucosidase was obtained through submerged fermentation. Response surface methodology was employed to optimize of fermentation medium. The higher production of β-glucosidase was obtained after 71h of fermentation in an optimized medium composed of 3.35% glucose, 1.78% yeast extract and 1% peptone. The optimum pH and temperature of enzymatic activity were 6.8 (citrate-phosphate buffer) and 71.7°C, respectively. Salts tested (10mM) CaCl2, Na2SO4 and ZnSO4 promotes the increase of 91%, 45% and 80% of activity, respectively. The enzyme retained 64% ± 2.3 of its initial activity after 1h heating at 90°C. The production of reducing sugars was 95.94% after 24h of hydrolysis and, with the addition of metal ions, this value increased more than 2 times. Among the supports analyzed for immobilization, chitosan showed higher residual activity during reuse. The immobilized enzyme showed higher activity at 60°C with pH 6 and preserved almost 100% of the initial activity after 30min at 70°C.

Cassava Bagasse as a Low-Cost Substrate for Cellulase and Organic Acid Production Using Co-Cultivated Fungi

Cassava bagasse has great potency as a substrate in the biorefinery industry. This paper proposes the valorisation of cassava bagasse into organic acids by cellulase through the co-cultivation of Aspergillus violaceofuscus and Trichoderma reesei RUT-C30 at the ratio 1:1. The optimised conditions for β-glucosidase production under submerged fermentation were pH 4.50, a tween 80 concentration of 0.05% (v/v), and a spore concentration of 7.18 × 107 spores·mL−1. We found base steam cassava bagasse (BSCB) to have high cellulose content, making it possible to replace avicel phosphoric acid swollen cellulose (PASC) as a substrate. The co-cultivation with the BSCB substrate had higher levels of β-glucosidase (1.72-fold), cellobiohydrolase (2.83-fold), and endoglucanase (2.82-fold) activity compared to that of the avicel PASC substrate. Moreover, acetic acid (7.41 g·L−1), citric acid (3.54 g·L−1), gluconic acid (0.30 g·L−1), and malic acid (0.37 g·L−1) were detected in the BSCB crude extract. These results demonstrate the considerable prospects of the A. violaceofuscus and T. reesei RUT-C30 co-cultivation approaches in the biorefinery industry.

Developing a novel selection method for alcoholic fermentation starters by exploring wine yeast microbiota from Greece.

The selection of native yeast for alcoholic fermentation in wine focuses on ensuring the success of the process and promoting the quality of the final product. The purpose of this study was firstly to create a large collection of new yeast isolates and categorize them based on their oenological potential. Additionally, the geographical distribution of the most dominant species, Saccharomyces cerevisiae, was further explored. Towards this direction, fourteen spontaneously fermented wines from different regions of Greece were collected for yeast typing. The yeast isolates were subjected in molecular analyses and identification at species level. RAPD (Random Amplified Polymorphic DNA) genomic fingerprinting with the oligo-nucleotide primer M13 was used, combined with Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. All yeast isolates were scrutinized for their sensitivity to killer toxin, production of non-desirable metabolites such as acetic acid and H2S, β-glucosidase production and resistance to the antimicrobial agent; SO2. In parallel, S. cerevisiae isolates were typed at strain level by interdelta - PCR genomic fingerprinting. S. cerevisiae strains were examined for their fermentative capacity in laboratory scale fermentation on pasteurized grape must. Glucose and fructose consumption was monitored daily and at the final point a free sorting task was conducted to categorize the samples according to their organoleptic profile. According to our results, among the 190 isolates, S. cerevisiae was the most dominant species while some less common non-Saccharomyces species such as Trigonopsis californica, Priceomyces carsonii, Zygosaccharomyces bailii, Brettanomyces bruxellensis and Pichia manshurica were identified in minor abundancies. According to phenotypic typing, most isolates were neutral to killer toxin test and exhibited low acetic acid production. Hierarchical Cluster Analysis revealed the presence of four yeast groups based on phenotypic fingerprinting. Strain level typing reported 20 different S. cerevisiae strains from which 65% indicated fermentative capacity and led to dry wines. Sensory evaluation results clearly discriminated the produced wines and consequently, the proposed yeast categorization was confirmed. A novel approach that employs biostatistical tools for a rapid screening and classification of indigenous wine yeasts with oenological potential, allowing a more efficient preliminary selection or rejection of isolates is proposed.

Characterization of β-glucosidases from Meridianimaribacter sp. CL38

The production of second-generation biofuel requires a huge amount of freshwater. It is estimated that at least three gal of freshwater is used to produce one gal of biofuel. The replacement of freshwater with seawater serves as a potential alternative in biofuel generation. Therefore, salt-tolerant enzymes play an important role in saccharification and fermentation process. Halophilic β-glucosidase is one of the key enzymes for the process. In this study, the β-glucosidase of halophile Meridianimaribacter sp. CL38 isolated from mangrove soil was characterized. Strain CL38 achieved maximum production of β-glucosidase at 12th hour of growth. The β-glucosidase showed highest activity at 2% (w/v) NaCl while highly stable at salt concentration ranging from 1-2% (w/v) (more than 96% of relative activity). Its β-glucosidase activity remained active in the presence of 5mM Mn2+, Mg2+, Ca2+ ions, and 1% (v/v) Tween-20 and Tween-80. The draft genome sequence of strain CL38 was retrieved from GenBank database and submitted to dbCAN meta server for CAZymes annotation. Strain CL38 harbors 44 GHs and GH3 are annotated as β-glucosidases. The β-glucosidases of Meridianimaribacter flavus (99.61%) and Mesoflavibacter sabulilitoris (97.44%) showed the closest identity with Bgl3a and Bgl3b protein sequences from strain CL38, respectively. Glycoside hydrolase family 3 domain was identified in both enzymes via InterPro scan server. The presence of signal peptides indicated that both enzymes were secreted extracellularly. Five motifs were identified in Bgl3a and Bgl3b, with the active site (nucleophile) found at Asp296 and Asp297, respectively. Collectively, these β-glucosidases could be potentially used in the biofuel production, in particular the lignocellulosic biomass pretreatment process. This is the first attempt to characterize the β-glucosidase in genus Meridianimaribacter as so far none of the lignocellulolytic enzymes from this genus were characterized.

Publisher Erratum: Improved Production and Properties of β-glucosidase Influenced by 2-deoxy-d-glucose in the Culture Medium of Termitomyces clypeatus

Publisher Erratum: Improved Production and Properties of β-glucosidase Influenced by 2-deoxy-d-glucose in the Culture Medium of Termitomyces clypeatus

Production of β-glucosidase by Rhodotorula oryzicola and use of enzyme for hydrolysis of sugarcane bagasse delignified.

Bioethanol is obtained by hydrolysis of sugarcane bagasse by cellulases. Commercial cellulases are expensive and have a low concentration of β-glucosidase (EC 3.2.1.21), which decrease hydrolysis efficiency. The present work aims to produce supernatant rich in β-glucosidase (BGL) using the yeast Rhodotorula oryzicola and apply it in the hydrolysis of delignified sugarcane bagasse. Yeast fermented in a modified YPD (Yeast Peptone Dextrose) medium with 0.5% (w/v) cellobiose and 1.0% (w/v) glucose produced BGL with a specific activity of 1.44 ± 0.013 U/mg. Partial purification of BGL by acetone showed a specific activity of 3.48 U/mg. The optimum pH and temperature were 6.02 and 65°C, respectively. BGL partially purified (BGLppR.oryzicola) by acetone showed tolerance to glucose, with a relative activity of 82.89 ± 0.11%. The activity increased with the addition of iron sulfate and zinc sulfate and decreased with manganese sulfate. BGL partially purified was thermal stable, with a relative activity of 85.59% after 60min at 90°C. BGL partially purified applied in the hydrolysis of sugarcane bagasse delignified with 3% (w/w) NaOH + 6% (w/w) Na2SO3 showed a conversion rate of 72.46 ± 1.60%. The results showed that BGL partially purified is a glucose tolerant cellulase of low-cost, promising the application of bioethanol production.

Selection of Debaryomyces hansenii isolates to improve quality of dry fermented sausage

ABSTRACT The objective of this experiment was to select D. hansenii isolates that are stable in dry fermented sausage (DFS) production and can positively affect the quality. D. hansenii isolates were examined for production of biogenic amines, β-glucuronidase, β-glucosidase, proteolytic, and lipolytic enzymatic activities. The acid and salt tolerance of D. hansenii isolates were also examined. DFS inoculated with selected D. hansenii isolates were analyzed for microorganisms, weight loss, Aw, and pH. DFS was also subjected to analyses of color, texture profile, reducing sugar, volatile basic nitrogen (VBN), lipid oxidation, and free fatty acid. Twenty-two D. hansenii isolates were isolated. None of these D. hansenii isolates produced biogenic amines, and all strains were negative for β-glucuronidase and β-glucosidase activities. After examining these strains for proteolysis, lipolysis, and salt and acid tolerance, D. hansenii isolates SMFM2021-C14, SMFM2021-S8, and SMFM2021-SL3 were selected to be used in DFS. During the processing of DFS, the groups treated with D. hansenii isolate SMFM2021-S8 or SMFM2021-SL3 showed lower (p < .05) hardness values of DFS. The sample treated with D. hansenii SMFM2021-S8 showed lower (p < .05) VBN and lipid oxidation contents than the others. The ratio of ω-6/ω-3 was lower (p < .05) in D. hansenii SMFM2021-SL3 treated group than in the other treatments. These results indicate that D. hansenii isolates SMFM2021-S8 and SMFM2021-SL3 might be useful in producing DFS to improve its quality.

Optimisation of β-Glucosidase Production in a Crude Aspergillus japonicus VIT-SB1 Cellulase Cocktail Using One Variable at a Time and Statistical Methods and its Application in Cellulose Hydrolysis.

Pulp and paper mill sludge (PPMS) is currently disposed of into landfills which are reaching their maximum capacity. Valorisation of PPMS by enzymatic hydrolysis using cellulases is an alternative strategy. Existing commercial cellulases are expensive and contain low titres of β-glucosidases. In this study, β-glucosidase production was optimised by Aspergillus japonicus VIT-SB1 to obtain higher β-glucosidase titres using the One Variable at a Time (OVAT), Plackett Burman (PBD), and Box Behnken design (BBD)of experiments and the efficiency of the optimised cellulase cocktail to hydrolyse cellulose was tested. β-Glucosidase production was enhanced from 0.4 to 10.13 U/mL, representing a 25.3-fold increase in production levels after optimisation. The optimal BBD production conditions were 6 days of fermentation at 20 °C, 125 rpm, 1.75% soy peptone, and 1.25% wheat bran in (pH 6.0) buffer. The optimal pH for β-glucosidase activity in the crude cellulase cocktail was (pH 5.0) at 50 °C. Optimal cellulose hydrolysis using the crude cellulase cocktail occurred at longer incubation times, and higher substrate loads and enzyme doses. Cellulose hydrolysis with the A. japonicus VIT-SB1 cellulase cocktail and commercial cellulase cocktails resulted in glucose yields of 15.12 and 12.33 µmol/mL glucose, respectively. Supplementation of the commercial cellulase cocktail with 0.25 U/mg of β-glucosidase resulted in a 19.8% increase in glucose yield.

English

β-glucosidases are found in all domains of life and has the ability to hydrolyse the glycosidic bond between carbohydrates and non-carbohydrates residues making it a potent enzyme from an industrial and agricultural perspective. The aim of the current study was isolation, screening, and identification of bacterial strains having maximum β-glucosidase activity from soil sample of Jacobabad, Pakistan. The isolates were screened primarily, and efficient isolates were confirmed for β-glucosidase production through secondary screening. Among the different isolated strains, 1j2 was selected as the best β-glucosidase producer. The isolate showed 99% similarity with Bacillus stercoris after 16S rRNA sequencing. Different culture conditions were optimized for effective β-glucosidase production. Enhanced level of β-glucosidase was achieved at 40°C, pH 8 at 150 rpm in a β-glucosidase production medium supplemented with a combination of agricultural residues of wheat bran 0.25% (w/v) and orange peel 0.25% (w/v), using 5% inoculum. Bacillus stercoris produced a higher yield (2.89U/mg) of β-glucosidase under optimized conditions. Partial purification of β-glucosidase was achieved after 80% ammonium sulfate precipitation. The enzyme was purified and had shown maximum specific activity of 53.2 U/mg with molecular weight of approximately 36 kDa. In all the tested metals Cu2+ was observed to have inhibitory effect while Ca2+ had not affected the enzyme activity significantly. While organic solvents and surfactants had significantly reduced the β-glucosidase activity. Based on the afore mentioned abilities of purified β-glucosidase from Bacillus stercoris strain it could be a promising player for the industrial applications of lignocellulose hydrolysis found in agricultural residues for the cheap production of biofuels.

Screening and oenological property analysis of ethanol-tolerant non-Saccharomyces yeasts isolated from Rosa roxburghii Tratt.

Ethanol tolerance is crucial for the oenological yeasts. Rosa roxburghii Tratt, a Rosaceae plant native to China, is rich in nutritional and medicinal ingredients. In this study, ethanol-tolerant non-Saccharomyces yeasts were screened, and their oenological properties were further evaluated. Three ethanol-tolerant yeast strains (designated as C6, F112, and F15), which could tolerate 12% (v/v) ethanol treatment, were isolated from R. roxburghii, and identified as Candida tropicalis, Pichia guilliermondii, and Wickerhamomyces anomalus, respectively. The winemaking condition tolerances of these ethanol-tolerant yeast strains were similar to those of Saccharomyces cerevisiae X16. However, their growth, sugar metabolic performance and sulphureted hydrogen activities, were different. The β-glucosidase production ability of strain W. anomalus F15 was lower than that of S. cerevisiae X16, and strains of C. tropicalis C6 and P. guilliermondii F112 were similar to S. cerevisiae X16. Electronic sensory properties of the R. roxburghii wines fermented using ethanol-tolerant yeasts together with S. cerevisiae showed no significant differences. However, the mixed inoculation of the ethanol-tolerant yeast strains with S. cerevisiae could regulate the volatile aroma characteristics of the fermented R. roxburghii wine, enriching and enhancing the aroma flavor. Therefore, the selected ethanol-tolerant yeasts have the potential for application in the production of unique R. roxburghii wine.

Intraspecific variability in growth and in vitro production of plant cell wall-degrading enzymes among argentinean isolates of Colletotrichum graminicola, a maize pathogen

Background and aims: Colletotrichum graminicola (Glomerellaceae, Glomerellales), the causal agent of maize (Zea mays) anthracnose, as many other fungal pathogens, relies on its battery of cell wall degrading enzymes (CWDEs) to make its way through the cell walls of the host, and thus the production of these enzymes is considered a virulence factor. Since this disease is relatively new in Argentina, little is known about the capacity of CWDEs production in native isolates. The aim of this work was to investigate if there is intraspecific variability in growth and in vitro production of several extracellular CWDEs among Argentinean fungal isolates of C. graminicola. M&amp;M: Eight isolates of C. graminicola (obtained from symptomatic maize plants growing in different geographical locations) were tested in vitro to evaluate growth capacity and polygalacturonase, polymethylgalacturonase, β-glucosidase and laccase production, using two different liquid culture media. Results: Polygalacturonase, polymethylgalacturonase and β-glucosidase production greatly varied among isolates and culture media. Laccase activity was detected only in three isolates. Utmost enzymatic titres attained were respectively 250, 280, 45 and 63 U/l. The observed intraspecific variability in CWDEs in vitro production is consistent with the high variability found at genetic level when assessing ISSR markers. Conclusions: The isolates of C. graminicola evaluated showed notable differences in CWDEs production, not associated with a differential growth. This indicates a large intraspecific variability, which might be considered when choosing a method to deal with this pathogen.

A glucose tolerant β-glucosidase from a newly isolated Neofusicoccum parvum strain F7: production, purification, and characterization

Cellulase-producing microorganisms produce low titres of β-glucosidases with low tolerance to glucose. This study aimed to improve production, purify, and characterize a β-glucosidase from a newly isolated Neofusicoccum parvum strain F7. β-Glucosidase production was significantly enhanced by a sequential statistical modelling approach from 1.5-fold in Plackett–Burman design to 2.5 U/ml in the Box–Behnken design compared to the preliminary one variable at a time experiments (1.6 U/ml). The optimal conditions for enzyme production by BBD were 12 days of fermentation at 20 °C, 175 rpm, 0.5% glycerol and 1.5% casein in pH 6.0 buffer. Three β-glucosidase isoforms referred to as Bgl1, Bgl2, Bgl3 were purified and characterized from the optimized crude extract displaying IC50 values of 2.6, 22.6 and 319.5 mM for glucose, respectively. Bgl3 with a molecular mass of approximately 65 kDa demonstrated the highest tolerance to glucose among the isoforms. The optimum activity and stability for Bgl3 was observed at pH 4.0 in 50 mM sodium acetate buffer with 80% β-glucosidase residual activity retained for three hours. This isoform also retained 60% residual activity at 65 °C for one hour which was then reduced to 40% which remained stable for another 90 min. The β-glucosidase activity of Bgl3 was not enhanced after the addition of metal ions in assay buffers. The Km and vmax for 4-nitrophenyl-β-d-glucopyranoside were 1.18 mM and 28.08 µmol/min, respectively indicating high affinity for the substrate. The ability to withstand the presence of glucose in conjunction with its thermophilic nature indicates promise for this enzyme in industrial application.

Tradeoffs of microbial life history strategies drive the turnover of microbial-derived organic carbon in coastal saline soils.

Stable soil organic carbon (SOC) formation in coastal saline soils is important to improve arable land quality and mitigate greenhouse gas emissions. However, how microbial life-history strategies and metabolic traits regulate SOC turnover in coastal saline soils remains unknown. Here, we investigated the effects of microbial life history strategy tradeoffs on microbial carbon use efficiency (CUE) and microbial-derived SOC formation using metagenomic sequencing technology in different salinity soils. The results showed that high-salinity is detrimental to microbial CUE and microbial-derived SOC formation. Moreover, the regulation of nutrients stoichiometry could not mitigate adverse effects of salt stress on microbial CUE, which indicated that microbial-derived SOC formation is independent of stoichiometry in high-salinity soil. Low-salinity soil is dominated by a high growth yield (Y) strategy, such as higher microbial biomass carbon and metabolic traits which are related to amino acid metabolism, carbohydrate metabolism, and cell processes. However, high-salinity soil is dominated by stress tolerance (S) (e.g., higher metabolic functions of homologous recombination, base excision repair, biofilm formation, extracellular polysaccharide biosynthesis, and osmolytes production) and resource acquisition (A) strategies (e.g., higher alkaline phosphatase activity, transporters, and flagellar assembly). These trade-offs of strategies implied that resource reallocation took place. The high-salinity soil microbes diverted investments away from growth yield to microbial survival and resource capture, thereby decreasing biomass turnover efficiency and impeding microbial-derived SOC formation. Moreover, altering the stoichiometry in low-salinity soil caused more investment in the A-strategy, such as the production of more β-glucosidase and β-N-acetyl-glucosaminidase, and increasing bacterial chemotaxis, which thereby reduced microbial-derived SOC formation. Our research reveals that shift the microbial community from S- and A- strategies to the Y-strategy is important to increase the microbial CUE, and thus enhance SOC turnover in coastal saline soils.