Biosynthesis and one-step enrichment process of potentially prebiotic cello-oligosaccharides produced by ß-glucosidase from Fusarium solani.

Cello-oligosaccharides (COS) become a new type of functional oligosaccharides. COS transglycosylation reactions were studied to enhance COS yield production. Seeking the ability of the free form of Fusarium solani ß-glucosidase (FBgl1) to synthesize COS under low substrate concentrations, we found out that this biocatalyst initiates this reaction with only 1 g/L of cellobiose, giving rise to the formation of cellotriose. Cellotriose and cellopentaose were detected in biphasic conditions with an immobilized FBgl1 and when increased to 50 g/L of cellobiose as a starter concentration. After the biocatalyst recycling process, the trans-glycosylation yield of COS was maintained after 5 cycles, and the COS concentration was 6.70 ± 0.35 g/L. The crude COS contained 20.15 ± 0.25 g/L glucose, 23.15 ± 0.22 g/L non-reacting substrate cellobiose, 5.25 ± 0.53 g/L, cellotriose and 1.49 ± 0.32 g/L cellopentaose. A bioprocess was developed for cellotriose enrichment, using whole Bacillus velezensis cells as a microbial purification tool. This bacteria consumed glucose, unreacted cellobiose, and cellopentaose while preserving cellotriose in the fermented medium. This study provides an excellent enzyme candidate for industrial COS production and is also the first study on the single-step COS enrichment process.

Combined effects of α-amylase, xylanase, and cellulase coproduced by Stachybotrys microspora on dough properties and bread quality as a bread improver.

This study aims to explore the feasibility of introducing, during the manufacture of bakery bread, an enzymatic cocktail coproduced by the fungus Stachybotrys microspora: α-amylases, xylanases and cellulases, using wheat bran as a nutrient source. Among the characteristics of the alveograph (dough tenacity "P" and dough extensibility "L"), the addition of a cocktail of enzymes at a concentration of 2 %, to weak wheat flour, has made it possible to significantly reduce its P/L ratio from 2.45 to 1.41. Furthermore, the use of enzyme cocktails at 2 %, 4 %, and 6 % concentrations increases the brown color of the bread crust. The great reduction in the rate of bread firmness, during storage over 5 days, was obtained in the presence of an enzyme cocktail in comparison with bread control (65.13 N for the control and 22.99 N, 23.24 N, and 18.24 N for bread enriched with enzyme cocktail at 2 %, 4 % and 6 % concentrations, respectively). In conclusion, the enzyme cocktail added can synergistically improve bread dough rheology and bread properties.

Stable and effective eco-enzyme cocktails in powder and liquid form of Stachybotrys microspora used as detergent additives.

Objective: The present work aims to optimize fermentation parameters for the simultaneous production of eco-enzymes: proteases, amylases, and endoglucanases from the same fungus Stachybotrys microspora, and to evaluate their stability in free form and formulated in lye as detergent additives. Methods: Initially, enzyme cocktail production was assayed in a medium comprising inexpensive waste biomass. Using the best substrate, we investigated the effect of its different concentrations and the NaCl concentration on the three enzymes co-production. Next, we studied the effect of several additives on the storage stability of the lyophilized enzyme cocktail (powder in liquid forms) free and incorporated in commercial laundry detergent. Finally, the washing efficiency analysis of the newly formulated enzyme cocktail was evaluated on dirty tissue pieces with different stains. Results: The highest enzymatic cocktail production was achieved at 30 °C for 96 h after adding 0.1% NaCl and 1.5% wheat bran as waste biomass in the basal culture medium. The effect of adding maltodextrin, sucrose, or polyethylene glycol 4000 during freeze-drying showed that maltodextrin is the best additive to protect the activities of proteases, amylases, and cellulases of liquid and powder enzyme form. Additionally, the liquid formulation of these enzymes showed excellent stability and compatibility with 1% maltodextrin and 10% glycerol. Interestingly, we have developed a new formulation of an enzyme cocktail (liquid and powder) stable and highly compatible with detergents. Comparing the washing performance of different formulations containing our enzyme cocktail to commercial ones showed significantly better removal of different types of stains. Conclusions: This research shows a cost-effective approach to simultaneously produce proteases, amylases, and endoglucanases from Stachybotrys microspora that could be considered a compatible detergent additive in the green detergent industry.

Halophilic filamentous fungi and their enzymes: Potential biotechnological applications.

Recently, interest in the study of microorganisms growing under extreme conditions, particularly halophiles, has increased due to their potential use in industrial processes. Halophiles are the class of microorganisms that grow optimally at high NaCl concentrations and are capable of producing halophilic enzymes capable of catalyzing reactions under harsh conditions. So far, fungi are the least studied halophilic microorganisms, even though they have been shown to counteract these extreme conditions by producing secondary metabolites with very interesting properties. This review highlights mechanisms that allow halophilic fungi to adapt high salinity and the specificity of their enzymes to a spectrum of action in industrial and environmental applications. The peculiarities of these enzymes justify the urgent need to apply green alternative compounds in industries.

Large-scale analysis of the genome of the rare alkaline-halophilic Stachybotrys microspora reveals 46 cellulase genes.

Fungi are of great importance in biotechnology, for example in the production of enzymes and metabolites. The main goal of this study was to obtain a high-coverage draft of the Stachybotrys microspora genome and to annotate and analyze the genome sequence data. The rare fungus S. microspora N1 strain is distinguished by its ability to grow in an alkaline halophilic environment and to efficiently secrete cellulolytic enzymes. Here we report the draft genome sequence composed of 3715 contigs, a genome size of 35 343 854 bp, with a GC content of 53.31% and a coverage around 20.5×. The identification of cellulolytic genes and of their corresponding functions was carried out through analysis and annotation of the whole genome sequence. Forty-six cellulases were identified using the fungicompanion bioinformatic tool. Interestingly, an S. microspora endoglucanase selected from those with a low isoelectric point was predicted to have a halophilic profile and share significant homology with a well-known bacterial halophilic cellulase. These results confirm previous biochemical studies revealing a halophilic character, which is a very rare feature among fungal cellulases. All these properties suggest that cellulases of S. microspora may have potential for use in the biofuel, textile, and detergent industries.

Neutral and alkaline cellulases: Production, engineering, and applications.

Neutral and alkaline cellulases from microorganisms constitute a major group of the industrial enzymes and find applications in various industries. Screening is the important ways to get novel cellulases. Most fungal cellulases have acidic pH optima, except some fungi like Humicola insolens species. However, new applications require the use of neutral and alkaline cellulases in food, brewery and wine, animal feed, textile and laundry, pulp and paper industries, agriculture as well in scientific research purposes. Indeed, the demand for these enzymes is growing more rapidly than ever before, and becomes the driving force for research on engineering the cellulolytic enzymes. Here, we present an overview of the biotechnological research for neutral and alkaline cellulases.

Trans-glycosylation capacity of a highly glycosylated multi-specific ß-glucosidase from Fusarium solani.

An extracellular ß-glucosidase from Fusaruim solani cultivated on wheat bran was purified by only two chromatographic steps. The purified enzyme exhibited optimal temperature and pH at 60 °C and pH 5, respectively. The purified ß-glucosidase behaves as a very large protein due to its high degree of glycosylation. More interestingly, the endoglycosidase H (Endo H) treatment led to 97.55% loss of its initial activity after 24 h of treatment. Besides, the addition of Tunicamycin (nucleoside antibiotic blocking the N-glycosylation first step) during the culture of the fungus affected seriously the glycosylation of the enzyme. Both treatments (endo H and Tunicamycin) strengthened the idea that the hyperglycosylation is involved in the ß-glucosidase activity and thermostability. This enzyme was also shown to belong to class III of ß-glucosidases (multi-specific) since it was able to act on either cellobiose, gentiobiose or sophorose which are disaccharide composed of two units of D-glucose connected by ß1-4, ß1-6 and ß1-2 linkage, respectively. The ß-glucosidase activity was strongly enhanced by ferrous ion (Fe2+) and high ionic strength (1 M KCl). The purified enzyme exhibited an efficient transglycosylation capacity allowing the synthesis of cellotriose and cellotetraose using cellobiose as donor.

Medium initial pH and carbon source stimulate differential alkaline cellulase time course production in Stachybotrys microspora.

The production profile of cellulases of the mutant strain A19 from the filamentous fungus Stachybotrys microspora was studied in the presence of various carbon sources (glucose, lactose, cellulose, carboxymethylcellulose (CMC), and wheat bran) and a range of medium initial pH (5, 7, and 8). Two extracellular cellulases from the Stachybotrys strain (endoglucanases and ß-glucosidases) were monitored by enzymatic assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and zymogram analysis. Glucose and lactose repressed CMCase time course production while they permitted a strong ß-glucosidase one. On Avicel cellulose, CMC, and wheat bran, both activities were highly produced. Wheat bran (WB) is the best carbon source with an optimum of production at days 5 and 6. The production kinetics of both activities were shown to depend on the medium initial pH, with a preference for neutral or alkaline pH in the majority of conditions. The exception concerned the ß-glucosidase which was much more produced at acidic pH, on glucose and cellulose. Most interestingly, a constitutive and conditional expression of an alkaline endoglucanase was revealed on the glucose-based medium at an initial pH of 8 units. The zymogram analysis confirmed such conclusions and highlighted that carbon sources and the pH of the culture medium directed a differential induction of various endoglucanases and ß-glucosidases.

Investigations on hydrolytic activities from Stachybotrys microspora and their use as an alternative in yeast DNA extraction.

Stachybotrys microspora is a filamentous fungus characterized by the secretion of multiple hydrolytic activities (cellulolytic and non-cellulolytic enzymes). The production of these biocatalysts was studied under submerged culture using glucose, cellulose, and wheat bran as carbon sources. Endoglucanases, pectinases, xylanases, ß-glucanases, chitinases, and proteases were induced on cellulose-based medium and repressed on glucose in both strains with higher amounts produced by the mutant. ß-glucosidases were roughly equally produced by both strains under glucose and cellulose conditions. The yield of chitinases, ß-glucanases, and proteases produced by Stachybotrys strains was as much higher than the commercialized lysing enzyme called "zymolyase," currently used in yeast DNA extraction. In this context, we showed that S. microspora hydrolases can be successfully applied in the extraction of yeast DNA.

Improvement of cellulase and xylanase production by solid-state fermentation of Stachybotrys microspora.

The current study investigated the production of cellulases and xylanases from the rare fungus Stachybotrys microspora under solid-state fermentation (SSF) on wheat bran (WB). A comparison of both activities was first performed in submerged cultures using various concentrations of WB, glucose, and cellulose as substrates. The maximal activity of ß-glucosidases and xylanases was obtained with 2% and 4% WB, respectively, whereas cellulose yielded the highest endoglucanase production. The SSF conditions were therefore consequently optimized. A moisture content of 70% gave the most significant levels of enzyme production. Inoculation by spores led to better results than by preculture, with 10(5) spores per gram of dried matter as the best inoculum dose for all activities tested. Interestingly, the WB-based medium need not to be supplemented by an exogeneous nitrogen source. Considering the richness of S. microspora secreted proteins as lytic hydrolases, the crude extracellular enzyme extracts were successfully tested in two different biotechnological fields: protoplasting of fungi and subsequent extraction of their DNA, paper pulp hydrolysis to produce fermentable sugars.