Heteroxylan hydrolysis by a recombinant cellulase-free GH10 xylanase from the alkaliphilic bacterium Halalkalibacterium halodurans C-125.

The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremely alkaliphilic bacterium Halalkalibacterium halodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60 °C. It also presented good stability with a half-life of 3 h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46 mg/mL and 0.54 mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24 h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54 mg/mL after 4 h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.

Improved Expression of a Thermostable GH18 Bacterial Chitinase in Two Different Escherichia coli Strains and Its Potential Use in Plant Protection and Biocontrol of Phytopathogenic Fungi.

Chitinases are enzymes that can break down chitin, a major component of the exoskeleton of insects and fungi. This feature makes them potential biopesticides in agriculture since they are considered a safe and environmentally friendly alternative to synthetic pesticides. In this work, we performed a comparative study between two different bacterial expression strains to produce a recombinant chitinase with improved stability. Escherichia coli strains Origami B and BL21 (DE3) were selected for their distinct cytosolic environment to express BhChitA chitinase of Bacillus halodurans C-125 and to investigate the role of disulfide bond formation and proper folding on its stability and activity. Expression of the recombinant BhChitA in bacterial strain containing oxidative cytosol (Origami B) improved its activity and stability. Although both expression systems have comparable biochemical properties (temperature range 20-80 °C and pH spectrum 3-10), BhChitA expressed in Origami strain seems more stable than expressed in BL21. Furthermore, the optimal expression conditions of the recombinant BhChitA has been carried out at 30 °C during 6 h for the Origami strain, against 20 °C during 2 h for BL21. On the other hand, no significant differences were detected between the two enzymes when the effect of metal ions was tested. These findings correlate with the analysis of the overall structure of BhChitA. The model structure permitted to localize disulfide bond, which form a stable connection between the substrate-binding residues and the hydrophobic core. This link is required for efficient binding of the chitin insertion domain to the substrate. BhChitA exhibited in vitro antifungal effect against phytopathogenic fungi and suppressed necrosis of Botrytis cinerea on detached tomato leaves. In vitro assays showed the influence of BhChitA on growth suppression of Botrytis cinerea (53%) Aspergillus niger (65%), Fusarium graminearum (25%), and Fusarium oxysporum (34%). Our results highlight the importance of the bacterial expression system with oxidative cytosol in producing promising biopesticides that can be applied for post-harvest processing and crop protection.

Effects of Growth Medium Variation on the Nutri-Functional Properties of Microalgae Used for the Enrichment of Ricotta.

Research background: Microalgae represent an emergent sustainable source of bioactive compounds such as antioxidants, vitamins, minerals and polyunsaturated fatty acids that can ameliorate the nutritional characteristics of foods. The biochemical composition of microalgae could be modulated by varying the culture conditions to enhance the accumulation of biomolecules of interest. The aim of this work is to optimise the nutri-functional properties of two microalgae that can be used in food production. Experimental approach: Nannochloropsis gaditana L2 and Chlorella sp. SM1 were screened for growth, biochemical composition and radical scavenging activity employing four different growth media (algal, BG-11, f/2 and Conway) with different nutrient composition. The feasibility of using Chlorella sp. SM1 cultivated in BG-11 medium, in an under-investigated Mediterranean dairy product ricotta cheese and its effect on the sensory attributes was investigated. Additionally, Arthrospira platensis was used as reference in sensory analysis. Results and conclusions: Nitrate- and phosphate-rich media (BG-11 and algal) enhanced the biomass productivity. However, the highest lipid production (23.10 and 11.86 mg/(L·day) by strains SM1 and L2 respectively) and carbohydrate content (34.79 and 44.84% by SM1 and L2 respectively) were obtained with the nitrate-deficient f/2 medium. Regardless of the used medium, the lipid profile of Chlorella sp. SM1 and N. gaditana L2 remained adequate for different applications with >50% C16-18 as the main fatty acids. Significant increase in oleic acid (C18:1) content was recorded in response to nitrogen deficiency, being the highest in SM1 in f/2 medium (34%). Nitrogen deficiency was also found to enhance phenolic compound (expressed as gallic acid equivalents, 48.8 and 35.1 mg/g in SM1 and L2 respectively) and carotenoid contents (2.2 and 2.0 mg/g in SM1 and L2 respectively). Due to its interesting antioxidant potential, Chlorella sp. SM1 was used at different mass fractions (0.2, 1 and 1.5%) to enrich the ricotta cheese. The sample with 0.2% Chlorella sp. SM1 was found to give the most appreciated product. Novelty and scientific contribution: This study presents the production of an innovative ricotta cheese using Chlorella sp. as a functional ingredient, without altering the manufacturing procedure, while maintaining acceptable sensorial characteristics. The biochemical composition of the used strains varied depending on the culture medium composition, which enabled the accumulation of phytonutrients of interest.

Technological Feasibility of Couscous-Algae-Supplemented Formulae: Process Description, Nutritional Properties and In Vitro Digestibility.

The aim of this work was to develop functional couscous in a traditional Tunisian manner (hand rolling), enriched in algae biomass (6% w/w). Four Chlorella vulgaris (C. vulgaris) biomasses and one mixture of C. vulgaris and two macroalgae biomasses (Ulva rigida and Fucus vesiculosus) were used. The C. vulgaris strain was subjected to random mutagenesis and different culture conditions (Allmicroalgae), resulting in different pigmentations and biochemical compositions. Couscous samples were characterized in terms of nutritional properties, oscillatory rheology properties and digestibility. All biomasses provided a significant supplementation of nutrients and excellent acceptance. The enrichment resulted in lower firmness, higher viscoelastic functions (G' and G″) and a significant improvement in the cooking quality. Major differences between couscous samples with different microalgae were observed in protein and mineral contents, fully meeting Regulation (EC) No. 1924/2006 requirements for health claims made on foodstuffs. The amount of digested proteins was also higher in algae-containing samples. The fatty acid profile of the enriched couscous varied in a biomass-specific way, with a marked increase in linolenic acid (18:3 ω3) and a decrease in the ω6/ω3 ratio. Sensory analysis revealed that microalgae-containing products could compete with conventional goods with an added advantage, that is, having an ameliorated nutritional value using algae as a "trendy" and sustainable ingredient.

Uncover aldehydes in biomass hydrolyzates: disproportionation of aldehydes in alkaline solution and subsequent measurement using an automated HPAEC-PAD method.

High-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) was used for developing a method for identifying and quantifying aldehydes in biomass hydrolyzates. This method was optimized to the requirements of HPAEC-PAD in order to allow for a simultaneous determination of aldehydes by respective Cannizzaro alcohols. To this end, sodium hydroxide concentration (0.1 to 5.0 mol/L), temperature (30 to 40 °C), and reaction time (0 to 24 h) were investigated for sufficient and reproducible disproportionation of the biomass-derived aldehydes. The optimized method for aldehyde disproportionation and subsequent measurement are 1 mol/L sodium hydroxide, 40 °C, and 1 h reaction time. The detection limits resulting from this method are lower than 68.55 mg/L and the sensitivity above 0.024 (nC min)/(mg/L) for 3,4-dimethoxybenzaldehyde. Linearity for aldehyde calibration always exceeded 0.98. Thus, HPAEC-PAD analysis allows for the quantification of biomass-derived compounds from all natural polymers and, therefore, it has exemplarily been used to quantify aldehyde concentration of beech wood, orange peel, and algae biomass hydrolyzates. Graphical abstract.

Draft Genome Sequence of Enterococcus faecalis Strain OB15, a Probiotic Strain Recently Isolated from Tunisian Rigouta Cheese.

Enterococcus faecalis OB15 is a probiotic strain that was isolated from rigouta, a popular traditional Tunisian fermented cheese. We report here the draft genome sequence of this strain, consisting of 2,912,159 bp, with an average G+C content of 37.49%.

Bioreactor Membranes for Laccase Immobilization Optimized by Ionic Liquids and Cross-Linking Agents.

A novel concept of membrane bioreactor based on polymeric ionic liquids laccase membrane has been implemented in batch process for decolorization of the anthraquinonic dye Remazol Brillant Blue R (RBBR). New laccase immobilization strategy has been optimized by casting the enzyme into a polymeric inclusion membrane (PIM) using ionic liquids (ILs) and polyvinyl chloride (PVC) leading to laccase polymeric IL membrane (PILM). Four different ILs (1-octyl-3-metylimidazolium bis(trifluoromethylsulfonyl)imide, [OMIM][NTF2]; cholinium bis(trifluoromethylsulfonyl)imide, [Ch ol][NTF2]; cholinium dihydrogenphosphate, [Chol][H2PO4] and hydroxyethylammonium formate, [HEA][Fo]) have been screened and mixed to constitute the active phase of the support of PIM. This strategy has been fully succeeded since high laccase immobilization rates were recorded (about 98%) when using the optimal mixture containing three ILs (45% [OMIM][NTf2]/5% [Chol][NTf2]/2.5% [HEA][Fo]) and supplemented by 0.5% glutaraldehyde. It was found that such mixture contributes to increase the stability and reusability of laccase-PILM during eight successive assays in a batch discontinued stirred reactor. Decolorization rate of 75% has been reached in the batch decolorization process of RBBR with high reusability yield. Graphical Abstract Decolorization of RBBR by PILM_laccase.

Efficient enzymatic saccharification of macroalgal biomass using a specific thermostable GH 12 endoglucanase from Aspergillus terreus JL1.

Stranded green macroalgae represents an important and renewable biomass that remains under valorized despite the numerous environmental problems generated by their accumulation in coastal regions. This work describes the isolation of a filamentous thermophile fungus identified as Aspergillus terreus JL1 that produces an efficient cellulolytic activity for green macroalgae saccharification. The characterization of the endoglucanase activity obtained after submerged fermentation showed a differential induction depending on the carbon source used with a unique isoform released when Ulva lactuca was used as inducer. The crude extract obtained hydrolyzed efficiently the untreated algal biomass (70.5%) compared to other cellulolytic extracts. The unique endoglucanase released was then purified to homogeneity (Yield: 49.6%; Specific activity: 30.1 U/mg; Purification fold: 4.36) and characterized biochemically. Its peptidic sequence was then determined and showed its belonging to the GH12. The described enzyme represents a promising biotechnological tool for algal biomass conversion.

Variations in Physicochemical Properties and Bioconversion Efficiency of Ulva lactuca Polysaccharides After Different Biomass Pretreatment Techniques.

Green macroalgae are an abundant and undervalued biomass with a specific cell wall structure. In this context, different pretreatments, namely ethanol organosolv (Org), alkaline, liquid hot water (LHW), and ionic liquid (IL) pretreatments, were applied to the green macroalgae Ulva lactuca biomass and then evaluated. Their effects on chemical composition, biomass crystallinity, enzymatic digestibility, and theoretical ethanol potential were studied. The chemical composition analysis showed that the Org and LHW pretreatments allowed the highest glucan recovery (80.8 ± 3.6 and 62.9 ± 4.4 g/100 g DM, respectively) with ulvan (80.0 and 99.1%) and hemicellulose (55.0 and 42.3%) removal. These findings were in agreement with both thermogravimetric analysis and scanning electron microscopy results that confirm significant structural changes of the pretreated biomasses. It was found that the employed pretreatments did not significantly affect the cellulose crystallinity; however, they both increased the whole crystallinity and the enzymatic digestibility. This later reached 97.5% in the case of LHW pretreatment. Our results showed high efficiency saccharification of Ulva lactuca biomass that will constitute the key step of the implementation of a biorefinery process.

Growth Parameters, Photosynthetic Performance, and Biochemical Characterization of Newly Isolated Green Microalgae in Response to Culture Condition Variations.

This work aimed to characterize two native microalgal strains newly isolated from South Mediterranean areas and identified as Chlorella sorokiniana ES3 and Neochloris sp. AM2. The growth properties and biochemical composition of these microalgae were evaluated in different culture media (Algal, BG-11, f/2, and Conway). Among the tested media, nitrate- and phosphate-rich Algal medium provided the maximum biomass productivities (85.5 and 111.5 mg l(-1) day(-1) for C. sorokiniana and Neochloris sp., respectively), while the nitrate- and phosphate-deficient f/2 medium resulted in the highest lipid productivities (24.1 and 35.8 mg l(-1) day(-1) for C. sorokiniana and Neochloris sp., respectively). The physiological state of both microalgae was investigated under different light and temperature levels using the pulse amplitude-modulated fluorometry. The better photosynthetic efficiency of C. sorokiniana was obtained at 23 °C with a light saturation of 156 µE m(-2) s(-1), while that of Neochloris sp. was achieved at 15 °C with a light saturation of 151 µE m(-2) s(-1). The analysis of fatty acid profile and biodiesel parameters revealed that C. sorokiniana, cultivated in Algal and f/2 media, can be considered as a suitable candidate for high-quality biodiesel production.

Industrial textile effluent decolourization in stirred and static batch cultures of a new fungal strain Chaetomium globosum IMA1 KJ472923.

The treatment of an industrial textile effluent (ITE) was investigated by using a mono-culture of a novel fungal strain Chaetomium globosum IMA1. This filamentous fungus was selected based on its capacity for dye removal via the biodegradation mechanism. The respirometric analysis showed that C. globosum IMA1 was resistant to an indigo concentration up to 700 mg equivalent COD/L. The decolourization of the ITE by C. globosum was performed in static and stirred batch systems. The better lignin peroxidase (LiP), laccase and the manganese peroxidase (MnP) productions were 829.9 U/L, 83 U/L and 247.8 U/L, respectively since 3-5 days under a stirred condition. Therefore, the chemical oxygen demand (COD) and colors (OD620) removal yields reached 88.4% and 99.8%, respectively. Fourier transforms infrared spectroscopy (FTIR) analysis of the treated effluent showed that the decolourization was due to the degradation and the transformation of dye molecules. However, spectrophotometric examination showed that the complete dye removal was through fungal adsorption (8%), followed by degradation (92%).

Physico-chemical characterization and enzymatic functionalization of Enteromorpha sp. cellulose.

Although green macro-algae represent a renewable and highly abundant biomass, they remain poorly exploited in terms of carbohydrate polymers compared to red and brown ones and other lignocellulosic materials. In this study, cellulose from the green macro-algae Enteromorpha sp. was isolated, physico-chemically characterized and enzymatically functionalized. The cellulose content was about 21.4% (w/w). FTIR analyses indicated an absence of acetyl or uronic esters confirming the absence of hemicellulose contamination. The 36% crystallinity index of the extracted cellulose revealed a high amorphous character as determined by X-ray diffraction. The moisture adsorption isotherms and specific surface measurements were respectively 42.87g/100g and 8.34m(2)/g. The Enteromorpha sp. cellulose was first enzymatically saccharified by a commercial cellulase preparation from Aspergillus niger with a hydrolysis yield of 70.4%. Besides, it was successfully functionalized based on the transglycosylation mechanism of the same enzymatic preparation, to produce highly added-value biosurfactants (butyl-glucoside) with a concentration of 8mM.

A whole biodiesel conversion process combining isolation, cultivation and in situ supercritical methanol transesterification of native microalgae.

A coupled process combining microalgae production with direct supercritical biodiesel conversion using a reduced number of operating steps is proposed in this work. Two newly isolated native microalgae strains, identified as Chlorella sp. and Nannochloris sp., were cultivated in both batch and continuous modes. Maximum productivities were achieved during continuous cultures with 318mg/lday and 256mg/lday for Chlorella sp. and Nannochloris sp., respectively. Microalgae were further characterized by determining their photosynthetic performance and nutrient removal efficiency. Biodiesel was produced by catalyst-free in situ supercritical methanol transesterification of wet unwashed algal biomass (75wt.% of moisture). Maximum biodiesel yields of 45.62wt.% and 21.79wt.% were reached for Chlorella sp. and Nannochloris sp., respectively. The analysis of polyunsaturated fatty acids of Chlorella sp. showed a decrease in their proportion when comparing conventional and supercritical transesterification processes (from 37.4% to 13.9%, respectively), thus improving the quality of the biodiesel.

Biocatalytic conversion of wheat bran hydrolysate using an immobilized GH43 beta-xylosidase.

To investigate the concept of a xylosidase-based process for the continuous production of xylose from arabinoxylan-containing feedstocks, a beta-xylosidase from Bacillus halodurans C-125 was immobilized and deployed in packed bed reactor (PBR). Among the several immobilization methods tested, glutaraldehyde-mediated immobilization on chitosan was the best both in terms of immobilization and activity yields (91% and 72.9%, respectively). In batch experiments the immobilized enzyme hydrolyzed wheat bran hydrolysates quite efficiently, consuming nearly all xylobiose and xylotriose after 6h. Its reusability showed only a 50% decrease of its activity after 92h. Using the chitosan-immobilized beta-xylosidase in a PBR, xylose productivity was 7.2g xylose l(-1)h(-1) and the conversion factor was 0.55 (derived from initial xylose in the substrate). The operational stability of the PBR was good, because only 25% of productivity was lost after the treatment of three batches of substrate over a 72-h period.

Expression in Escherichia coli and characterization of beta-xylosidases GH39 and GH-43 from Bacillus halodurans C-125.

To develop xylosidases as tools for the hydrolysis of wheat bran arabinoxylans, two beta-xylosidases from Bacillus halodurans C-125 have been cloned and expressed in Escherichia coli. The recombinant (His)(6)-tagged enzymes, designated as XylBH39 and XylBH43, were efficiently purified using Ni(2+)-affinity chromatography. Determination of native molecular masses indicated that XylBH43 is dimeric in solution, whereas a similar analysis of XylBH39 did not allow differentiation between the dimeric and trimeric states. Both enzymes had similar pH and temperature optima (pH 7.5 and 55 degrees C for XylBH39 and pH 8 and 60 degrees C for XylBH43) and were relatively stable over the pH range of 3.5-8.5. In contrast, XylBH39 was more thermostable. At 60 degrees C, XylBH39 and XylBH43 displayed approximate half-life values of 2.40 and 0.05 h, respectively. The comparison of the ratio k (cat)/K (M) revealed that XylBH43 hydrolyzed p-nitrophenyl-beta-D: -xyloside more efficiently (4.6-fold) than XylBH39. Similarly, while XylBH43 was 18-fold less active on p-nitrophenyl-alpha-L: -arabinofuranoside, XylBH39 was essentially inactive on this substrate. Using either p-nitrophenyl-beta-D: -xyloside or xylotriose, XylBH39 performed transglycosylation, while xylobiose proved to be a poor substrate for both hydrolysis and transglycosylation. The use of XylBH39 and XylBH43 for the posttreatment of endoxylanase-generated wheat bran hydrolysates revealed that XylBH43 efficiently produced xylose monomers (385 microg/ml after 330 min incubation). Its activity was improved by the simultaneous deployment of an alpha-L: -arabinofuranosidase. Together, these enzymes were able to release 521 microg/ml of xylose after 330 min. This constitutes an approximate yield improvement of 35%.