Enhanced extracellular ammonium release in the plant endophyte Gluconacetobacter diazotrophicus through genome editing.

IMPORTANCE: Our results demonstrate increased extracellular ammonium release in the endophyte plant growth-promoting bacterium Gluconacetobacter diazotrophicus. Strains were constructed in a manner that leaves no antibiotic markers behind, such that these strains contain no transgenes. Levels of ammonium achieved by cultures of modified G. diazotrophicus strains reached concentrations of approximately 18 mM ammonium, while wild-type G. diazotrophicus remained much lower (below 50 µM). These findings demonstrate a strong potential for further improving the biofertilizer potential of this important microbe.

Tannin Removal of Cashew Apple Juice by Powdered Gelatin Treatment and Its Utilization in Bacterial Cellulose Production.

In this study, cashew apple juice was treated with different levels of powdered gelatin (2%, 5%, and 10%) to remove tannins. The results showed that the addition of 5% gelatin removed 99.2% of condensed tannins while did not affect reducing sugars of juice. Subsequently, tannin-free cashew apple juice (CA) was aerobically fermented for 14 days with Komagataeibacter saccharivorans strain 1.1 (KS) and Gluconacetobacter entanii HWW100 (GE) in comparison with Hestrin-Schramm (HS) medium as control. The dry weight of bacterial cellulose (BC) obtained from the KS strain (2.12 and 1.48 g/L for CA and HS media, respectively) was higher than that from the GE strain (0.69 and 1.21 g/L for CA and HS media, respectively). Although GE showed low BC production yield, its viability in both media after 14-day fermentation was notable (6.06-7.21 log CFU/mL) compared to KS strain (1.90-3.30 log CFU/mL). In addition, the XRD and FT-IR analysis showed that there was no significant difference in the crystallinity and functional groups of BC films when cultured on CA and HS medium, while the morphology by SEM exhibited the phenolic molecules on the film surface. Cashew apple juice has been shown to be a viable and cost-effective medium for the BC production.

Structures and roles of BcsD and partner scaffold proteins in proteobacterial cellulose secretion.

Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. Although bacterial cellulose (BC) was first described in the 19th century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing ß- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.

Improved production of bacterial cellulose using Gluconacetobacter sp. LYP25, a strain developed in UVC mutagenesis with limited viability conditions.

Bacterial cellulose (BC), a natural polymer synthesized by bacteria, has received considerable attention owing to its impressive physicomechanical properties. However, the low productivity of BC-producing strains poses a challenge to industrializing this material and making it economically viable. In the present study, UV-induced random mutagenesis of Gluconacetobacter xylinus ATCC 53524 was performed to improve BC production. Sixty mutants were obtained from the following mutagenesis procedure: the correlation between UVC fluence and cell death was investigated, and a limited viability condition was determined as a UVC dose to kill 99.99 %. Compared to the control strain, BC production by the mutant strains LYP25 and LYP23 improved 46.4 % and 44.9 %, respectively. Fermentation profiling using the selected strains showed that LYP25 was superior in glucose consumption and BC production, 13.8 % and 41.0 %, respectively, compared to the control strain. Finally, the physicochemical properties of LYP25-derived BC were similar to those of the control strain; thus, the mutant strain is expected to be a promising producer of BC in the bio-industry based on improved productivity.

Engineering Gluconobacter cerinus CGMCC 1.110 for direct 2-keto-L-gulonic acid production.

Gluconobacter is a potential strain for single-step production of 2-keto-L-gulonic acid (2-KLG), which is the direct precursor of vitamin C. Three dehydrogenases, namely, sorbitol dehydrogenase (SLDH), sorbose dehydrogenase (SDH), and sorbosone dehydrogenase (SNDH), are involved in the production of 2-KLG from D-sorbitol. In the present study, the potential SNDH/SDH gene cluster in the strain Gluconobacter cerinus CGMCC 1.110 was mined by genome analysis, and its function in transforming L-sorbose to 2-KLG was verified. Proteomic analysis showed that the expression level of SNDH/SDH had a great influence on the titer of 2-KLG, and fermentation results showed that SDH was the rate-limiting enzyme. A systematic metabolic engineering process, which was theoretically suitable for increasing the titer of many products involving membrane-bound dehydrogenase from Gluconobacter, was then performed to improve the 2-KLG titer in G. cerinus CGMCC 1.110 from undetectable to 51.9 g/L in a 5-L bioreactor after fermentation optimization. The strategies used in this study may provide a reference for mining other potential applications of Gluconobacter. KEY POINTS: • The potential SNDH/SDH gene cluster in G. cerinus CGMCC 1.110 was mined. • A systematic engineering process was performed to improve the titer of 2-KLG. • The 2-KLG titer was successfully increased from undetectable to 51.9 g/L.

Gluconacetobacter diazotrophicus Gene Fitness during Diazotrophic Growth.

Plant growth-promoting (PGP) bacteria are important to the development of sustainable agricultural systems. PGP microbes that fix atmospheric nitrogen (diazotrophs) could minimize the application of industrially derived fertilizers and function as a biofertilizer. The bacterium Gluconacetobacter diazotrophicus is a nitrogen-fixing PGP microbe originally discovered in association with sugarcane plants, where it functions as an endophyte. It also forms endophyte associations with a range of other agriculturally relevant crop plants. G. diazotrophicus requires microaerobic conditions for diazotrophic growth. We generated a transposon library for G. diazotrophicus and cultured the library under various growth conditions and culture medium compositions to measure fitness defects associated with individual transposon inserts (transposon insertion sequencing [Tn-seq]). Using this library, we probed more than 3,200 genes and ascertained the importance of various genes for diazotrophic growth of this microaerobic endophyte. We also identified a set of essential genes. IMPORTANCE Our results demonstrate a succinct set of genes involved in diazotrophic growth for G. diazotrophicus, with a lower degree of redundancy than what is found in other model diazotrophs. The results will serve as a valuable resource for those interested in biological nitrogen fixation and will establish a baseline data set for plant free growth, which could complement future studies related to the endophyte relationship.

Transcriptomic Response of the Diazotrophic Bacteria Gluconacetobacter diazotrophicus Strain PAL5 to Iron Limitation and Characterization of the fur Regulatory Network.

Gluconacetobacter diazotrophicus has been the focus of several studies aiming to understand the mechanisms behind this endophytic diazotrophic bacterium. The present study is the first global analysis of the early transcriptional response of exponentially growing G. diazotrophicus to iron, an essential cofactor for many enzymes involved in various metabolic pathways. RNA-seq, targeted gene mutagenesis and computational motif discovery tools were used to define the G. diazotrophicusfur regulon. The data analysis showed that genes encoding functions related to iron homeostasis were significantly upregulated in response to iron limitations. Certain genes involved in secondary metabolism were overexpressed under iron-limited conditions. In contrast, it was observed that the expression of genes involved in Fe-S cluster biosynthesis, flagellar biosynthesis and type IV secretion systems were downregulated in an iron-depleted culture medium. Our results support a model that controls transcription in G. diazotrophicus by fur function. The G. diazotrophicusfur protein was able to complement an E. colifur mutant. These results provide new insights into the effects of iron on the metabolism of G. diazotrophicus, as well as demonstrate the essentiality of this micronutrient for the main characteristics of plant growth promotion by G. diazotrophicus.

Liquid-crystalline ordering in bacterial cellulose produced by Gluconacetobaсter hansenii on glucose-containing media.

This research is dedicated to the studies of the microscale morphology of bacterial cellulose (BC) obtained by means of static cultivation of Gluconacetobacter hansenii GH-1/2008. We found that the microscale morphology depended on the BC production rate that was varied by using different glucose concentrations in the cultivation medium. It was revealed that at higher production rates, BC fibrils were aligned in a liquid-crystalline-like (LC-like) order. The observed helical alignment was always left-handed. The half-periods of the helix varied from 50 µm to 150 µm depending on the cultivation conditions. The mechanical and water absorption properties of the obtained BC pellicles were measured. The former correlated mainly with the density of the samples; the latter were the best for films with layered structure, where the BC had segregated into fleece sheets separated by gaps with low density of fibrils.

Stoichiometric Analysis and Production of Bacterial Cellulose by Gluconacetobacter liquefaciens using Borassus flabellifer L. Jaggery.

The objective of the work is to examine the potential utilization of Palmyra palm jaggery (PPJ) for the enhancement of bacterial cellulose (BC) production by Gluconacetobacter liquefaciens. To evaluate the culturing condition, the production of BC fermentation was carried out in batch mode using different carbon sources namely glucose, sucrose and PPJ. PPJ in the HS medium (PHS medium) resulted maximum concentration of BC (14.35 ± 0.18 g/L) under shaking condition than other carbon sources in HS medium. The influence of different medium variables including initial pH and nitrogen sources on BC production was investigated using PHS medium under shaking condition. The maximum BC concentration of 17.79 ± 2.4 g/L was obtained in shaking condition at an initial pH of 5.6 using yeast extract as nitrogen source. Stoichiometric equation for the cell growth and BC synthesis was developed using elemental balance approach. The metabolic heat of reaction (40 kcal generated per liter of medium) was evaluated using electron balance approach. Based on the process economic analysis and the yield of BC during the fermentation, PHS medium without nitrogen source could be a promising cost-effective nutrient than HS medium. Thermal stability, crystallinity index and structural characterizations of produced BC using PPJ medium were evaluated using TGA, XRD and FTIR and the obtained results were compared with HS medium containing glucose and sucrose.

Essential role of extracytoplasmic proteins in the resistance of Gluconacetobacter diazotrophicus to cadmium.

Cadmium (Cd) is a heavy metal used as raw material for several fertilizers and pesticides. The increase of Cd concentration in soils has been observed in cultivated areas, affecting animals, plants, and microorganisms. Gluconacetobacter diazotrophicus is a plant growth-promoting bacterium able to survive under adverse environmental conditions. Here, we investigated key mechanisms involved with the resistance of G. diazotrophicus to Cd. Proteomic analyses revealed that the main pathways regulated in response to Cd are nutrient uptake, multidrug efflux pumps, response to oxidative stress, and protein quality control system. Extracytoplasmic proteins related to multidrug efflux pumps were up-accumulated, while several proteins related to nutrients uptake were down-accumulated. The relevance of these pathways for bacterial resistance to Cd was investigated by reverse genetic analysis using mutants defective for nutrient uptake (tdbr, ompW, and oprB), multidrug efflux (czcC), response to oxidative stress (ggt), and protein quality control system (clpX). Our data demonstrated the essential role of the tdbr and czcC genes for resistance to Cd in G. diazotrophicus. These results contribute to a better understanding of the resistance mechanisms to Cd in G. diazotrophicus, shedding light on responses associated with extracytoplasmic compartments.

Dissection and Reconstitution Provide Insights into Electron Transport in the Membrane-Bound Aldehyde Dehydrogenase Complex of Gluconacetobacter diazotrophicus.

Acetic acid bacteria catalyze the two-step oxidation of ethanol to acetic acid using the membrane-bound enzymes pyrroloquinoline quinone-dependent alcohol dehydrogenase and molybdopterin-dependent aldehyde dehydrogenase (ALDH). Although the reducing equivalents from the substrate are transferred to ubiquinone in the membrane, intramolecular electron transport in ALDH is not understood. Here, we purified the AldFGH complex, the membrane-bound ALDH that is physiologically relevant to acetic acid fermentation in Gluconacetobacter diazotrophicus strain PAL5. The purified AldFGH complex showed acetaldehyde:ubiquinone (Q2) oxidoreductase activity. c-type cytochromes of the AldFGH complex (in the AldF subunit) were reduced by acetaldehyde. Next, we genetically dissected the AldFGH complex into AldGH and AldF units and reconstituted them. The AldGH subcomplex showed acetaldehyde:ferricyanide oxidoreductase activity but not Q2 reductase activity. The ALDH activity of AldGH was not found in membranes but was found in the soluble fraction of the recombinant strain, suggesting that the AldF subunit is responsible for membrane binding of the AldFGH complex. The absorption spectra of the purified AldGH subcomplex suggested the presence of an [Fe-S] cluster, which can be reduced by acetaldehyde. The AldFGH complex reconstituted from the AldGH subcomplex and AldF showed Q2 reductase activity. We propose a model in which electrons from the substrate are abstracted by a molybdopterin in the AldH subunit and transferred to the [Fe-S] cluster(s) in the AldG subunit, followed by electron transport to c-type cytochrome centers in the AldF subunit, which is the site of ubiquinone reduction in the membrane. IMPORTANCE Two membrane-bound enzymes of acetic acid bacteria, pyrroloquinoline quinone-dependent alcohol dehydrogenase and molybdopterin-dependent aldehyde dehydrogenase (ALDH), are responsible for vinegar production. Upon the oxidation of acetaldehyde, ALDH reduces ubiquinone in the cytoplasmic membrane. ALDH is an enzyme complex of three subunits. Here, we tried to understand how ALDH works by using a classical biochemical approach and genetic engineering to dissect the enzyme complex into soluble and membrane-bound parts. The soluble part had limited activity in vitro and did not reduce ubiquinone. However, the enzyme complex reconstituted from the soluble and membrane-bound parts showed ubiquinone reduction activity. The proposed working model of ALDH provides a better understanding of how the enzyme works in the vinegar fermentation process.

Systematic optimization of exopolysaccharide production by Gluconacetobacter sp. and use of (crude) glycerol as carbon source.

The usage of polysaccharides as biodegradable polymers is of growing interest in the context of a sustainable and ecofriendly economy. For this, the production of exopolysaccharides (EPS) by Gluconacetobacter sp. was investigated. Glycerol as carbon source revealed to be beneficial compared to glucose. In addition, pure glycerol could be substituted by a crude glycerol waste stream from biodiesel production. Systematic analysis of the peptone and phosphate concentrations in glycerol-based media indicated a strong effect of peptone. Optimized parameters resulted in a titer of 25.4 ± 2.4 g/L EPS with a productivity of 0.46 ± 0.04 g*(L*h)-1. With decreasing peptone, a variation in the monomer ratios was observed. An accompanying change in molecular size distribution indicated the production of two different polysaccharides. Intensified analysis revealed the main polysaccharide to be composed of glucose (Glc), galactose (Gal), mannose (Man) and glucuronic acid (GlcA), and the minor polysaccharide of Gal, Man, ribose (Rib).

Differential Gene Expression of Brachypodium distachyon Roots Colonized by Gluconacetobacter diazotrophicus and the Role of BdCESA8 in the Colonization.

Alternatives to synthetic nitrogen fertilizer are needed to reduce the costs of crop production and offset environmental damage. Nitrogen-fixing bacterium Gluconacetobacter diazotrophicus has been proposed as a possible biofertilizer for monocot crop production. However, the colonization of G. diazotrophicus in most monocot crops is limited and deep understanding of the response of host plants to G. diazotrophicus colonization is still lacking. In this study, the molecular response of the monocot plant model Brachypodium distachyon was studied during G. diazotrophicus root colonization. The gene expression profiles of B. distachyon root tissues colonized by G. diazotrophicus were generated via next-generation RNA sequencing, and investigated through gene ontology and metabolic pathway analysis. The RNA sequencing results indicated that Brachypodium is actively involved in G. diazotrophicus colonization via cell wall synthesis. Jasmonic acid, ethylene, gibberellin biosynthesis. nitrogen assimilation, and primary and secondary metabolite pathways are also modulated to accommodate and control the extent of G. diazotrophicus colonization. Cellulose synthesis is significantly downregulated during colonization. The loss of function mutant for Brachypodium cellulose synthase 8 (BdCESA8) showed decreased cellulose content in xylem and increased resistance to G. diazotrophicus colonization. This result suggested that the cellulose synthesis of the secondary cell wall is involved in G. diazotrophicus colonization. The results of this study provide insights for future research in regard to gene manipulation for efficient colonization of nitrogen-fixing bacteria in Brachypodium and monocot crops.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Biofabrication of a Hyaluronan/Bacterial Cellulose Composite Nanofibril by Secretion from Engineered Gluconacetobacter.

Naturally occurring polysaccharides, such as cellulose, hemicellulose, and chitin, have roles in plant skeletons and/or related properties in living organisms. Their hierarchically regulated production systems show potential for designing nanocomposite fabrication using engineered microorganisms. This study has demonstrated that genetically engineered Gluconacetobacter hansenii (G. hansenii) individual cells can fabricate naturally composited nanofibrils by simultaneous production of hyaluronan (HA) and bacterial cellulose (BC). The cells were manipulated to contain hyaluronan synthase and UDP-glucose dehydrogenase genes, which are essential for HA biosynthesis. Fluorescence microscopic observations indicated the production of composited nanofibrils and suggested that HA secretion was associated with the cellulose secretory pathway in G. hansenii. The gel-like nanocomposite materials produced by the engineered G. hansenii exhibited superior properties compared with conventional in situ nanocomposites. This genetic engineering approach facilitates the use of G. hansenii for designing integrated cellulose-based nanomaterials.

Combination of osmotic stress and sugar stress response mechanisms is essential for Gluconacetobacter diazotrophicus tolerance to high-sucrose environments.

Sugar-rich environments represent an important challenge for microorganisms. The osmotic and molecular imbalances resulting from this condition severely limit microbial metabolism and growth. Gluconacetobacter diazotrophicus is one of the most sugar-tolerant prokaryotes, able to grow in the presence of sucrose concentrations up to 30%. However, the mechanisms that control its tolerance to such conditions remain poorly exploited. The present work investigated the key mechanisms of tolerance to high sugar in G. diazotrophicus. Comparative proteomics was applied to investigate the main functional pathways regulated in G. diazotrophicus when cultivated in the presence of high sucrose. Among 191 proteins regulated by high sucrose, regulatory pathways related to sugar metabolism, nutrient uptake, compatible solute synthesis, amino acid metabolism, and proteolytic system were highlighted. The role of these pathways on high-sucrose tolerance was investigated by mutagenesis analysis, which revealed that the knockout mutants zwf::Tn5 (sugar metabolism), tbdr::Tn5 (nutrient uptake), mtlK::Tn5 (compatible solute synthesis), pepN::Tn5 (proteolytic system), metH::Tn5 (amino acid metabolism), and ilvD::Tn5 (amino acid metabolism) became more sensitive to high sucrose. Together, our results identified mechanisms involved in response to high sugar in G. diazotrophicus, shedding light on the combination of osmotolerance and sugar-tolerance mechanisms. KEY POINTS: • G. diazotrophicus intensifies glycolysis to metabolize the excess of sugar. • G. diazotrophicus turns down the uptake of nutrients in response to high sugar. • G. diazotrophicus requires amino acid availability to resist high sugar.

Effect of chia seed mucilage/bacterial cellulose edible coating on bioactive compounds and antioxidant activity of strawberries during cold storage.

This study aimed to investigate the effect of chia seed mucilage (CSM) - bacterial cellulose nano-fiber (CNF) edible coating on bioactive compounds and antioxidant enzyme activity of strawberries. Strawberries were coated with CSM containing 0.6 and 8.0% (w/w) of CNF. The content of total phenol, flavonoids, anthocyanin, ascorbic acid, protein content, antioxidant activity and the activity of polyphenol oxidase, peroxidase, superoxide dismutase and phenylalanine ammonia-lyase enzymes were evaluated. The use of CSM - CNF edible coatings further preserved the phenolic, flavonoid, ascorbic acid and antioxidant activity of strawberries, and this effect was more evident in the CSM-coated sample containing CNF; However, the accumulation of anthocyanins in the coated samples was lower than the control sample. The activity of polyphenol oxidase and peroxidase enzymes, which lead to the degradation of phenolic compounds and brown color in the product, was also effectively controlled by the edible coating.

Relocation of dehydroquinate dehydratase to the periplasmic space improves dehydroshikimate production with Gluconobacter oxydans strain NBRC3244.

3-Dehydroshikimate (3-DHS) is a key intermediate for the synthesis of various compounds, including the antiviral drug oseltamivir. The Gluconobacter oxydans strain NBRC3244 intrinsically oxidizes quinate to produce 3-dehydroquinate (3-DHQ) in the periplasmic space. Even though a considerable activity is detected in the recombinant G. oxydans homologously overexpressing type II dehydroquinate dehydratase (DHQase) encoded in the aroQ gene at a pH where it grows, an alkaline shift of the culture medium is required for 3-DHS production in the middle of cultivation. Here, we attempted to adopt type I DHQase encoded in the aroD gene of Gluconacetobacter diazotrophicus strain PAL5 because the type I DHQase works optimally at weak acid, which is preferable for growth conditions of G. oxydans. In addition, we anticipated that subcellular localization of DHQase is the cytoplasm, and therefore, transports of 3-DHQ and 3-DHS across the cytoplasmic membrane are rate-limiting steps in the biotransformation. The Sec- and TAT-dependent signal sequences for secretion were attached to the N terminus of AroD to change the subcellular localization. G. oxydans that expresses the TAT-AroD derivative achieved 3-DHS production at a tenfold higher rate than the reference strain that expresses wild-type AroD even devoid of alkaline shift. Enzyme activity with the intact cell suspension and signal sequence cleavage supported the relocation of AroD to the periplasmic space. The present study suggests that the relocation of DHQase improves 3-DHS production in G. oxydans and represents a proof of concept for the potential of enzyme relocation in metabolic engineering. KEY POINTS: • Type-I dehydroquinate dehydratase (DHQase) was expressed in Gluconobacter oxydans. • Cytoplasmic DHQase was relocated to the periplasmic space in G. oxydans. • Relocation of DHQase in G. oxydans improved 3-dehydroshikimate production.

Hybrid Living Capsules Autonomously Produced by Engineered Bacteria.

Bacterial cellulose (BC) has excellent material properties and can be produced sustainably through simple bacterial culture, but BC-producing bacteria lack the extensive genetic toolkits of model organisms such as Escherichia coli (E. coli). Here, a simple approach is reported for producing highly programmable BC materials through incorporation of engineered E. coli. The acetic acid bacterium Gluconacetobacter hansenii is cocultured with engineered E. coli in droplets of glucose-rich media to produce robust cellulose capsules, which are then colonized by the E. coli upon transfer to selective lysogeny broth media. It is shown that the encapsulated E. coli can produce engineered protein nanofibers within the cellulose matrix, yielding hybrid capsules capable of sequestering specific biomolecules from the environment and enzymatic catalysis. Furthermore, capsules are produced which can alter their own bulk physical properties through enzyme-induced biomineralization. This novel system uses a simple fabrication process, based on the autonomous activity of two bacteria, to significantly expand the functionality of BC-based living materials.

Enzymatic synthesis of phlorizin fructosides.

Phlorizin is a low soluble dihydrochalcone with relevant pharmacological properties. In this study, enzymatic fructosylation was approached to enhance the water solubility of phlorizin, and consequently its bioavailability. Three enzymes were assayed for phlorizin fructosylation in aqueous reactions using sucrose as fructosyl donor. Levansucrase (EC 2.4.1.10) from Gluconacetobacter diazotrophicus (Gd_LsdA) was 6.5-fold more efficient than invertase (EC 3.2.1.26) from Rhodotorula mucilaginosa (Rh_Inv), while sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) from Schedonorus arundinaceus (Sa_1-SST) failed to modify the non-sugar acceptor. Gd_LsdA synthesized series of phlorizin mono- di- and tri-fructosides with maximal conversion efficiency of 73 %. The three most abundant products were identified by ESI-MS and NMR analysis as ß-D-fructofuranosyl-(2→6)-phlorizin (P1a), phlorizin-4'-O-ß-D-fructofuranosyl-(2→6)-D-fructofuranoside (P2c) and phlorizin-4-O-monofructofuranoside (P1b), respectively. Purified P1a was 16 times (30.57 g L-1 at 25 °C) more soluble in water than natural phlorizin (1.93 g L-1 at 25 °C) and exhibited 44.56 % free radical scavenging activity. Gd_LsdA is an attractive candidate enzyme for the scaled synthesis of phlorizin fructosides in the absence of co-solvent.

Enzymatic and Chemical Cross-Linking of Bacterial Cellulose/Fish Collagen Composites-A Comparative Study.

This article compares the properties of bacterial cellulose/fish collagen composites (BC/Col) after enzymatic and chemical cross-linking. In our methodology, two transglutaminases are used for enzymatic cross-linking-one recommended for the meat and the other proposed for the fish industry-and pre-oxidated BC (oxBC) is used for chemical cross-linking. The structure of the obtained composites is characterized by scanning electron microscopy, thermogravimetric analysis, X-ray diffraction, and Fourier transform infrared spectroscopy, and their functional properties by mechanical and water barrier tests. While polymer chains in uncross-linked BC/Col are intertwined by H-bonds, new covalent bonds in enzymatically cross-linked ones are formed-resulting in increased thermal stability and crystallinity of the material. The C2-C3 bonds cleavage in D-glucose units, due to BC oxidation, cause secondary alcohol groups to vanish in favor of the carbonyl groups' formation, thus reducing the number of H-bonded OHs. Thermal stability and crystallinity of oxBC/Col remain lower than those of BC/Col. The BC/Col formation did not affect tensile strength and water vapor permeability of BC, but enzymatic cross-linking with TGGS improved them significantly.