Self-immobilized recombinant Acetobacter xylinum for biotransformation

Biofilms have been successfully applied for biotransformation for decades, especially in the area of bioremediation due to the feature of harsh reaction condition resistance. Acetobacter xylinum is known for its cellulose pellicle forming capability. Like biofilm, A. xylinum cells are immobilized by simultaneously produced cellulose nanofibers in the pellicle. A recombinant A. xylinum was constructed with the expectation that the cells could be self-immobilized and achieve a desired and stable biotransformation. d-Amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as the model enzyme to be expressed in the recombinant A. xylinum. The constructed recombinant A. xylinum not only successfully produced DAAO activity but also self-immobilized by cellulose nanofibers in both the static and shaken culture. Although self-immobilized cells demonstrated a DAAO activity approximately 10% of the cell crude extract activity, it provided the benefits of improved thermal stability, operational stability, and easy retrieval for repeated use.     

SYNTHESIS OF CELLULOSE BY ACETOBACTER XYLINUM : VIII. On the Formation and Orientation of Bacterial Cellulose Fibrils in the Presence of Acidic Polysaccharides

The transfer of the glucosyl moiety from uridine diphosphate glucose in the presence of Acetobacter xylinum cell-free extracts led to the formation of a mixture of alkali-soluble and -insoluble cellodextrins. Typical cellulose fibrils could not be detected by electron microscopy in this product. Immediately after release into the medium, cellulose formed by whole cells is in a "prefibrous" form which passes through Millipore filters of 0.45 and 0.8 µ pore diameter. Non-filtrable fibrils arise from this material probably by a process of crystallization involving no extracellular enzymes. Fibrils formed in shaken cell suspensions intertwine and form aggregates visible to the naked eye. In quiet suspensions pellicles are formed which float on the surface. Soluble Na-carboxymethylcellulose (CMC) is incorporated into cellulose fibrils formed in its presence, probably by a process of co-crystallization. Aggregation of fibrils containing CMC is delayed because of electrostatic repulsion between carboxylic groups. The aggregation time depends on the amount of CMC incorporated, its degree of substitution, the pH of the medium, and the ionic strength. The amount of CMC incorporated depends on the relative concentration CMC/cellulose and on the similarity of the CMC and the cellulose molecules i.e. in molecular weight and the number of carboxyl substitutions. Cellulose pellicles formed in the presence of CMC by unshaken cell suspensions consist of crossed, superimposed layers of parallel oriented cellulose fibrils. The same phenomenon is observed when phosphomannan, but not levan, is substituted for CMC. The biogenesis of oriented cellulose fibrils is envisaged as a process comprising the following steps: polymerization of the monomeric precursor, diffusion of the molecule to crystallization sites, crystallization, and orientation. It is proposed that charged polysaccharides play a role similar to that of CMC in affecting the orientation of cellulose fibrils in the plant cell wall.     

Cellulose synthesis by Acetobacter xylinum III. Matrix,primer and lipid requirements and heat stability of the cellulose-forming enzymes

The addition of soluble cellodextrins of increasing size to a cell envelope preparation of Acetobacter xylinum stimulated cellulose synthesis from UDPG. This stimulation was attributed to both acceptor and activator effects. Enzymes required for cellulose synthesis were found to be heat-unstable and those required for synthesis of glycosylated lipid components from UDPG, heat-stable. Both heat-inactivated envelope fragments and supernatant fluid from whole cells were necessary for cellulose synthesis from UDPG. Cellulose was not formed from UDPG in the presence of either supernatant fluid alone or heat-inactivated envelopes alone.The combined results of this and previous studies suggest that either the cell envelope is necessary for synthesis of a more immediate precursor to cellulose than UDPG, or that the synthesis from UDPG requires a matrix. The former suggestion and its possible link with lipid intermediate involvement was strengthened by the observation of inefficient glycoxylated lipid formation by a celluloseless mutant strain of A. xylinum. The possible locations of various enzyme activities required for the synthesis of the cellulose precursor are indicated and a possible microfibril nucleation process is discussed.     

Oxygen Isotope Exchange between Metabolites and Water during Biochemical Reactions Leading to Cellulose Synthesis

Cellulose was produced heterotrophically from different carbon substrates by carrot tissue cultures and Acetobacter xylinum (a cellulose-producing bacterium) and by castor bean seeds germinated in the dark, in each case in the presence of water having known concentration of oxygen-18 (¹⁸O). We used the relationship between the amount of ¹⁸O in the water and in the cellulose that was synthesized to determine the number and ¹⁸O content of the substrate oxygens that exchanged with water during the reactions leading to cellulose synthesis. Our observations support the hypothesis that oxygen isotope ratios of plant cellulose are determined by isotopic exchange occurring during hydration of carbonyl groups of the intermediates of cellulose synthesis.     

Isolation and Structure Elucidation of α Substance-IB,a Hexapeptide Inducing Sexual Agglutination in Saccharomyces cerevisiae

Cellulose triacetate prepared from bacterial cellulose of Acetobacter xylinum subsp. sucrofermentans BPR3001A showed a higher degree of polymerization and higher mechanical strength than that from the cotton linter. The fine fibrils of bacterial cellulose required only a short time for acetylation which preserved the high degree of polymerization.     

Colonization of Crystalline Cellulose by Clostridium cellulolyticum ATCC 35319

Cellulose colonization by Clostridium cellulolyticum was studied by using [methyl-³H]thymidine incorporation. The colonization process indicated that a part of the bacterial population was released from cellulose to the liquid phase before binding and colonizing another adhesion site of the cellulose. We postulate that cellulose colonization occurs according to the following process: adhesion, colonization, release, and readhesion.     

Cellulose synthesis by acetobacter xylinum II. Investigation into the relation between cellulose synthesis and cell envelope components

Cell envelope fractions, capable of cellulose synthesis from uridine diphosphate glucose, α-glucose-1-phosphate, glucose-6-phosphate and glucose, have been isolated from Acetobacter xylinum suspensions and various enzymatic properties examined.Essential enzymes were found to be distributed throughout the cell envelope region, with both inner (cytoplasmic) and outer (cell wall) membranes contributing to cellulose synthesis. The central role of UDPG in cellulose synthesis was confirmed and the results indicated that the nucleoside diphosphate sugar functions solely in the cell envelope region of whole cells. A comparison of properties of the cell envelope system with those of different preparations used by other workers, suggested that the method of cell disruption may influence substrate specificity.     

Production of cellulose from glucose by Acetobacter xylinum

Acetobacter xylinum 1FO 13693 was selected as the best cellulose-producing bacterium among 41 strains belonging to the genus Acetobacter and Agrobacterium. Cellulose was found to be produced at the liquid surface in static liquid cultivation. The rate of cellulose production depended proportionally on the surface-area of the culture medium and was unaffected by the depth and volume of the medium. The optimum pH for cellulose production was 4.0 to 6.0. Glucose, fructose and glycerol were preferred carbon sources for cellulose production. The yield of cellulose, relative to the glucose consumed, decreased with an increase in initial glucose concentration, and gluconic acid accumulated at a high initial glucose concentration. The decrease in cellulose yield could be due to some glucose being metabolized to gluconic acid. However, the accumulated gluconic acid did not affect cellulose production. The culture conditions of the bacterium for cellulose production were optimized. The maximum production rate of cellulose was 36 g/d·m², with a yield of 100% for added glucose under the optimal conditions.     

Cellulose synthesis by Acetobacter xylinum I. Low molecular weight compounds present in the region of synthesis

An analysis has been made of the low molecular weight fraction present in the region of cellulose synthesis in Acetobacter xylinum suspensions.A number of nucleic acid bases, nucleosides and nucleotides, together with α-glucose 1-phosphate and UDPG, were detected in various extracts of washed cells supplied with glucose. Since glucose-6-P could be detected in extracts of ultrasonically disrupted cells, but not in extracts of whole cells, it was concluded that separate pools of hexose phosphate exist in A. xylinum. Preferential release of α-glucose-1-P, UDPG and nucleotides was observed during ethanol and EDTA treatment of bacteria. Electron microscopic examination of treated and untreated cells revealed that extensive modification of the cell wall region occured during such treatments. The results support the proposal that α-glucose-1-P, UDPG and nucleotide pools are localised in the cell envelope region, possibly in the periplasm, and that A. xylinum possesses a second permeability barrier outside the cytoplasmic membrane. Nucleic acid bases and nucleosides were observed to diffuse freely through the cell wall and accumulate in the medium, probably as the result of nucleic acid breakdown. The results imply that the effects of cell damage caused by the isolation of the bacteria from the surface pellicle of the culture medium, together with nutrient deprivation, should be considered in work using the non-proliferating system. A study of the variation in concentration with time of α-glucose-1-P and UDPG, during cellulose synthesis, indicated that both components mau play an immediate role in cellulose synthesis.Glycosylated lipid compounds were detected in both cell wall extracts and supernatant fluid, but it is not certain whether these compounds are constituents of the supernatant fluid in vivo.     

The Synthesis of Cellulose Microfibrils by Particulate Fractions Released from Acetobacter xylinum by Washing

Washing A. xylinum whole cells in 0.01 m phosphate-0.003 m citrate,pH 6.2, leads to the release of particulate fractions whichretain the ability to synthesize cellulose microfibrils fromglucose and from UDPG. These fractions contain both outer andcytoplasmic membrane fragments. Frccze-etching shows the microfibrilsto arise from outer membrane vesicles. A scheme to describethe synthesis of cellulose microfibrils by A. xylinum is proposed.     

Taxonomic significance of cellulosic cell walls in the bangiales (Rhodophyta)

Cellulose has been characterized from isolated cell walls of the conchocelis phases of both Porphyra umbilicalis and P. leucostricta. Evidence for cellulose II (regenerated cellulose) in Schweitzer's reagent extracts was provided by X-ray powder analysis and paper chromatography of partial hydrolyzates. The presence of cellulose in the conchocelis phase of species of Porphyra provides evidence for the continuity of cell wall composition within the Rhodophyta. Adoption of a classification scheme incorporating consolidation of all red algal orders under the single class Rhodophyceae is proposed.     

Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL⁻¹ CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL⁻¹, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.     

Isolation and Characterization of Two Cellulose Morphology Mutants of Gluconacetobacter hansenii ATCC23769 Producing Cellulose with Lower Crystallinity

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.     

Synthetic Medium for Acetobacter xylinum That Can Be Used for Isolation of Auxotrophic Mutants and Study of Cellulose Biosynthesis

Acetobacter xylinum is a bacterium that can synthesize cellulose when grown in an undefined medium containing glucose. We developed a defined minimal medium for A. xylinum that contains 1% glucose, 0.1% NH₄Cl, 0.115% citric acid, 0.33% Na₂HPO₄, 0.01% KCl, 0.025% MgSO₄. 7H₂O, and 7.5 mg of nicotinamide per liter which both allows cellulose synthesis and can be used to isolate a variety of auxotrophic mutants.     

Synthesis of 20α- and 20β-Amino-3,5-pregnadiene Derivatives and Antimicrobial Activity

Cellulose production by Acetobacter strains is enhanced by the addition of a small amount of cellulose to the production culture. The effect of an endo-β-1, 4-glucanase from Bacillus subtilis on the cellulose production by Acebohacter xylinum BPR2001 was examined by adding various amounts of the purified glucanase to the culture. The addition of a small amount of this glucanase enhanced cellulose production. Furthermore, it reduced the amount of a polysaccharide called acetan produced. However, an active-site mutant enzyme of the glucanase, which showed no enzyme activity but still had cellulose-binding ability, had no effect on cellulose production. It was concluded, therefore, that the endoglucanase activity itself, but not the cellulose-binding ability, was essential for the enhancement of cellulose production. The structural properties of the cellulose produced in the presence of the endoglucanase were found to be almost identical to those of native bacterial cellulose.     

In vitro assembly of cellulose/xyloglucan networks: ultrastructural and molecular aspects

Features of the interaction between cellulose and xyloglucan have been studied using the cellulose-producing bacterium Acetobacter aceti ssp. xylinum (ATCC 53524) and tamarind seed xyloglucan. Direct microscopic evidence is provided for the generation of cross-bridges between cellulose ribbons produced in the presence of xyloglucan but not carboxymethyl-cellulose. Cross-bridge lengths are very similar to those observed for de-pectinated onion cell walls. Similar cross-bridge lengths are observed following mixing of isolated A. xylinum cellulose and xyloglucan, showing that network formation can be an abiotic process. The level of incorporation of xyloglucan in an actively growing system (ca. 38% of cellulose) is an order of magnitude higher than that observed in mixtures of isolated polymers and is comparable with cell wall levels. NMR spectroscopy suggests that 80–85% of incorporated xyloglucan is segmentally rigid with the backbone adopting an extended ‘cellulosic’ conformation and probably aligned with cellulose chains. The remaining xyloglucan is more mobile and is assigned to cross-bridges with, on average, a twisted backbone conformation. No evidence for specific involvement of side-chain residues in binding is found, and the observation of cross-bridges with a non-fucosylated xyloglucan shows that fucose residues are not essential for network formation. Xyloglucan causes cellulose ribbons to become more amorphous and to have a decreased 1α/1β crystallite ratio without any significant alteration in ribbon diameter. Based on the findings that levels of xyloglucan incorporation, the presence and lengths of cross-bridges, and the modification of cellulosic molecular organization are all similar to those found in plant cell walls, we suggest that A. aceti ssp. xylinum is a more useful model for primary plant cell walls and their assembly than has previously been appreciated.     

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

Influence of 1‐methylcyclopropene (1‐MCP) on the production of bacterial cellulose biosynthesized by Acetobacter xylinum under the agitated culture

Aims: Bacterial cellulose is an extracellular polysaccharide secreted by Acetobacter xylinum, which has become a novel material increasingly used in food and medical industries. However, its broad application is limited by its low yield and high cost. 1‐Methylcyclopropene (1‐MCP) is a potent inhibitor to either exogenous or endogenous ethylene during the biological senescence of plants, which has been broadly applied in commercial preservation of fruits and vegetables. The purpose of this study was to investigate the effects of 1‐MCP on both the growth of Acet.  xylinum and its cellulose production to demonstrate the potential enhancement of bacterial cellulose yield. Methods and Results: Three groups of samples were fermented under agitated culture with 125 rev min^?1 rotational speed. To the culture media, 0·14 mg of 1‐MCP contained in 100 mg dextrose powder was added on assigned days or on the first culture day only. Results from the measurement of bacterial cell concentration and bacterial cellulose yield at the end of a 12‐day culture demonstrated that cultures excluding 1‐MCP displayed a higher cell concentration and a lower cellulose production, while cultures containing 1‐MCP produced 15·6% more cellulose (1‐MCP added on day 1) and 25·4% (1‐MCP added on each assigned day) with less biomass. Conclusions: 1‐MCP was able to affect the growth of Acet. xylinum cells and resulted in increasing bacterial cellulose yield up to 25·4% over controls, which did not contain 1‐MCP. Significance and Impact of the Study: This was the first study to use the growth inhibitor of plants to investigate its effects on bacterial growth and production. It also demonstrated a significant enhancement of bacterial cellulose yield by the addition of 1‐MCP during the common agitated culture of Acet. xylinum.     

Oxaloacetate decarboxylase from Acetobacter aceti

An oxaloacetate (OAA) decarboxylase (EC 4.1.1.3) has been purified 72-fold from Acetobacter aceti cells grown on ethanol, and its molecular weight was estimated to be about 80,000 by gel filtration. Several properties distinguished this enzyme from the OAA decarboxylase from A. xylinum:

1. It was not a constitutive enzyme; the activity was 6- to 20-fold higher in cells grown on a C₂ substrate (acetate or ethanol) than in cells grown on a C₃ compound (pyruvate or propionate).
2. The optimum pH was 7.5; a value of 5.6 was reported for the enzyme from A. xylinum.
3. The enzyme did not need a divalent cation and was not inhibited by EDTA.
4. The K _mvalue for OAA was found to be 0.22 mM. It was not affected by the addition of nicotinamide adenine dinucleotide.
5. The enzyme activity was neither inhibited by acetate nor by L-malate.

In addition, the OAA decarboxylase from A. aceti was insensitive to monovalent cations, avidin or acetyl coenzyme A.     

Cellulose component of Acetobacter xylinum pellicle: morphology and its significance for the mechanism of microfibril formation

The cellulose component of native, minimally disturbed pellicles of Acetobacter sylinum has a three-dimensional, microfibrillar, interconnected, ‘brush-wood’ structure. This structure could not originate from a spinneret or extrusion mechanism of cellulose microfibril formation. It may be produced by a mechanism of spontaneous association and post factor crystallization of preformed, transient I → 4β glucans.