Engineered Escherichia coli for short-chain-length medium-chain-length polyhydroxyalkanoate copolymer biosynthesis from glycerol and dodecanoate.

Short-chain-length medium-chain-length polyhydroxyalkanoate (SCL-MCL PHA) copolymers are promising as bio-plastics with properties ranging from thermoplastics to elastomers. In this study, the hybrid pathway for the biosynthesis of SCL-MCL PHA copolymers was established in recombinant Escherichia coli by co-expression of ß-ketothiolase (PhaARe) and NADPH-dependent acetoacetyl-CoA reductase (PhaBRe) from Ralstonia eutropha together with PHA synthases from R. eutropha (PhaCRe), Aeromonas hydrophila (PhaCAh), and Pseudomonas putida (PhaC2Pp) and with (R)-specific enoyl-CoA hydratases from P. putida (PhaJ1Pp and PhaJ4Pp), and A. hydrophila (PhaJAh). When glycerol supplemented with dodecanoate was used as primary carbon source, E. coli harboring various combinations of PhaABCJ produced SCL-MCL PHA copolymers of various monomer compositions varying from C4 to C10. In addition, polymer property analysis suggested that the copolymers produced from this recombinant source have thermal properties (lower glass transition and melting temperatures) superior to polyhydroxybutyrate homopolymer.

Poly(3-hydroxybutyrate) production by isolated Halomonas sp. KM-1 using waste glycerol.

Biodiesel fuel is favored as a type of carbon neutral energy. To popularize its usage, by-product waste glycerol utilization is a critical problem. We tried to isolate waste glycerol utilizing bacteria, and obtained the alkalo- and halophile bacteria Halomonas sp. KM-1. This strain produced bioplastic poly(3-hydroxybutyrate) (PHB) in a simple medium and diluted waste glycerol as a sole carbon source.

A novel soft and cotton-like chitosan-sugar nanoscaffold.

A novel type of chitosan nanoscaffold with a soft and cotton-like appearance is proposed. The key to success is based on two points: (i) the change in morphology of chitin whisker to chitosan nanoscaffold and (ii) the surface modification of the nanoscaffold chitosan with a sugar unit. Simple deacetylation of chitin whisker gives a colloidal solution of chitosan, of which the chitosan is in a nanoscaled scaffold. Surface functionalization of the chitosan nanoscaffold with lactose or maltose via a heterogeneous system in water at room temperature results in a soft and cotton-like chitosan containing mesopores. As all steps are organic solvent free, this chitosan-sugar nanoscaffold might be a promising material for biopolymer-supported tissue engineering.

Depolymerization of beta-chitin to mono- and disaccharides by the serum fraction from the para rubber tree, Hevea brasiliensis.

The serum fraction of latex from Hevea brasiliensis, the para rubber tree, is known to contain an endo-chitinolytic enzyme, hevamine. Herein the activity of the rubber serum towards beta-chitin is investigated. The serum contained 6 mg/mL of protein and a chitinolytic activity of 18 mU permg of protein. The optimum ratio of enzyme to chitin was 0.22 mU/mg, and the optimum substrate concentration was 60 mg/mL. The optimum pH range was pH2-4, and the optimum temperature was 45 degrees C. At these conditions both (GlcNAc)2 and GlcNAc were produced in a molar ratio of approximately 2:1. The hydrolysis of 300 mg of chitin with 64 mU of the rubber serum for 8 days under the optimum conditions gave 39 mg of GlcNAc and 108 mg of (GlcNAc)2 as determined by HPLC. Mixing the rubber serum preparation with an Aspergillus niger pectinase preparation containing beta-N-acetylhexosaminidase can be used to produce almost exclusively the GlcNAc monomer in about 50% yield.

Michael reaction of chitosan with various acryl reagents in water.

A Michael reaction of chitosan was conducted in water containing acetic acid with various acryl reagents. The degree of substitution could be controlled by temperature, reaction time, and the amount of acryl reagents. Although the modified chitosan derivatives with acrylic acid esters showed water-solubility, that with poly(ethylene glycol) acrylate, however, turned to water-insoluble material by lyophilization. Good biodegradation was observed in modified chitosan derivatives by standard activated sludge.

Synthesis of a chitosan-dendrimer hybrid and its biodegradation.

Chitosan-dendrimer hybrids having various functional groups such as carboxyl, ester, and poly(ethylene glycol) groups were prepared successfully using dendrimer acetal by reductive N-alkylation. The synthetic procedure could be accomplished by one-step reaction without organic solvent. The degree of substitution of dendrimer was 0.13-0.18 evaluated by (1)H NMR. A perfectly or partially water-soluble chitosan-dendrimer hybrid could be obtained. By standard activated sludge, good biodegradation was observed in these hybrids.

Chemical modification of chitosan. Part 15: synthesis of novel chitosan derivatives by substitution of hydrophilic amine using N-carboxyethylchitosan ethyl ester as an intermediate.

The Michael type reaction of chitosan with ethyl acrylate has been investigated. Although this reaction was quite slow in the case of chitosan, the reiteration of the reaction was an effective means for increasing the degree of substitution (DS) of ethyl ester. The N-carboxyethylchitosan ethyl ester as an intermediate was successfully substituted with various hydrophilic amines, although the simultaneous hydrolysis of the ester to carboxylic acid also occurred. Water-soluble chitosan derivatives were obtained by substitution with hydroxyalkylamines and diamines.

Production of N-acetyl-D-glucosamine from alpha-chitin by crude enzymes from Aeromonas hydrophila H-2330.

The selective and efficient production of N-acetyl-D-glucosamine (GlcNAc) was achieved from flake type of alpha-chitin by using crude enzymes derived from Aeromonas hydrophila H-2330.

Quantitative production of 2-acetamido-2-deoxy-D-glucose from crystalline chitin by bacterial chitinase.

Finely powdered alpha- and beta-chitin can be completely hydrolyzed with chitinase (EC 3.2.1.14) and beta-N-acetylhexosaminidase (EC 3.2.1.52) for the production of 2-acetamido-2-deoxy-D-glucose (GlcNAc). Crude chitinase from Burkholderia cepacia TU09 and Bacillus licheniformis SK-1 were used to digest alpha- and beta-chitin powder. Chitinase from B. cepacia TU09 produced GlcNAc in greater than 85% yield from beta- and alpha-chitin within 1 and 7 days, respectively. B. licheniformis SK-1 chitinase completely hydrolyzed beta-chitin within 6 days, giving a final GlcNAc yield of 75%, along with 20% of chitobiose. However, only a 41% yield of GlcNAc was achieved from digesting alpha-chitin with B. licheniformis SK-1 chitinase.

Utilization of commercial non-chitinase enzymes from fungi for preparation of 2-acetamido-2-deoxy-D-glucose from beta-chitin.

Commercial non-chitinase enzymes from Aspergilus niger, Acremonium cellulolyticus and Trichoderma viride were investigated for potential utilization in the preparation of 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine, GlcNAc) from chitin. Among the tested enzymes, cellulase A. cellulolyticus exhibited highest chitinolytic activity per weight toward amorphous chitin and beta-chitin from squid pen. The optimum pH of the enzyme was 3 where it produced two major hydrolytic products, GlcNAc and N,N'-diacetylchitobiose ([GlcNAc](2)). The product ratio, GlcNAc:[GlcNAc](2), increased while the total yield decreased as the pH was raised from 3. All of the [GlcNAc](2) produced at pH 3 can be converted in situ to GlcNAc by mixing cellulase A. cellulolyticus with one of several other enzymes from A. niger resulting in a higher yield of GlcNAc. An appropriate mixing ratio of cellulase A. cellulolyticus to another enzyme was 9:1 (w/w) and an optimum substrate concentration was 20 mg/mL.