A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme.

Polylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalyst-mediated chemical polymerization of lactate (LA) supplied by microbial fermentation. To establish the one-step microbial process for synthesis of LA-based polyesters, we explored whether polyhydroxyalkanoate (PHA) synthase would exhibit polymerizing activity toward a LA-coenzyme A (CoA), based on the fact that PHA monomeric constituents, especially 3-hydroxybutyrate (3HB), are structurally analogous to LA. An engineered PHA synthase was discovered as a candidate by a two-phase in vitro polymerization system previously developed. An LA-CoA producing Escherichia coli strain with a CoA transferase gene was constructed, and the generation of LA-CoA was demonstrated by capillary electrophoresis/MS analysis. Next, when the engineered PHA synthase gene was introduced into the resultant recombinant strain, we confirmed the one-step biosynthesis of the LA-incorporated copolyester, P(6 mol% LA-co-94 mol% 3HB), with a number-average molecular weight of 1.9 x 10(5), as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.

In vivo effects of isolated implantation of salmon-derived crosslinked atelocollagen sponge into an osteochondral defect.

We have developed crosslinked salmon-derived atelocollagen (SC) sponge, which has a denaturation temperature of 47°C. Sixty-four knees of 32 mature rabbits were randomly divided into 4 groups after creating an osteochondral defect in the femoral trochlea. Defects in Groups I, II, and III were filled with the crosslinked SC sponge, the crosslinked porcine collagen (PC) sponge, and the non-crosslinked PC sponge, respectively. In Group IV, defects were left untreated as the control. At 12 weeks after implantation, the histological score showed that Group I was significantly greater than Groups III (P = 0.0196) and IV (P = 0.0021). In addition, gene expression of type-2 collagen, aggrecan, and SOX9 was the greatest in Group I at 12 weeks. The fundamental in vivo properties of the crosslinked SC sponge showed that this is a promising biomaterial, specifically as a scaffold for cartilage tissue engineering.

Purification, crystallization and preliminary X-ray studies of AxCesD required for efficient cellulose biosynthesis in Acetobacter xylinum.

AxCesD protein required for bacterial cellulose biosynthesis in Acetobacter xylinum was overexpressed in E. coli, purified and crystallized. Single crystals of SeMet-substituted AxCesD were obtained by the sitting-drop vapor-diffusion method. The crystal belongs to the primitive trigonal space group P3 2, with unit-cell parameters a = b = 77.7 A, and c = 213.9 A. The asymmetric unit in the crystal was assumed to contain 8 protein molecules giving the Matthews coefficient (VM) of 2.54 A3 Da(-1). Se-MAD data were collected to 2.3 A resolution using synchrotron radiations.

Biological properties of crosslinked salmon collagen fibrillar gel as a scaffold for human umbilical vein endothelial cells.

Collagen derived from chum salmon (Oncorhynchus keta) was crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) during collagen fibrillogenesis and applied to an in vitro cell culture to evaluate its potential use as a scaffold for vascular tissue engineering. Human umbilical vein endothelial cells (HUVEC) were cultured on the crosslinked salmon collagen fibrillar gel (EDC-SC gel), and their growth rates and production levels of cytokines, including platelet-derived growth factor-BB and von Willebrand factor, were measured. Comparison was also made with bovine collagen gel crosslinked with EDC (EDC-BC gel). The growth and cytokine production of the HUVEC cultured on the EDC-SC gel were higher than those on the EDC-BC gel. In addition, HUVEC were found to attach to the EDC-BC gel through alpha2beta1 integrin for native collagen, whereas they attached to the EDC-SC gel through alphavbeta3 integrin for denatured collagen as well as the alpha2beta1 integrin, indicating that HUVEC recognized denatured domains in the EDC-SC gel. In conclusion, the EDC-SC gel can be used as a scaffold to support HUVEC growth, although the integrin-mediated attachment manner differs between the two gels.

In vitro growth and differentiated activities of human periodontal ligament fibroblasts cultured on salmon collagen gel.

Marine-derived collagen is expected to be a much safer alternative to calf collagen, which in medical applications carries the risk of bovine spongiform encephalopathy. In this study, acid-soluble collagen was extracted from salmon skin and crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide during fibril formation to produce a crosslinked salmon collagen (SC) gel. The growth rates and the differentiated functions of human periodontal ligament fibroblasts (HPdLFs) cultured on the SC gel were investigated. Growth was faster on the SC gel than on porcine collagen (PC) gel. In addition, the HPdLFs cultured on the SC gel exhibited higher alkaline phosphatase (ALP) activity than those cultured on the PC gel. Quantitative RT-PCR revealed higher mRNA expression of type I collagen, ALP, and osteocalcin in the HPdLFs cultured on the SC gel. HPdLFs had a flat shape on the SC gel and a spindle shape on the PC gel, as revealed by observation with scanning electron microscopy and immunostaining with cytoskeletal protein and vinculin. The results showed that HPdLFs could grow and show highly differentiated activity on the SC gel as well as on the PC gel.

Preparation and characterization of multilayered hydroxyapatite/silk fibroin film.

We prepared multilayered films consisting of silk fibroin (SF) and hydroxyapatite (HAp) by alternating lamination using untreated SF and HAp-deposited SF films. Untreated SF films were prepared from a regenerated SF solution by air drying. HAp-deposited SF films were prepared by soaking methanol-treated SF films containing >5 wt% CaCl2 in a simulated body fluid with the ion concentration 1.5-fold higher than that of the standard one. The multilayered HAp/SF films had HAp layers with approximate thicknesses of 3-5 microm and SF layers with thicknesses of 40-70 microm. The bonding strength between the SF and HAp layers was significantly affected by temperature and compression time under the lamination method. The optimal conditions for achieving the maximum T-peel strength and beta-sheet contents were determined to be 130 degrees C for 4 min. The Young's modulus of the multilayered films (133.4 MPa) was higher than that of the films consisting of SF alone (92.5 MPa) under swollen conditions. The biocompatibility of the HAp-deposited SF films was analyzed by culturing of osteoblasts (MC3T3-E1) on a film. The results indicate that HAp-deposited SF films and SF films show similar degrees of cell adhesion and alkaline phosphatase activities.

Structural characterization of the Acetobacter xylinum endo-beta-1,4-glucanase CMCax required for cellulose biosynthesis.

Previous studies have demonstrated that endoglucanase is required for cellulose biosynthesis both in bacteria and plants. However, it has yet to be elucidated how the endoglucanases function in the mechanism of cellulose biosynthesis. Here we describe the crystal structure of the cellulose biosynthesis-related endo-beta-1,47-glucanase (CMCax; EC 3.2.1.4) from the cellulose-producing Gramnegative bacterium, Acetobacter xylinum (= Gluconacetobacter xylinus), determined at 1.65-A resolution. CMCax falls into the glycoside hydrolase family 8 (GH-8), and the structure showed that the overall fold of the CMCax is similar to those of other glycoside hydrolases belonging to GH-8. Structure comparison with Clostridium thermocellum CelA, the best characterized GH-8 endoglucanase, revealed that sugar recognition subsite +3 is completely missing in CMCax. The absence of the subsite +3 leads to significant broadness of the cleft at the cellooligosaccharide reducing-end side. CMCax is known to be a secreted enzyme and is present in the culture medium. However, electron microscopic analysis using immunostaining clearly demonstrated that a portion of CMCax is localized to the cell surface, suggesting a link with other known membrane-anchored endoglucanases that are required for cellulose biosynthesis.

Crystallization and preliminary crystallographic analysis of the cellulose biosynthesis-related protein CMCax from Acetobacter xylinum.

The cellulose biosynthesis-related protein CMCax from Acetobacter xylinum was overexpressed in Escherichia coli, purified and crystallized. Single crystals of selenomethionine (SeMet) substituted CMCax were obtained by the hanging-drop vapour-diffusion method at 293 K, primarily using polyethylene glycol 4000 as a precipitant. The crystals belong to the primitive hexagonal space group P6(1) or P6(5), with unit-cell parameters a = b = 89.1, c = 94.2 A. The predicted Matthews coefficient (VM) value is 3.0 A3 Da(-1) for one CMCax monomer in the asymmetric unit. A single-wavelength anomalous dispersion (SAD) data set was collected to a resolution of 2.3 A using synchrotron radiation.

Cloning of cellulose synthesis related genes from Acetobacter xylinum ATCC23769 and ATCC53582: comparison of cellulose synthetic ability between strains.

About 14.5 kb of DNA fragments from Acetobacter xylinum ATCC23769 and ATCC53582 were cloned, and their nucleotide sequences were determined. The sequenced DNA regions contained endo-beta-1,4-glucanase, cellulose complementing protein, cellulose synthase subunit AB, C, D and beta-glucosidase genes. The results from a homology search of deduced amino acid sequences between A. xylinum ATCC23769 and ATCC53582 showed that they were highly similar. However, the amount of cellulose production by ATCC53582 was 5 times larger than that of ATCC23769 during a 7-day incubation. In A. xylinum ATCC53582, synthesis of cellulose continued after glucose was consumed, suggesting that a metabolite of glucose, or a component of the medium other than glucose, may be a substrate of cellulose. On the other hand, cell growth of ATCC23769 was twice that of ATCC53582. Glucose is the energy source in A. xylinum as well as the substrate of cellulose synthesis, and the metabolic pathway of glucose in both strains may be different. These results suggest that the synthesis of cellulose and the growth of bacterial cells are contradictory.

Effects of irradiation on cementum matrix cytokins function during periodontal regeneration.

The influence of gamma-ray irradiation on a cementum-impregnated gelatine membrane (CGM) was analyzed with emphasis on its function during periodontal regeneration. In brief, proteins were extracted from gamma-ray irradiated cementum (gammaC). With the gammaC protein, sample cells (gingival fibroblasts, periodontal ligament cells, and alveolar bone cells) were co-cultured, and cytological parameters (cell attachment, cell differentiation and alkaline phosphatase activity) were analyzed. Additionally, kinetics of some gene expression was analysed using reverse transcript RT-PCR, which included osteoproteogerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) mRNA. BMP-2 and osteonectin were resistant to gamma-rays, and other cytokines involved in regeneration were decreased. Thus, the attachment activity of osteoblasts to gammaC protein was higher than that of non-irradiated cementum (control C). The expression of OPG/OCIF mRNA was lower in co-cultured cells with gammaC protein than those with in control C protein. Together the results imply that some cytokine in intact cementum prevents the attachment (differentiation) of bone cells onto the root surface, which may explain why the introduction of CGM following gingival flap surgery induces new cementum, new ligament and new bone formation, but CGM irradiated with gamma-rays for clinical use causes ankylosis.

Preparation and characterization of collagen from soft-shelled turtle (Pelodiscus sinensis) skin for biomaterial applications.

Collagen was isolated from the skin of soft-shelled turtle (Pelodiscus sinensis) by acid solubilization with pepsin. The yield of soft-shelled turtle collagen (STC) was 12.1% on a dry weight basis. The electrophoresis assay showed that STC consisted of a alpha(1)alpha(2) heterodimer similar to porcine collagen (PC). Amino-acid composition analysis showed that the hydroxyproline content of STC was 7.8%, which was lower than that of PC (9.5%). The denaturation temperature of STC was 36 degrees C from optical rotation analysis. An accelerated fibrillogenesis of STC was observed in phosphate-buffered saline at 25 degrees C. The resulting STC fibrillar gel had microfibrillar network with fibril diameter of ca. 124 nm, as revealed by observation with scanning electron microscopy. The compressive moduli of the STC gel and the PC gel were 3.2 +/- 0.8 kPa and 3.6 +/- 0.3 kPa, respectively. The potential of the STC gel for biomaterial applications was investigated by in vitro cell culture. Human dermal fibroblasts were three-dimensionally cultured in the STC gel and their growth was evaluated by DNA content measurement. Steady growth was observed in the STC gel for a 6-day culture period, although the growth rate was slower than in the PC gel. In conclusion, STC could be used as a novel collagen source for biomaterial applications.

Chemo-enzymatic synthesis of polyhydroxyalkanoate by an improved two-phase reaction system (TPRS).

In our previous paper, we synthesized poly-3-hydroxybutyrate [P(3HB)] by using the water-organic solvent two-phase reaction system (TPRS), in which thiophenyl (R)-3-hydroxybutyrate [(R)-3HBTP] was used as a precursor of 3HBCoA. We have developed an improved TPRS for the chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA). In this method, acetyl thioester of ethyl thioglycolate (AcETG) was used as a precursor of acetylCoA (AcCoA), which was a donor of CoA. The AcCoA was formed by the ester exchange reaction between CoA in the water phase and AcETG in the organic solvent phase. The AcCoA and free 3-hydroxybutyrate (3HB) in the water phase were converted into 3HBCoA and acetate by a CoA transfer reaction of propionylCoA transferase (PCT). The synthesized 3HBCoA was polymerized sequentially by PHA synthase, and P(3HB) was successfully formed. The maximal yield of P(3HB) was 1.2 g/l under the optimal reaction condition; this is comparable to that of in vivo PHA production. Furthermore, the number of enzymes was reduced and enzyme preparation was simplified by the construction of a fusion protein, PCT-PhaC. The chemo-enzymatic synthesis of P(3HB-co-3-hydroxypropionate) and P(3HB-co-3-mercaptopropionate) was also achieved by the improved TPRS using the fusion protein.

Regulation of endoglucanase gene (cmcax) expression in Acetobacter xylinum.

Although cellulose is the most abundant biopolymer in nature, the detailed mechanisms of cellulose biosynthesis remain unknown. Acetobacter xylinum is one of the best-studied model organisms for cellulose biosynthesis. Interestingly, the over-expression of the cmcax gene cause enhancement of cellulose production in A. xylinum, while its product (CMCax) has cellulose degradation activity. The addition of CMCax into medium also promotes cellulose production, suggesting that CMCax is involved in cellulose synthetic pathway. In the present study, we reveal the regulation mechanism of cmcax expression in A. xylinum. First, we treated cells with four kinds of beta-glucodisaccharide. Using an enzyme assay and real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we observed an increase in CMCax activity and an induction of cmcax expression by gentiobiose treatment. Therefore, we concluded that gentiobiose induced cmcax expression. Although gentiobiose does not originally exist in the cultivation medium, we have revealed that membrane and intra-cellular proteins extracted from A. xylinum produce gentiobiose from glucose, which is one of the components in the cultivation medium. Furthermore, we confirmed that cmcax expression in a wild-type strain increased gradually after 5 d cultivation using real-time qRT-PCR. These results have led us to conclude that the increase in cmcax expression after 5 d cultivation is caused by the increase in gentiobiose, which could be synthesized by a condensation reaction in A. xylinum. Since CMCax plays a pivotal role in the cellulose production system, our results will contribute to the elucidation of mechanisms of cellulose biosynthesis.

Novel elastic material from collagen for tissue engineering.

Elastic collagen gel (e-gel) was prepared from salmon atelocollagen fibrillar gel reinforced by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) mediated cross-linking (f-gel). The preparation consisted of a simple heat treatment of the f-gel at 80 degrees C, in which the f-gel drastically shrank and the collagen fibril structure was deformed. The e-gel obtained showed rubber-like elasticity; its stress-strain behavior little changed through repeated stretching. The elongation at the breaking point was approximately 230%. Furthermore, normal human osteoblasts showed good attachment and proliferation on the e-gel. These results suggest its potential to be utilized for the development of tissue engineering.