Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.
Biocatalysis ; Chitin ; analogs & derivatives ; Chitinases ; Hydrogen-Ion Concentration ; Polymerase Chain Reaction

OBJECTIVETo optimize the preparation of genistein chitosan microspheres with central composite design (CCD).

METHODThe chitosan microspheres were prepared by the O/W/O multiple emulsion method. Second-order polynomial and linear equations were fitted to the data, and the resulting equations were used to produce response surface graphs and the best experiment conditions.

RESULTThe theoretical drug content was 13%-15%, the concentration of organic phase was 30%-40% and the concentration of oil phase was 68%-72%.

CONCLUSIONThe best experiment conditions can be obtained by central composite design and response surface methodology. The observed values agree well with model predicted values.

Antineoplastic Agents ; administration & dosage ; Chitin ; analogs & derivatives ; Chitosan ; Delayed-Action Preparations ; Genistein ; administration & dosage ; Mathematics ; Microspheres

OBJECTIVETo prepare microbubble, made of N-carboxymethyl chitosan, as ultrasound contrast agent and evaluate its characteristics and acoustic effects in vivo.

METHODSOil-Water-Oil multiple emulsion/solvent evaporation method was used to prepare the microbubble contrast agent. Both optical micrography and scanning electron micrography were performed to determine the bubble size and morphology. The acoustic effect of the N-carboxymethyl chitosan echo contrast agent was evaluated in vivo in rabbit. Liver echo images were recorded with ultrasound machine before and after intravenous bolus injecting 0.5 ml of the agent.

RESULTSThe novel N-carboxymethyl chitosan echo contrast agent was formulated as lyophilized product, with a mean diameter of 2-3 microm and a shell thickness of 250-300 nm. Its size is relatively uniform. The imaging effect was remarkably enhanced with the ultrasonic contrast agent when applied in rabbit livers.

CONCLUSIONIt is feasible to prepare excellent microbubble ultrasound contrast agent with N-carboxymethyl chitosan as membrane components.

Animals ; Chitin ; analogs & derivatives ; chemical synthesis ; chemistry ; Contrast Media ; Liver ; diagnostic imaging ; Microbubbles ; Rabbits ; Ultrasonics ; Ultrasonography

Chitosan-carboxymethyl-chitosan complex film was prepared by freeze drying. Some tests in vivo and in animal were employed, in order to evaluate it on biology. All results indicated that the film has not only good surface compatibility but also good structural compatibility. It can be more suitable for GTR technology.
Animals ; Biocompatible Materials ; chemical synthesis ; pharmacology ; Chitin ; analogs & derivatives ; Chitosan ; Materials Testing ; Membranes, Artificial ; Rabbits ; Rats ; Skin Irritancy Tests

OBJECTIVETo observe the skin regeneration after hair follicle bulb cells were implanted into collagen/chitosan porous scaffolds in vitro.

METHODSThe cultured dorsal hair follicle bulb cells of 4d-old C57BL/6J mice were implanted into collagen/chitosan porous scaffolds in vitro. The skin regeneration was observed.

RESULTThe skin-like structure was formed on the collagen/chitosan porous scaffolds where were cultured the hair follicle bulb cells before 4th passages.

CONCLUSIONThe skin-like structure is generated in vitro when early passages of cultured hair bulb cells are implanted into collagen/chitosan porous scaffolds.

Animals ; Chitin ; analogs & derivatives ; Chitosan ; Collagen ; Hair Follicle ; cytology ; Mice ; Mice, Inbred C57BL ; Regeneration ; Skin ; cytology ; Tissue Engineering

In this work a system which consists of chitosan (CS) microcores entrapped within enteric polymer is presented. Vitamin D2, used as a model drug, was efficiently entrapped within CS microcores using spray-drying and then microencapsulated into ethylic cellulose(EC). The morphology and release properties of microcapsules were tested. The influential factors of preparation conditions included molecular weight of chitosan, concentration of chitosan solution, concentration of acetic acid, loading of vitamin D2 were discussed. The results of in vitro release studies showed that the microcapsules prepared in this article could realize sustained release in intestine.
Capsules ; Cellulose ; analogs & derivatives ; pharmacology ; Chitin ; analogs & derivatives ; pharmacology ; Chitosan ; Delayed-Action Preparations ; Drug Compounding ; Drug Delivery Systems ; Ergocalciferols ; pharmacology ; In Vitro Techniques

Objective proteins synthesized from genetically recombined Escherichia coli strain (E. coli) have been successfully produced by microbe fermentation, but complicated separation and purification steps always make against the maintenance of activities as well as increase the cost. Aiming at simplifying the process, an idea of administrating directly the microencapsulated genetically recombined E. coli is proposed. In this paper, study on culture of E. coli DH5 alpha immobilized in alginate/chitosan (ACA) microcapsule is presented. It was found that E. coli DH5 alpha grew well in the microcapsule with stable growth period longer than that of suspension culture, and cell aggregation phenomenon was observed. In vivo experiments showed that ACA microcapsules with E. coli DH5 alpha stayed over 48 h in mouse intestine, and the morphology of microcapsules was kept intact. These preliminary results have demonstrated that administration of microencapsulated E. coli DH5 alpha is safe, which laied the foundation for microencapsulated genetically recombined E. coli as carriers of gene engineering drugs.
Alginates ; Animals ; Capsules ; Chitin ; analogs & derivatives ; Chitosan ; Drug Delivery Systems ; Escherichia coli ; growth & development ; physiology ; Gene Transfer Techniques ; Glucuronic Acid ; Hexuronic Acids ; Mice

AIMTo investigate the formation mechanism, macromolecular drug loading capacity and release property of alginate-chitosan microcapsules (ACM).

METHODSACM was prepared by emulsification-gelation method and its formation mechanism was studied by DSC analysis. Using bovine serum albumin (BSA) as model drug, the drug loading and release properties of the microcapsules on macromolecular drug were investigated.

RESULTSThe results of DSC analysis showed that there is electrostatic interaction between materials encapsulated in the microcapsule. With the increase of BSA microcapsule ratio, the BSA loading percentage rose from 9.20% to 35.08%; and with the ascent of chitosan (CTS) concentration, the BSA loading percentage increased from 30.29% to 38.12%. The BSA microcapsules whowed a two-phase release in both 0.1 mol.L-1 HCl and phosphate buffer saline (pH 7.4). With the increase of CTS concentration, the BSA release more and more slowly in 0.1 mol.L-1 HCl.

CONCLUSIONSpheric and well-dispersed alginate-chitosan microcapsules were prepared. The microcapsule showed good loading capacity to BSA as well as sustained release to a certain degree.

Alginates ; chemistry ; Biopolymers ; Calorimetry, Differential Scanning ; Capsules ; Chitin ; analogs & derivatives ; chemistry ; Chitosan ; Delayed-Action Preparations ; Drug Carriers ; Drug Delivery Systems ; Serum Albumin, Bovine ; administration & dosage ; Technology, Pharmaceutical ; methods

Animals ; Cations ; chemistry ; Chitin ; administration & dosage ; analogs & derivatives ; chemistry ; Chitosan ; DNA ; administration & dosage ; Gene Transfer Techniques ; Genetic Therapy ; Liposomes ; administration & dosage ; chemistry ; Polylysine ; administration & dosage ; chemistry

OBJECTIVESTo investigate the effect of chitosan biodegradable external stent (CES) on the early changes of rabbit vein graft (VG).

METHODSRabbit vein grafting models were divided into S group (with perivenous CES) and NS group (without perivenous CES). The VG were harvested in 1 week, 2 weeks, 4 weeks after operation, respectively. The expression of proliferating cell nuclear antigen (PCNA) was used for evaluating the proliferation of the smooth muscle cell (SMC). The thickness, area of neointima and media of the VG were calculated by computer imaging analysis system.

RESULTSCES began to degrade in 2 weeks after operation. The thickness, area of both neointima and media of the VG in S group, increased mildly in 1 week after operation, and kept steady in 1 or 2 weeks after grafting, which was significantly less than NS group (both P < 0.01), then increased mildly in 4 weeks after grafting but still less than NS group (P < 0.05). The expression of PCNA of SMC decreased significantly in comparison with NS group though increasing mildly in four weeks after operation. Both neointimal formation and cell proliferation in the graft wall were significantly reduced by external stenting as compared to the results with unstented grafts.

CONCLUSIONSCES may reduce early intimal and medial hyperplasia, and may be beneficial in improving the long term patency of the VG. The biodegradable characteristics of the CES may influence its effect.

Animals ; Biodegradation, Environmental ; Chitin ; administration & dosage ; analogs & derivatives ; Chitosan ; Female ; Humans ; Hyperplasia ; Male ; Proliferating Cell Nuclear Antigen ; analysis ; Rabbits ; Stents ; Tunica Intima ; pathology ; Veins ; pathology ; transplantation