Yasuyuki Takiguchi

Preparation of crustacean chitin

Publisher Summary This chapter describes the isolation of chitin from crustacean cuticles (shells). To isolate chitin from crustacean shells, the following three steps are required: (1) demineralization with dilute hydrochloric acid or with ethylenediaminetetraacetic acid (EDTA), (2) deproteinization with aqueous sodium hydroxide or by use of the proteolytic activity of a bacterium, and (3) elimination of lipids with organic solvent(s). The chapter tabulates composition and molecular weight of various chitin preparations from abdominal shell of Penaeus japonicus (kuruma prawn). The chapter also describes the preparation of colloidal chitin. The procedure consists of the dissolution of chitin into and the reprecipitation from concentrated hydrochloric acid and the dispersion of the reprecipitated chitin into water. Colloidal chitin serves as a substrate of chitinase and a carbon and nitrogen source of chitinolytic microorganisms.

Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u

Four kinds of thermostable chitinase were isolated from the cell-free culture broth of Bacillus licheniformis X-7u by successive column chromatographies on Butyl-Toyopearl, Q-Sepharose, and Sephacryl S-200. We named the enzymes chitinases I(89 kDa), II(76 kDa), III(66 kDa) and IV(59 kDa). Chitinases II, III and IV possessed extremely high optimum temperatures (70-80 degrees C), showing remarkable heat stability. Chitinases II, III and IV produced (GlcNAc)2 and GlcNAc from colloidal chitin and chitinase I predominantly produced (GlcNAc)2. The action pattern of chitinase I on PN-(GlcNAc)4 also showed a stronger propensity to cleave off the (GlcNAc)2 unit from the non-reducing end than the other three chitinases. Chitinases II, III and IV catalyzed a transglycosylation reaction that converted (GlcNAc)4 into (GlcNAc)6.

Peracetylated chitobiose: Preparation by specific degradations of chitin, and chemical manipulations

Abstract Chitobiose octaacetate (3) was preparable in moderate yield from chitin by microbial degradation followed by acetylation, or by modified chemical degradation. Compound 3 was chemically manipulated to give various compounds, including an oxazoline derivative, glycosides, and partially O-benzylated derivatives. The conformation of the oxazoline derivative is discussed.

Evaluation of the Effectiveness and Safety of Chitosan Derivatives as Adjuvants for Intranasal Vaccines

Intranasal immunization is currently used to deliver live virus vaccines such as influenza. However, to develop an intranasal vaccine to deliver inactivated virus, a safe and effective adjuvant is necessary to enhance the mucosal immune response. Here, we demonstrate the effectiveness of a chitosan microparticle (1-20 μm, 50 kDa, degree of deacetylation=85%) and a cationized chitosan (1000 kDa, degree of deacetylation=85%) derived from natural crab shells as adjuvants for an intranasal vaccine candidate. We examined the effectiveness of chitosan derivatives as an adjuvant by co-administering them with ovalbumin (OVA) intranasally in BALB/c mice, polymeric Ig receptor knockout (pIgR-KO) mice, and cynomolgus monkeys (Macaca fascicularis). pIgR-KO mice were used to evaluate S-IgA production on the mucosal surface without nasal swab collection. Administration of OVA with chitosan microparticles or cationized chitosan induced a high OVA-specific IgA response in the serum of pIgR-KO mice and a high IgG response in the serum of BALB/c mice and cynomolgus monkeys. We also found that administration of chitosan derivatives did not have a detrimental effect on cynomolgus monkeys as determined by complete blood count, blood chemistries, and gross pathology results. These results suggest that chitosan derivatives are safe and effective mucosal adjuvants for intranasal vaccination.

CHEMICAL COMPOSITION AND SOME PROPERTIES OF CRUSTACEAN CHITIN PREPARED BY USE OF PROTEOLYTIC ACTIVITY OF PSEUDOMONAS MALTOPHILIA LC102

A proteolytic bacterium Pseudomonas maltophilia LC102 was cultured at 30 °C in a medium consisted of K 2 HPO 4 and Penaeus japonicus cuticle previously decalcified in 0.1 M EDTA solution at pH 7.5. Protein content of the cuticle decreased from initial 25.4% to 0.7% within 24 hr. After this period, the velocity of deproteinization diminished markedly and complete removal of the protein was not attained. Protein content of the cuticle from 7-day culture was 0.3%. Decalcified cuticles of other Decapoda species , Panulirus japonicus, Paralithodes camtschatecus, Chionoecetes opilio, and Porutunus trituber-culatus, were more slowly deproteinized. Their cuticles from 7-day cultures contained protein in the range from 3 to 5%.