Mitsuaria chitosanitabida gen. nov., sp. nov., an aerobic, chitosanase-producing member of the 'Betaproteobacteria'.

Four strains (3001(T), 2, 12 and 13), which were isolated as chitosanase-producing bacteria from soil from Matsue city (Japan), were studied phenotypically, genotypically and phylogenetically. Based on sequence analysis of 16S rRNA genes, DNA G+C content (67.4-69.2 mol%), quinone type (UQ-8), major fatty acid composition (3-OH 10:0, 3-OH 14:0) and other phylogenetic studies, strains 3001(T), 12 and 13 were found to occupy a separate position in the 'Betaproteobacteria'. Roseateles depolymerans, Rubrivivax gelatinosus and Ideonella dechloratans were their closest neighbours (93-95% 16S rRNA gene sequence similarity). The 16S rRNA gene sequence and other characteristics suggested that strain 2 belonged to the genus Flavobacterium. DNA-DNA hybridization experiments supported the conclusion that strains 3001(T), 12 and 13 were of the same species (72-78% DNA hybridization) and only distantly related to I. dechloratans and R. gelatinosus. It is proposed that strains 3001(T), 12 and 13 represent a novel genus and species for which the name Mitsuaria chitosanitabida gen. nov., sp. nov. is proposed. The type strain of Mitsuaria chitosanitabida is 3001(T) (=IAM 14711(T)=ATCC BAA-476(T)).

The new BMI criteria for asians by the regional office for the western pacific region of WHO are suitable for screening of overweight to prevent metabolic syndrome in elder Japanese workers.

Obesity occurs less frequently in Japanese than in various other ethnic populations. A person with abnormal glucose tolerance is often found to have one or more of the other cardiovascular disease risk factors, such as obesity, hypertension and hyperlipidemia. This clustering has been labeled as metabolic syndrome (WHO, 1998). It was suggested that Japanese, categorized as having normal weight (BMI of less than 25.0), as defined by the WHO (2000), have an increasing tendency toward metabolic syndrome. Our objective was to analyze metabolic syndrome in "Overweight" with BMI of 23.0-24.9 in Japanese workers, and to assess the suitability for Asians of the Regional Office for the Western Pacific Region of WHO criteria pertaining to obesity (WPRO criteria, 2000). We conducted a cross-sectional study in the workplace setting and investigated the relationship between BMI classification based on WPRO criteria and metabolic syndrome by gender and age group (18-44 yr vs. 45-60 yr). Three hundred seventy-nine men and 432 women Japanese workers participated in this study. BMI were categorized as 20% "Overweight" (23.0-24.9 BMI), 20% "Obese I" (25.0-29.9 BMI) and 2% "Obese II" (over 30.0 BMI), based on WPRO criteria. Graded increases in BMI were positively associated with body fat percentage, waist circumference, hip circumference and waist/hip ratio in both genders and age groups. A progressively increasing BMI category in the elder group aged 45-60 yr in both genders was positively related with parameters constituting metabolic syndrome. Graded increases in BMI classes in elder workers based on WPRO criteria were positively associated with prevalence of metabolic syndrome, and "Overweight" elder women had significantly higher prevalence of metabolic syndrome. The present investigation, based on the increasing risks of "Overweight" with a BMI of 23.0-24.9, suggests that WPRO criteria are suitable for Japanese workers aged over 45 yr.

Functional expression of chitinase and chitosanase, and their effects on morphologies in the yeast Schizosaccharomyces pombe.

The chitinase A (chiA) gene from Enterobacter sp. G-1 and the chitosanase A (choA) gene from Matsuebacter chitosanotabidus 3001 were expressed separately and simultaneously in the yeast Schizosaccharomyces pombe. The chiA gene was placed under the transcriptional control of the nmt1 promoter with the glutathione S transferase (GST), and the choA gene was expressed by the human cytomegalovirus (hCMV) promoter with the P factor secretion signal (P3). The expressed proteins of ChoA and GST-ChiA were enzymatical active and were detected as 34-kDa and 80-kDa, respectively, by Western blot analysis. The transformant that expressed the choA gene was able to secrete ChoA efficiently into the culture medium with the specific activity of 102.36 U/mg protein. When the chiA gene was expressed in S. pombe, yeast cells grew slowly and cells became elongated, but when the choA gene was expressed, cells became swollen. Expression of both the chiA and the choA genes resulted in elongated and fat cells.

Two glutamic acids in chitosanase A from Matsuebacter chitosanotabidus 3001 are the catalytically important residues.

Chitosanase is the glycolytic enzyme that hydrolyzes the glucosamine GlcN-GlcN bonds of chitosan. To determine the catalytically important residues of chitosanase A (ChoA) from Matsuebacter chitosanotabidus 3001, we performed both site-directed and random mutagenesis of choA, obtaining 31 mutants. These mutations indicated that Glu-121 and Glu-141 were catalytically important residues, as mutation at these sites to Ala or Asp drastically decreased the enzymatic activity to 0.1-0.3% of that of the wild type enzyme. Glu-141 mutations remarkably decreased kinetic constant k(cat) for hydrolysis of chitosan, meanwhile Glu-121 mutations decreased the activities to undeterminable levels, precluding parameter analysis. No hydrolysis of (GlcN)(6) was observed with the purified Glu-121 mutant and extremely slow hydrolysis with the Glu-141 mutant. We also found that Asp-139, Asp-148, Arg-150, Gly-151, Asp-164, and Gly-280 were important residues for enzymatic activities, although they are not directly involved in catalysis. In addition, mutation of any of the six cysteine residues of ChoA abrogated the enzymatic activity, and Cys-136 and Cys-231 were found to form a disulfide bond. In support of the significance of the disulfide bond of ChoA, chitosanase activity was impaired on incubation with a reducing agent. Thus, ChoA from M. chitosanotabidus 3001 uses two glutamic acid residues as putative catalytic residues and has at least one disulfide bond.