Mannanase (endo 1,4 D mannanase, mannan endo 1,4 mannosidase; EC 3.2.1.78) is a hydrolase that catalyzes the endo type hydrolysis of 1,4 mannosidic linkages in the main chain of naturally occurring mannan and heteromannans, such as locust bean gum, konjak mannan, guar gum, and ivory nut. 1) Many mannanases have been found from various origins, and they are grouped into glycoside hydrolase (GH) families 5 and 26 (http: afmb.cnrs mrs. fr CAZY ).Mannanases have many possible applications, for instance, for use in kraft pulp bleaching, reduction of viscosity of instant coffee, and clarification of fruit juices and wines. 2) An alkaline mannanase was found by Horikoshi and his colleagues from alkaliphilic Bacillus sp. strain AM 001 for the first time. 3,4) And then an alkalitolerant enzyme was found in Bacillus stearothermophilus. 5) Recently, alkaline mannanases were found to remove gum stains of cosmetics and or foods and thus be effective additives for laundry detergents (WO 99 09128 WO 99 09133). Therefore, alkaline mannanase is a promising enzyme and can be used widely, especially in the detergent industry as well as in the paper industry.There is little information on the genetics, physicochemical, and catalytic properties of alkaline mannanases.In this report, we describe the cloning and sequencing of the gene for an alkaline mannanase (amn5A) produced by a novel species of an alkaliphilic Bacillus (strain JAMB 602) isolated from a sample of soils. Moreover, we show that the recombinant enzyme (Ramn5A) could be hyper exoproduced using a proteases deficient Bacillus subtilis as the host and clarify its physicochemical and catalytic properties. MATERIALS AND METHODS Materials.Unless otherwise stated, all chemicals used were from Wako Pure Chemical. N Bromosuccinimide (NBS) was obtained from Sigma. Iodoacetate, iodoacetamide, and 1 ethyl 3 (3 dimethyl aminopropyl)carbonate were obtained from Nacalai Tesque. Dithiothreitol was purchased from Amersham Biosciences. Coomassie Brilliant Blue R 250 (CBB), SDS, EDTA and EGTA are the products of Bio Rad. D Mannose (M1; Sigma), locust bean gum ( Sigma ) , ivory nut ( Megazyme ) , konjak mannan (Kanto Chemical) and guar gum (Sigma) were obtained from the manufacturers in parentheses. Manno oligosaccharides mannobiose (M2) through mannohexaose (M6) were the products of Megazyme.Bacterial strains and culture conditions. Strain JAMB 602 was one of the stock cultures in this laboratory as a mannanase (Amn5A) producer, isolated from a soil sample. It was grown at 30 C for several days and isolated on 1.5% (w v) agar containing 0.2% locust bean gum, 0.5% Polypepton S (Nihon Seiyaku), 0.5% yeast extract (Difco), 0.1% K2HPO4, 0.02% MgSO4 7H2O and 0.5% Na2CO3 (separately autoclaved). Escherichia coli HB101 (F supE44 hsdS20 recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 leuB6 thi-1) was used as the host for cloning and routinely grown at 37 C in Luria Bertani (LB) broth (Difco) supplemented with 100 g Abstract: A novel species of alkaliphilic Bacillus (JAMB-602), isolated from a ...

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