Production of fructosylxyloside by the new fructosyltransferase from Bacillus macerans EG-6

The new fructosyltransferase (FTase) from Bacillus maceransEG-6 showed a broad acceptor specificity, and resulted in the formation of fructosylxyloside (FX) with d-xylose being the most effective acceptor. The optimal FTase concentration for FX production was 0.6 unit per g sucrose, which gave the highest transfer ratio, 83%, of fructosyl moiety from sucrose to d-xylose. Maximum yield of FX was 114 g l^–1with 200 g sucrose l^–1and 300 g d-xylose l^–1.     

Catabolism of protocatechuate by Bacillus macerans.

An aerobic endospore-forming bacterium, tentatively identified as a strain (JJ-lb) of Bacillus macerans, was isolated by enrichment on 4-hydroxybenzoate (4HBA), using as an inoculum soil taken from a 50 degrees C Iadho hot spring. Enzymatic analyses of cells grown on succinate and 4HBA indicated that strain JJ-1b degrades 4HBA by way of the novel protocatechuate (PCA) 2,3-dioxygenase pathway. Purification of the PCA 2,3-dioxygenase by affinity chromatography allowed the first observation of the immediate ring fission product of PCA, namely, 5-carboxy-2-hydroxymuconic semialdehyde (CHMS; labmda max at pH 7.0 = 348 nm). An affinity column fraction was obtained that decarboxylated CHMS to 2-hydroxymuconic semialdehyde (HMS; lambdamax at pH 7.0 = 375 nm). Thus, conversion of PCA to HMS is accomplished in two steps, 2,3-fission of the PCA ring followed by enzymatic decarboxylation of the ring fission product, forming HMS.     

Catabolism of protocatechuate by Bacillus macerans.

An aerobic endospore-forming bacterium, tentatively identified as a strain (JJ-lb) of Bacillus macerans, was isolated by enrichment on 4-hydroxybenzoate (4HBA), using as an inoculum soil taken from a 50 degrees C Iadho hot spring. Enzymatic analyses of cells grown on succinate and 4HBA indicated that strain JJ-1b degrades 4HBA by way of the novel protocatechuate (PCA) 2,3-dioxygenase pathway. Purification of the PCA 2,3-dioxygenase by affinity chromatography allowed the first observation of the immediate ring fission product of PCA, namely, 5-carboxy-2-hydroxymuconic semialdehyde (CHMS; labmda max at pH 7.0 = 348 nm). An affinity column fraction was obtained that decarboxylated CHMS to 2-hydroxymuconic semialdehyde (HMS; lambdamax at pH 7.0 = 375 nm). Thus, conversion of PCA to HMS is accomplished in two steps, 2,3-fission of the PCA ring followed by enzymatic decarboxylation of the ring fission product, forming HMS.     

New cyclomaltodextrin-glucanotransferases, produced by Bacillus macerans

For the first time, microorganisms producing cyclomaltodextrin glucan transferases (CGT, EC 2.4.1.19) were isolated from soil samples of various ecogeographical regions. These microorganisms were identified as Bacillus macerans. The enzymes were purified by affinity chromatography on a alpha-cyclodextrin polymer and gel filtration on Biogel P-450 and proved to be electrophoretically homogeneous. Some of their physicochemical and biochemical properties are reported.     

Production of a novel transfructosylating enzyme from Bacillus macerans EG-6

A new bacterium producing a novel transfructosylating enzyme was isolated from soil and designated as Bacillus macerans EG-6. Various culture conditions for enzyme production were optimized in a flask culture. 1% (w/v) sucrose as a carbon source and a mixed nitrogen source (1% yeast extract, 1% polypeptone, and 0.5% ammonium chloride) gave the best enzyme production. Addition of phosphate and magnesium ion into the medium enhanced the enzyme yield. Optimum culture pH and temperature were 7.0 and 37?°C, respectively. Under optimal culture conditions, transfructosylating enzyme was rapidly produced in the early growth period, thereafter invertase activity was predominant as the culture proceeded. Using the culture filtrate, production of fructooligosaccharides from sucrose was preliminarily carried out. In a low sucrose concentration (200?g/l), transfructosylating activity competes with invertase activity in sucrose utilization. Subsequently, low fructooligosaccharide yield (20%) was achieved due to liberation of high amounts of glucose and fructose. The best oligosaccharide yield (43%) was achieved when 500?g/l sucrose was utilized.     

Germination requirements of Bacillus macerans spores

2-Phenylacetamide is an effective germinant for spores of five strains of Bacillus macerans, particularly in the presence of fructose. Benzyl penicillin, the phenyl acetamide derivative of penicillin, and phenylacetic acid are also good germinants. l-Asparagine is an excellent germinant for four strains. alpha-Amino-butyric acid is moderately effective. Pyridoxine, pyridoxal, adenine, and 2,6-diaminopurine are potent germinants for NCA strain 7X1 only. d-Glucose is a powerful germinant for strain B-70 only. d-Fructose and d-ribose strongly potentiate germination induced by other germinants (except l-asparagine) but have only weak activity by themselves. Niacinamide and nicotinamide-adenine dinucleotide, inactive by themselves, are active in the presence of fructose or ribose. Effects of pH, ion concentration, and temperature are described.     

Molecular characterization of cycloinulooligosaccharide fructanotransferase from Bacillus macerans

Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cyclooligosaccharides of beta-(2-->1)-linked D-fructofuranose by catalyzing an intramolecular transfructosylation reaction. The CFTase gene was cloned and characterized from Bacillus macerans CFC1. The CFTase gene encoded a polypeptide of 1,333 amino acids with a calculated Mr of 149,563. Western blot and zymography analyses revealed that the CFTase with a molecular mass of 150 kDa (CFT150) was processed (between Ser389 and Phe390 residue) to form a 107-kDa protein (CFT107) in the B. macerans CFC1 cells. The processed CFT107 was similar in its mass to the previously purified CFTase from B. macerans CFC1. The CFT107 enzyme was produced by B. macerans CFC1 but was not detected from the recombinant Escherichia coli cells, indicating that the processing event occurred in a host-specific manner. The two CFTases (CFT150 and CFT107) exhibited the same enzymatic properties, such as influences of pH and temperature on the enzyme activity, the intermolecular transfructosylation ability, and the ability of hydrolysis of cycloinulooligosaccharides produced by the cyclization reaction. However, the thermal stability of CFT107 was slightly higher than that of CFT150. The most striking difference between the two enzymes was observed in their Km values; the value for CFT150 (1.56 mM) was threefold lower than that for CFT107 (4.76 mM). Thus, the specificity constant (kcat/Km) of CFT150 was about fourfold higher than that of CFT107. These results indicated that the N-terminal 358-residue region of CFT150 played a role in increasing the enzyme's binding affinity to the inulin substrate.     

Monoclonal antibodies to cyclodextrin-glycosyltransferase from Bacillus macerans]

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was isolated from the cell-free culture medium of Bacillus macerans strain BKMB-506. The purified enzyme was used to immunize BALB/c mice. Polyclonal antibodies (PAbs) and 15 hybridoma cells producing monoclonal antibodies (MAbs) against CGTase were obtained. The properties of MAbs were studied using ELISA and immunoblotting. A method for detecting small amounts of CGTase was developed. The interactions of PAbs and MAbs with CGTase of Bac. circulans var. alkalophilis were investigated.     

Modified Two-Phase System for Partition of Bacillus macerans Spores

An aqueous two-phase system made with polyethylene glycol (PEG) 1000 and potassium phosphate gives much higher recovery of Bacillus macerans spores in the upper phase (PEG rich) than does a similar system utilizing PEG 4000. The upper phase completely rejects vegetative cells, which collect at the interface. The system may be useful in purifying spores of other species. Scanning electron micrographs of B. macerans spores cleaned in this system show no obvious attached sporangial fragments. The micrographs show that the ridged coat may form polygonal structures at the poles, as previously observed in Bacillus polymyxa spores.     

Effect of PEG and other additives on cyclodextrin production by Bacillus macerans cyclomaltodextrin-glycosyl-transferase

Summary The effect of PEG and other polyols additives on cyclodextrins (CDs) production by Bacillus macerons cyclomaltodextrin-glycosyl-transferase (CGTase) was investigated. Mannitol, glycerol and PEG-200 (20%,v/v) enhanced the enzymatic production yield regardless of substrate concentration. Furthermore, the PEG-200 addition increased the thermostability of the CGTase.     

Expression of Bacillus macerans cyclodextrin glucanotransferase gene in Saccharomyces cerevisiae

Cyclodextrin glucanotransferase (CGTase) gene of Bacillus macerans was subcloned down-stream of yeast ADH1 promoter and expressed in Saccharomyces cerevisiae. Most of the CGTase expressed was in the extracellular medium with a maximum activity of about 0.28 unit ml^–1 after 48 h cultivation. The recombinant CGTase was secreted as an N-linked-glycosylated form and predominantly produced -cyclodextrin from starch.