The amylopullulanase of Bacillus sp. DSM 405.

The amylopullulanse produced by Bacillus sp. DSM 405 was purified to homogeneity. It exhibited dual activity, cleaving the alpha 1-4 bonds in starch, releasing a range of malto-oligosaccharides, and also cleaving the alpha 1-6 bonds in pullulan, releasing maltotriose as the sole end-product. The enzyme was a glycoprotein and had a relative molecular mass of 126,000 and an isoelectric point of 4.3. While the enzyme was optimally active on starch at pH 6.5 and at pH 6.0 on pullulan, activity on both substrates was maximal at 70 degrees C. Kinetic analyses of the enzyme in a system that contained both starch and pullulan as two competing substrates demonstrated the dual specificity of the enzyme. Chemical modification of the carboxyl groups within the active centre of the protein showed that one active site was responsible for hydrolysis of the alpha 1-4 and alpha 1-6 bonds in starch and pullulan respectively. This is the first comprehensive investigation of an amylopullulanse produced by an aerobic bacterium, showing a single active site responsible for both activities.

Effect of growth rate on alpha-amylase production by Streptomyces sp. IMD 2679.

The alpha-amylase of Streptomyces sp. IMD 2679 was subject to catabolite repression. Four different growth rates were achieved when the organism was grown at 40 degrees C and 55 degrees C in the presence and absence of cobalt, with an inverse relationship between alpha-amylase production and growth rate. Highest alpha-amylase yields (520 units/ml) were obtained at the lowest growth rate (0.062 h-1), at 40 degrees C in the absence of cobalt, while at the highest growth rate (0.35 h-1), at 55 degrees C in the presence of cobalt, alpha-amylase production was decreased to 150 units/ml. As growth rate increased, the rate of specific utilisation of the carbon source maltose also increased, from 46 to 123 micrograms maltose (mg biomass)-1 h-1. The pattern and levels of alpha-glucosidase (the enzyme degrading maltose) detected intracellularly in each case, indicate that growth rate effectively controls the rate of feeding of glucose to the cell, and thus catabolite repression.

The high maltose-producing alpha-amylase of the thermophilic actinomycete, Thermomonospora curvata.

The alpha-amylase of Thermomonospora curvata catalyses the formation of very high levels of maltose from starch (73%, w/w) without the attendant production of glucose. The enzyme was produced extracellularly in high yield during batch fermentation in a 5-1 fermentor. Purification was achieved by ammonium sulphate fractionation, Superose-12 gel filtration and DEAE-Sephacel ion-exchange chromatography. The enzyme exhibited maxima for activity at pH 6.0 and 65 degrees C, had a relative molecular mass of 60,900-62,000 and an isoelectric point at 6.2. The exceptionally high levels of maltose produced and the unique action pattern exhibited on starch and related substrates indicate a very unusual maltogenic system. The predominance of maltose as the final end-product may be explained by the participation of reactions other than simple hydrolysis and the preferential cleavage of maltotriose from higher maltooligosaccharides. The enzyme exhibits very low affinity for maltotriose (Km = 7.7 x 10(-3) M) and its conversion to maltose is achieved by synthetic followed by hydrolytic events, which result in the very high levels of maltose observed and preclude glucose formation. This system is distinguished from other very high maltose-producing amylases by virtue of its high temperature maximum, very low affinity for maltotriose and the absence of glucose in the final saccharide mixture.

Investigation of the mechanisms of irreversible thermoinactivation of Bacillus stearothermophilus alpha-amylase.

Bacillus stearothermophilus NCIB 11412 produces a highly thermostable alpha-amylase. The enzyme displayed half-lives of irreversible thermoinactivation at 90 degrees C of 1.9 min and 12.5 min at pH 5.0 and pH 8.0, respectively. Molecular mechanisms of irreversible thermoinactivation were investigated. At both pH 5.0 and pH 8.0 irreversible thermoinactivation was due to heat-induced breakdown of non-covalent interaction within the protein molecule, resulting in unfolding and consequent formation of altered structures. Hydrophobic interactions were shown to be the most important non-covalent mechanisms involved in this phenomenon. Although not dramatically effecting the rates of irreversible thermoinactivation, electrostatic interactions, including hydrogen bonding, were also shown to have a contributory role in this process. At pH 8.0 a covalent mechanism, that of oxidation of thiols was also shown to be of secondary importance to hydrophobic interactions in the irreversible thermoinactivation of this enzyme.

Dialysis culture for the production of Pseudomonas saccharophila alpha-amylase.

Growth and alpha-amylase synthesis of Pseudomonas saccharophila was shown to be inhibited by the accumulation of a mixture of nonvolatile fatty acids during nondialysis cultivation. Using dialysis culture a 9-fold increase in the level of biomass and an 8.5-fold increase in alpha-amylase yield was achieved.

Production and properties of polygalacturonate lyase by an alkalophilic microorganism Bacillus sp. RK9.

Bacillus sp. RK9 was isolated from soil and produced a constitutive polygalacturonate lyase. Production of the enzyme required the presence of complex nitrogen (peptone and yeast extract). Highest activity was obtained with an initial pH of 9.7. The organism was alkalophilic. No growth occurred below pH 7.5. The enzyme was purified by salt precipitation and diethylaminoethyl (DEAE) cellulose ion-exchange chromatography. The pH optimum for activity was 10.0 in 0.01 M glycine-NaOH buffer. Calcium alone, of divalent cations, activated the enzyme by 2.9-fold. Complete inhibition of enzyme activity was achieved by 1 mM ethylenediaminetetraacetic acid (EDTA). Hydrolysis of substrate occurred in a random fashion and the enzyme was 50% more active towards acid soluble pectic acid (ASPA) than towards sodium polypectate.

Production and Purification of Thermostable Amylase and Protease of Thermomonospora viridis.

Maximum yields of amylase were produced by the thermophilic actinomycete Thermomonospora viridis in a modified Simpson and McCoy medium containing 1.5% corn starch and 0.5% mycological peptone with an initial pH 7.0. Best yields of amylase were obtained after incubation for 48 h, when the pH of the medium had risen to 8.2. Amylase was purified 313-fold by precipitation with n-propyl alcohol, dialysis against tap water, adsorption on Ca(3)(PO(4))(2), and fractionation on Sephadex G-100. Protease was produced in nutrient broth containing 0.5% starch and 1.0% corn steep liquor and at an initial pH 7.0. Maximum yields of protease were produced after 42 h. The protease was purified 54-fold by precipitation with n-propyl alcohol, dialysis against tap water, adsorption on Ca(3)(PO(4))(2), and fractionation on Sephadex G-200.

Polygalacturonate lyase production by Bacillus subtilis and Flavobacterium pectinovorum.

Nutritional factors relating to the production of polygalacturonate lyases by strains of Bacillus subtilis and Flavobacterium pectinovorum were examined. Studies were carried out in shake flask cultures. In the case of B. subtilis the enzyme was produced constitutively, whereas in the case of F. pectinovorum it was only produced in quantity in the presence of pectic substances. Glucose was the most suitable carbon source for production of the polygalacturonate lyase of B. subtilis; of the nitrogen sources examined, the highest activities per milliliter of supernatant and per milligram of cells were obtained with glutamine and ammonium sulfate, respectively. The pattern of enzyme production and growth was similar although enzyme production ceased at pH 5.3. Sodium polypectate was the best inducer of polygalacturonate lyase with F. pectinovorum. Highest activity per milliliter of cell-free supernatant was obtained with skin milk powder as nitrogen source, although ammonium sulfate gave highest enzyme production per unit of biomass. Growth of F. pectinovorum occurred between pH 5.7 and 7.2. Enzyme production occurred during active growth and was independent of the pH of the medium.