Catherine T. Kelly

Recent Advances in Microbial Amylases

A wide variety of microorganisms produce, and in most cases secrete extracellularly, amylases having different specificities and some rather interesting properties (Fogarty & Kelly, 1980; Fogarty, 1983). Gram-positive bacteria, and particularly the genus Bacillus, are prolific producers of amylases, although very few are found among Gram-negative bacteria. A wide range of moulds produce amylases, particularly the genus Aspergillus. In recent years interest and awareness in the amylolytic activities of yeasts has generated a number of studies of these systems. It is not the purpose of this review to provide an exhaustive or detailed treatise of microbial amylases. Other publications have dealt with the subject area extensively (Fogarty & Kelly, 1979, 1980; Fogarty, 1983) and this work has as its aim an update of developments which have taken place in the area in recent years.

The alkaline amylase of the alkalophilic Bacillus sp. IMD 370

A novel extracellular amylase of alkalophilic Bacillus sp. IMD 370 was purified to homogeneity and displayed maxima for activity at pH 10.0 and 40°C. It had an isoelectric point of 4.9 and a relative molecular mass of 159,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was inhibited (47%) by ethylenediaminetetraacetic acid (1 mM). The α-amylase of Bacillus sp. IMD 370 was quite distinct by virtue of its mechanism of action and its inability to fit into the conventional classification scheme of amylolytic enzymes. It had a mode of action intermediate between an endo-acting enzyme, α-amylase, and a typical exo-acting enzyme, amyloglucosidase. The sugars produced on hydrolysis of starch had the α-configuration, but no oligosaccharide higher than maltotetraose was obtained at any stage during starch hydrolysis.

Thermostable extracellular α-amylase and α-glucosidase ofLipomyces starkeyi

SummaryThermostable, extracellular α-amylase and α-glucosidase were produced byLipomyces starkeyi CBS 1809 in a medium containing maize starch and soya bean meal. Contrary to published findings which suggested a single cell-bound amylolytic system for another strain ofL. starkeyi, this study revealed the presence of two enzymes — an α-amylase and an α-glucosidase inL. starkeyi CBS 1809. The enzymes were separated by solvent and salt precipitation and ion-exchange chromatography on DEAE-Biogel-A. The α-amylase and α-glucosidase had pH optima at 4.0 and 4.5 and temperature optima at 70°C and 60°C, respectively. While the low pH optima are not unique the enzymes are very distinctive in yeasts in having very high temperature optima. The α-glucosidase had highest activities on maltose and isomaltose (100) with relative rates of activity on maltotriose, isomaltotriose and p-nitrophenyl-α-d-glucoside of 59, 48 and 22, respectively. It was inactive towards sucrose. Both the α-amylase and α-glucosidase ofL. starkeyi were located extracellularly and had molecular weights of 76,000 and 35,000, respectively.

Production studies on the alkaline amylases of three alkalophilic Bacillus spp.

SummaryBacillus alcalophilus subsp. halodurans ATCC 21591, Bacillus sp. NCIB 11203 and Bacillus sp. IMD370 are obligate alkalophiles and produce alkaline α-amylases with maxima for activity at pH 10.0. All three organisms yielded maximum amylase activity in a medium containing starch as a carbon source and yeatex as a nitrogen source with an initial pH 10.0.

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of β-cyclodextrin (17.5 mM).

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

The α-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 ionexchange chromatography. The enzyme exhibited maxima for activity at pH 6.0 and 65°C, had a relative molecular mass of 60900–62000 and an isoelecric 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 × 10−3m) 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.

Purification and characterization of the α-amylase of Bacillus flavothermus

Abstract Bacillus flavothermus α-amylase was purified to homogeneity using a combination of ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The enzyme displayed maximal activity on starch at pH 5.5–6.0 and 60°C and had an isoelectric point of 8.4 and a Km of 2.2 mg ml−1. Diethyl pyrocarbonate inactivated the amylase at pH 6.6 and 20°C in a monomolecular reaction with a second-order rate constant of 250 m −1 min−1. The influence of pH on the rate of inactivation suggested the participation of a residue with a pKa of 6.7. Spectrophotometric studies and reactivation in the presence of hydroxylamine suggested the modification of histidine(s). A single histidine residue appeared to be essential and the substrate afforded complete protection indicating its location at the active site of the enzyme.

The isomaltulose synthesising enzyme ofSerratiaplymuthica

SummaryAn intracellular enzyme was located inSerratiaplymuthica which produced isomaltulose from sucrose. The enzyme was purified giving a preparation with a specific activity of 1,285. It has pH and temperature optima of 6.0 and 30°C, respectively. The enzyme was stable retaining 100% activity after 2 weeks at 30°C. It had an isoelectric point at pH 9.0, a Mr of 79,500 and the Km for sucrose was 65.3mM. The enzyme converted 40% (w/v) sucrose to isomaltulose with an efficiency of 87%.

A novel maltohexaose-forming α-amylase from Bacillus caldovelox: patterns and mechanisms of action

SummaryThe acidophilic, thermostable α-amylase of Bacillus caldovelox displays a unique end-product profile and action pattern on starch. Maltohexaose is preferentially produced, with a maximum yield of 40–44% (w/w) from 35% (w/v) starch and dextrins (DE 9 and DE 18). Maltohexaose, the initial product of 1% (w/v) starch and 35% (w/v) dextrin (DE 42) hydrolysis, is subsequently converted into maltopentaose with a maximum yield of 30% (w/w). This reaction does not involve glucose production. Substrates were hydrolysed from the non-reducing end by either a uni- or multimolecular mechanism, with no hydrolysis of maltohexaose or smaller sugars. The Km values for soluble starch and maltoheptaose were 4.68 mg/ml and 2.13 × 10−2M, respectively.

The high maltose-producing α-amylase of Penicillium expansum

SummaryAn α-amylase capable of producing exceptionally high levels of maltose (74%) from starch has been identified from a strain of Penicillium expansum. The enzyme is produced extracellularly and was purified to homogeneity by starch adsorption and Sephadex gel filtration chromatography. P. expansum α-amylase has a pH optimum of 4.5 and is stable in the pH range of 3.6–6.0. Other properties include a temperature optimum of 60° C, a molecular weight of 69 000 and an isoelectric point of 3.9. The most outstanding feature of the P. expansum enzyme is its ability to yield 14% more maltose and 17.1% less maltotriose than a currently used commercial enzyme. This may be partly explained by the greater affinity of this new enzyme for maltotriose (Km=0.76 mM) relative to the commerical enzyme, Fungamyl (Km=2.9 mM). The enzyme reported here is unique among fungal α-amylases in being able to produce such high levels of maltose and its physicochemical properties suggest that it has potential for commercial development.