Purification and Characterization of Two Alkaline, Thermotolerant α-Amylases fromBacillus halodurans38C-2-1 and Expression of the Cloned Gene inEscherichia coli

Abstract

A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant alpha-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named alpha-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50-60 degrees C and pH 10-11, and 42 and 38% relative activities at 30 degrees C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding alpha-amylase I was cloned and named amyI. Production of AmyI with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl beta-D(-)-thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.

Studies on the production of alkaline α-amylase from Bacillus subtilis CB-18

“…Temperature stabilities for α-amylases were at 55-60°C (Hagihara et al, 2001); 75-80°C (Mamo and Gessesse, 1999) and 50-60°C (Murakami et al, 2007).…”

“…Alkaline α-amylase has high catalytic effi ciency and stability at alkaline pH ranging from 9 to 11 (Burhan et al, 2003) and have potential applications to hydrolyse starch under high pH conditions in the starch and textile industries and as ingredients in detergents (Murakami et al, 2007). This present investigation deals with the isolation of Bacillus subtilis CB-18 and some properties of its α-amylase.…”

Purification and Characterization of a Liquefying α-Amylase from Alkalophilic ThermophilicBacillussp. AAH-31

“…These results indicate that the structure of AmyL is considerable different from those of LAMY and AmyK38. The molecular mass and the specific activity of AmyL are similar to an alkaline -amylase, B. halodurans 38C-2-1 -amylase I, 10) and a maltohexaose-forming -amylase from Bacillus sp. Culture supernatants of soil bacteria exhibiting extracellularamylase activity ( ) were incubated with 0.5% soluble starch in 80 mM glycine-NaOH buffer (pH 10) containing 1 mM CaCl 2 at 50 C for 2 h, and the hydrolysis rate and blue values were calculated based on the production of reducing sugar and the decrease in iodine-staining power respectively.…”

A Thermophilic Alkalophilic α-Amylase fromBacillussp. AAH-31 Shows a Novel Domain Organization among Glycoside Hydrolase Family 13 Enzymes

“…AAH-31, first isolated from Japanese paddy soil, produces extracellular liquefying -amylase (AmyL), which is highly stable at high pH and temperatures. 5) Many alkaline -amylases have been identified in various alkalophilic bacteria, [6][7][8][9][10][11] but AmyL is more stable at high temperature than the known alkalophilic enzymes. Furthermore, this enzyme is stable in the presence of chelating reagents, including EDTA, nitrilotriacetic acid, and sodium tripolyphosphate.…”

“…These properties of AmyL are useful in automatic dishwashing detergents, because an automatic dishwashing machine generally operates at high pH at temperatures above 60 C. On SDS-PAGE analysis, the molecular mass of AmyL was estimated to be 91 kDa, considerably higher than those of the typical bacterial -amylases of GH family 13 (approximately 60 kDa) and close to enzymes possessing starch-binding domains. 11,12) In the present study, the gene encoding AmyL was cloned to elucidate its primary structure, and the recombinant enzyme, produced in Escherichia coli, was characterized. Furthermore, a site-directed mutational study at the starchbinding site, which was identified through sequence analysis, was carried out.…”