Dataset Information

Cloning of the aapT gene and characterization of its product, alpha-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. strain XAL601.

ABSTRACT: A thermophilic and alkaliphilic Bacillus sp. strain, XAL601, was isolated from soil. It produces a thermostable and alkaline-stable enzyme with both alpha-amylase and pullulanase activities. The alpha-amylase-pullulanase gene (aapT) from this Bacillus strain was cloned, and its nucleotide sequence was determined (GenBank accession number D28467). A very large open reading frame composed of 6,096 bases, which encodes 2,032 amino acid residues with an M(r) of 224,992, was found. The deduced amino acid sequence revealed that the four highly conserved regions that are common among amylolytic enzymes were well conserved. These include an active center and common substrate-binding sites of various amylases. In the C-terminal region, a six-amino-acid sequence (Gly-Ser-Gly-Thr-Thr-Pro) is repeated 12 times. The aapT gene was then subcloned in Escherichia coli and overexpressed under the control of the lac promoter. Purification of AapT from this recombinant E. coli was performed, and it was shown that the aapT gene product exhibits both alpha-amylase and pullulanase activities with one active site. The optimum temperature and pH for enzyme activity were found to be 70 degrees C and pH 9, respectively. Furthermore, AapT was found to strongly adsorb to crystalline cellulose (Avicel) and raw corn starch. Final hydrolyzed products from soluble starch range from maltose (G2) to maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. The enzyme also hydrolyzes raw starch under a broad range of conditions (60 to 70 degrees C and pH 8 to 9).

SUBMITTER: Lee SP

PROVIDER: S-EPMC201885 | biostudies-literature | 1994 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cloning of the aapT gene and characterization of its product, alpha-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. strain XAL601.

Lee S P SP Morikawa M M Takagi M M Imanaka T T

Applied and environmental microbiology 19941001 10

A thermophilic and alkaliphilic Bacillus sp. strain, XAL601, was isolated from soil. It produces a thermostable and alkaline-stable enzyme with both alpha-amylase and pullulanase activities. The alpha-amylase-pullulanase gene (aapT) from this Bacillus strain was cloned, and its nucleotide sequence was determined (GenBank accession number D28467). A very large open reading frame composed of 6,096 bases, which encodes 2,032 amino acid residues with an M(r) of 224,992, was found. The deduced amino ...[more]

PMID: 7986049

Similar Datasets

Project description:The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and beta-cyclodextrin (beta-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and beta-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched beta-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the alpha-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.

Project description:Polylactic acid (PLA) is one important chemical building block that is well known as a biodegradable and a biocompatible plastic. The traditional lactate fermentation processes need CaCO3 as neutralizer to maintain the desired pH, which results in an amount of insoluble CaSO4 waste during the purification process. To overcome such environmental issue, alkaliphilic organisms have the great potential to be used as an organic acid producer under NaOH-neutralizing agent based fermentation. Additionally, high optical purity property in D-lactic acid is now attracting more attention from both scientific and industrial communities because it can improve mechanical properties of PLA by blending L- or D-polymer together. However, the use of low-price nitrogen source for D-lactate fermentation by alkaliphilic organisms combined with NaOH-neutralizing agent based process has not been studied. Therefore, our goal was the demonstrations of newly simplify high-optical-purity D-lactate production by using low-priced peanut meal combined with non-sterile NaOH-neutralizing agent based fermentation.In this study, we developed a process for high-optical-purity D-lactate production using an engineered alkaliphilic Bacillus strain. First, the native L-lactate dehydrogenase gene (ldh) was knocked out, and the D-lactate dehydrogenase gene from Lactobacillus delbrueckii was introduced to construct a D-lactate producer. The key gene responsible for exopolysaccharide biosynthesis (epsD) was subsequently disrupted to increase the yield and simplify the downstream process. Finally, a fed-batch fermentation under non-sterile conditions was conducted using low-priced peanut meal as a nitrogen source and NaOH as a green neutralizer. The D-lactate titer reached 143.99 g/l, with a yield of 96.09 %, an overall productivity of 1.674 g/l/h including with the highest productivity at 16 h of 3.04 g/l/h, which was even higher than that of a sterile fermentation. Moreover, high optical purities (approximately 99.85 %) of D-lactate were obtained under both conditions.Given the use of a cheap nitrogen source and a non-sterile green fermentation process, this study provides a more valuable and favorable fermentation process for future polymer-grade D-lactate production.

Dataset Information

Cloning of the aapT gene and characterization of its product, alpha-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. strain XAL601.

Publications

Cloning of the aapT gene and characterization of its product, alpha-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. strain XAL601.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets