Thermostable Alpha-amylase Research Articles

Modification of length, hydrophobic properties and electric charge of Bacillus subtilis α-amylase signal peptide and their different effects on the production of secretory proteins in B. subtilis and Escherichia coli cells

Bacillus subtilis alpha-amylase signal peptide, which consists of 33 amino acids, is functional in Escherichia coli cells. Lysine, glutamic acid, leucine, leucyl-leucine, or leucyl-leucyl-leucine was inserted between positions 28 and 29 of the alpha-amylase signal peptide using site directed mutagenesis. DNAs encoding the wild-type and modified signal peptides were then fused in-frame to DNAs encoding the mature regions of the beta-lactamase of pBR322 and a thermostable alpha-amylase. The secretion of beta-lactamase in E. coli cells was more inhibited by the modified signal peptides than that in B. subtilis cells, although the degree of inhibition varied and the inhibitory effect of each signal peptide was found to be similar in the two strains. In contrast, the difference in the inhibitory effect of each modified signal peptide was no longer detected in the case of the production of thermostable alpha-amylase, except for the insertion of glutamic acid. Nearly 50% of thermostable alpha-amylase in the precursor form was accumulated in the intracellular fraction of E. coli cells containing the DNAs for the modified signal peptides. The insertion of glutamic acid inhibited the secretion of the two enzymes in both B. subtilis and E. coli cells.

Some Factors Affecting the Copy Number of Specific Plasmids in Bacillus Species

Some factors affecting the copy number of specific plasmids in different hosts were presented. A low copy number (one copy per chromosome) Kmr Tcr plasmid pTB19 was isolated from thermophilic bacillus. We have constructed 22 derivatives from pTB19 and their copy numbers range from 1 to 214 per chromosome in B. subtilis and B. stearothermophilus. Some recombinant plasmids containing the specific 1.0-MDa EcoRI fragment exhibited high transformation frequency and low copy number in B. stearothermophilus and were stable in the thermophile; on the other hand, those plasmids were unstable in different host B. subtilis, as mentioned earlier. By selecting the best combination of vector plasmid and host strain, both molecular cloning of various enzyme genes (i.e., penicillinase, thermostable alpha-amylase, and thermostable neutral protease) and enhancement of the enzyme production could be easily achieved.

A novel bacterial thermostable alpha-amylase system produced under solid state fermentation

A novel thermostable alpha-amylase system produced byBacillus HOP-40 at neutral pH under solid state fermentation gave three peaks in descending order at pH 5.0, 8.5–9.0 and 7.0.

Πолучаване на частично пречистена термостабилна α-амилаза от Bacillus licheniformis 44 МВ 82

Click to increase image sizeClick to decrease image sizeРЕЗЮМЕИсследованы условия выделения термостабильной α-амилазы из культуральной жидкости Вас. licheniformis 44 МВ 82 и ее частичной очистки. Часть балластных веществ выделена путем обработки культуральной жидкости CaCl2 и Na2НРО4 в подходящих концентрациях при рН 7,0–8,0 и последующим фильтрованием суспензии через полотна с 0,2% перлнта. Затем проводили ультрафильтрацию осветленной жидкости через мембраны, пропускающие вещества с молекулярной массой ниже 20 000 D.Одновременно с концентрированием жидкости по объему обнаруживалось трехкратное увеличение удельной активности препарата nри сохранении его термостабильности.SUMMARYInvestigations were carried out for determination of the conditions for separation and partialy purification of a thermostable alpha-amylase from the cultural medium of Bacillus licheniformis 44 MB 82. Some of the balast substances were separated by the addition of CaCl2 and Na2HPO4 into the cultural medium in suitable concentration at pH 7,0–8,0 and saccessive filtration on coat of 0,2% perlite. The clear filtrate was subjected to ultrafiltration through membranes with porosity of about 20000 D. By this procedure was achieved 3 fold increasing of the specific activity together with the concentration of the fluid and without any loss of the thermostability of alpha-amylase.

Structural genes encoding the thermophilic alpha-amylases of Bacillus stearothermophilus and Bacillus licheniformis.

The genes encoding the thermostable alpha-amylases of Bacillus stearothermophilus and B. licheniformis were cloned in Escherichia coli, and their DNA sequences were determined. The coding and deduced polypeptide sequences are 59 and 62% homologous to each other, respectively. The B. stearothermophilus protein differs most significantly from that of B. licheniformis in that it possesses a 32-residue COOH-terminal tail. Transformation of E. coli with vectors containing either gene resulted in the synthesis and secretion of active enzymes similar to those produced by the parental organisms. A plasmid was constructed in which the promoter and the NH2-terminal two-thirds of the B. stearothermophilus coding sequence was fused out of frame to the entire mature coding sequence of the B. licheniformis gene. Approximately 1 in 5,000 colonies transformed with this plasmid was found to secrete an active amylase. Hybridization analysis of plasmids isolated from these amylase-positive colonies indicated that the parental coding sequences had recombined by homologous recombination. DNA sequence analysis of selected hybrid genes revealed symmetrical, nonrandom distribution of loci at which the crossovers had resolved. Several purified hybrid alpha-amylases were characterized and found to differ with respect to thermostability and specific activity.

4493893 Process for cloning the gene coding for a thermostable alpha-amylase into escherichia coli and bacillus subtilis: Jonathan R Mielenz, Susan Mickel assigned to CPC International Inc

4493893 Process for cloning the gene coding for a thermostable alpha-amylase into escherichia coli and bacillus subtilis: Jonathan R Mielenz, Susan Mickel assigned to CPC International Inc

Cloning in [formula omitted] of an extremely thermostable alpha amylase: Comparison with other cloned heatstable alpha amylases

A heatstable alpha amylase gene was shotgun cloned from Bacillus licheniformis RP01 into Bacillus subtilis . Restriction endonuclease analysis of the recombinant plasmid revealed a map which was identical to a previously cloned alpha amylase from B. licheniformis FD02 and very similar to the restriction map of a high temperature amylase from Bacillus coagulans . The thermostability and temperature optimum of the cloned alpha amylase was measureably different from those of the previously reported cloned alpha amylases.

Bacillus stearothermophilus contains a plasmid-borne gene for alpha-amylase.

The gene for thermostable alpha-amylase from the thermophilic bacterium Bacillus stearothermophilus has been cloned and expressed in Escherichia coli. Each alpha-amylase-producing colony contained at least a 9.7-kilobase-pair (kb) chimeric plasmid composed of the vector pBR322 and a common 5.4-kb HindIII fragment of DNA. B. stearothermophilus contains four plasmids with sizes from 12 kb to over 108 kb. Restriction endonuclease analysis of these naturally occurring plasmids showed they also contain a 5.4-kb HindIII fragment of DNA. Cloning experiments with the four plasmids yielded alpha-amylase-producing E. coli that contained the same 9.7-kb chimeric plasmid. Restriction endonuclease analysis and further recombinant DNA experiments identified a 26-kb plasmid that contains the gene for alpha-amylase. A spontaneous mutant of B. stearothermophilus unable to produce alpha-amylase was missing the 26-kb plasmid but contained a 20-kb plasmid. A 6-kb deletion within the region of the 5.4-kb HindIII fragment yielded the 20-kb plasmid unable to code for alpha-amylase. A nick-translated probe for the alpha-amylase coding region did not hybridize to either plasmid or total cellular DNA from this mutant strain of B. stearothermophilus. These results demonstrate the gene for alpha-amylase is located exclusively on a 26-kb plasmid in B. stearothermophilus with no genetic counterpart present on the chromosome.

Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli.

A partial EcoRI fragment of Bacillus coagulans DNA cloned in an Escherichia coli K12 bacteriophage lambda host-vector system was shown to direct the synthesis of a thermostable alpha-amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 3.31 kb EcoRI fragment containing the active gene with its own promoter was subcloned in pBR322; in the new clone, called pAMY2, the amylase was shown to accumulate in the periplasmic space. The molecular weight of the enzyme, confirmed by in vivo labelling of plasmid products in minicells, was estimated to be 60000. The restriction map of the plasmid was determined for five restriction enzymes and two new plasmids with smaller DNA inserts were constructed, both directing the synthesis of amylase; one of them with a 2.2 kb PstI insert was shown to be responsible for the synthesis of a fused beta-lactamase-alpha-amylase protein with amylase activity.

Effect of oilseed cakes on alpha-amylase production by Bacillus licheniformis CUMC305.

The effects of oilseed cakes on extracellular thermostable alpha-amylase production by Bacillus licheniformis CUMC305 was investigated. Each oilseed cake was made of groundnut, mustard, sesame, linseed, coconut copra, madhuca, or cotton. alpha-Amylase production was considerably improved in all instances and varied with the oilseed cake concentration in basal medium containing peptone and beef extract. Maximum increases were effected by a low concentration (0.5 to 1.0%) of groundnut or coconut, a high concentration (3%) of linseed or mustard, and an Rintermediate concentration (2%) of cotton, madhuca, or sesame. The oilseed cakes made of groundnut or mustard could completely replace the conventional peptone-beef extract medium as the fermentation base for the production of alpha-amylase by B. licheniformis. The addition of corn steep liquor to cotton, linseed, sesame, or madhuca cake in the medium improved alpha-amylase production.

Molecular weight of thermostable alpha-amylase from B. stearothermophilus.

The molecular weights of two α-amylase isozymes [EC 3.2.1.1] (55°c α-amylases I and II) from B. stearothermophilus grown at 55°c and α-amylase [EC 3.2.1.1] (43°c α-amylase) from the same bacterium grown at 43°c were determined by different methods (gel filtration and sedimentation equilibrium). The α-amylase produced at 43°c contained both monomeric and dimeric forms of the enzyme. To elucidate the effects arising from dimerization, the enzymes were reduced and carboxymethylated and then the molecular weight was measured in 6.1M guanidine hydrochloride solution. The molecular weights of 43°c α-amylase and 55°c α-amylases I and II were determined by sedimentation equilibrim to be 44,000, 46,000, and 43,000 and by gel filtration (Sepharose 6B) to be 41,000, 44,000, and 41,000, respectively.