Alpha-amylase Synthesis Research Articles

The alpha-amylase gene amyH of the moderate halophile Halomonas meridiana: cloning and molecular characterization.

Two types of Tn1732-induced mutants defective in extracellular amylase activity were isolated from the moderate halophile Halomonas meridiana DSM 5425. Type I mutants displayed amylase activity in the periplasm, and were unable to use any of the carbon sources tested, including starch and its hydrolysis product maltose. The type II mutant was affected in the gene responsible for the synthesis of the extracellular alpha-amylase. This gene (amyH) was isolated by functional complementation of mutant II and sequenced. The deduced protein (AmyH) showed a high degree of homology to a proposed family of alpha-amylases consisting of enzymes from Alteromonas (Pseudoalteromonas) haloplanktis, Thermomonospora curvata, streptomycetes, insects and mammals. AmyH contained the four highly conserved regions in amylases, as well as a high content of acidic amino acids. The amyH gene was functional in the moderate halophile Halomonas elongata and, when cloned in a multicopy vector, in Escherichia coli. AmyH is believed to be the first extracellular-amylase-encoding gene isolated from a moderate halophile, a group of extremophiles of great biotechnological potential. In addition, H. meridiana and H. elongata were able to secrete the thermostable alpha-amylase from Bacillus licheniformis, indicating that members of the genus Halomonas are good candidates for use as cell factories to produce heterologous extracellular enzymes.

Production of alpha-amylase in fed-batch cultures of vgb+ and vgb- recombinant Escherichia coli: some observations.

Synthesis and excretion of Bacillus stearothermophilus alpha-amylase is analyzed in fed-batch cultivations of Escherichia coli JM103[pMK79] and E. coli JM103[pMK57], the former strain containing the plasmid-encoded Vitreoscilla hemoglobin (VHb) gene (vgb) and the latter strain being devoid of this gene. Fed-batch operation is observed to be substantially superior to batch operation as concerns the alpha-amylase production rate and the extent of excretion of the enzyme. Faster feeding of a nutrient medium (LB or M9) discourages synthesis of alpha-amylase. While synthesis of alpha-amylase in the vgb(-) strain is discouraged when oxygen availability is reduced, the reverse is the case with the vgb(+) strain, the promotion of alpha-amylase synthesis in the latter strain being linked to the synthesis of VHb. Increased availability of the principal carbon source (glucose) in a defined medium leads to overproduction of both alpha-amylase and VHb under oxygen limitation, which may be responsible for the segregational instability observed with the vgb(+) strain. The very high extents of excretion of alpha-amylase attained in fed-batch cultures are encouraging for downstream processing of the recombinant protein.

Overexpression of BiP in tobacco alleviates endoplasmic reticulum stress.

To study the role of the lumenal binding protein (BiP) in the transport and secretion of proteins, we have produced plants with altered BiP levels. Transgenic plants overexpressing BiP showed dramatically increased BiP mRNA levels but only a modest increase in BiP protein levels. The presence of degradation products in BiP overproducers suggests a regulatory mechanism that increases protein turnover when BiP is abundant. Antisense inhibition of BiP synthesis was not successful, demonstrating that even a minor reduction in the basal BiP level is deleterious to cell viability. Overexpression of BiP leads to downregulation of the basal transcript levels of endogenous BiP genes and greatly reduces the unfolded protein response. The data confirm that BiP transcription is regulated via a feedback mechanism that involves monitoring of BiP protein levels. To test BiP activity in vivo, we designed a functional assay, using the secretory protein alpha-amylase and a cytosolic enzyme as a control for cell viability. During tunicamycin treatment, an overall reduction of alpha-amylase synthesis was observed when compared with the cytosolic marker. We show that the tunicamycin effect is due to the depletion of BiP in the endoplasmic reticulum because coexpressed BiP alone is able to restore efficient alpha-amylase synthesis. This is a novel assay to monitor BiP activity in promoting secretory protein synthesis in vivo.

Significance of HPr in catabolite repression of alpha-amylase.

CcpA and HPr are presently the only two proteins implicated in Bacillus subtilis global carbon source catabolite repression, and the ptsH1 mutation in the gene for the HPr protein was reported to relieve catabolite repression of several genes. However, alpha-amylase synthesis by B. subtilis SA003 containing the ptsH1 mutation was repressed by glucose. Our results suggest HPr(Ser-P) may be involved in but is not required for catabolite repression of alpha-amylase, indicating that HPr(Ser-P) is not the sole signaling molecule for CcpA-mediated catabolite repression in B. subtilis.

Induction and repression of alpha-amylase production in batch and continuous cultures of Aspergillus oryzae.

The intra- and extracellular concentrations of alpha-amylase in Aspergillus oryzae have been measured during batch culture of a wild-type strain and two recombinant strains. The mean intracellular level for the two recombinant strains was about four to five times the level of the wild-type strain. The recombinant strains also had a higher alpha-amylase productivity, whereas the residence time of the intracellular alpha-amylase pool was approximately the same for the three strains. At high glucose concentrations there was a low constitutive synthesis of alpha-amylase, whereas at low glucose concentrations derepression resulted in an increased production rate. Shifts from a glucose- to a maltose-limited chemostat showed that maltose induces both the production and secretion of alpha-amylase. Finally, from immunoblots, both a glycosylated and an unglycosylated alpha-amylase have been detected.

The -16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli.

The promoter (amyP) of the Bacillus subtilis alpha-amylase gene, which is recognized by E sigma A, has a three out of six match to the consensus promoter in both the -35 and -10 hexamers. Oligonucleotide-directed mutagenesis was used to identify important bases for promoter utilization in the spacer sequence between the hexamers. Mutations in the sequence TGTG extending from positions -18 to -15 (the -16 region) caused a 5-94-fold decrease in alpha-amylase production. A G-C transversion at position -15 was the most detrimental mutation: it essentially eliminated amyP utilization in B. subtilis and in Escherichia coli. Mutating the -35 and -10 hexamers to the E sigma A consensus promoter increased alpha-amylase production 56-fold in B. subtilis and fivefold in E. coli. Introducing the -15 G to C transversion into the consensus promoter reduced alpha-amylase production threefold, in contrast to the 94-fold reduction for the wild-type promoter in B. subtilis. The -15 G to C transversion did not reduce alpha-amylase synthesis directed by the consensus promoter in E. coli. The alpha-amylase gene is subject to two forms of transcriptional regulation: catabolite repression and temporal regulation. None of the mutants constructed in this study had any effect on either type of regulation. The -16 region, especially the G at position -15, appears to be moderately conserved in B. subtilis and in other Gram-positive organisms and weakly conserved in E. coli. The evidence suggests that the -16 region is an additional region of E sigma A promoters in B. subtilis and E sigma 70 promoters in E. coli, essential in some weak promoters such as the alpha-amylase promoter but, of little benefit to very strong promoters.

Co-overexpression of prlF increases cell viability and enzyme yields in recombinant Escherichia coli expressing Bacillus stearothermophilus alpha-amylase.

The effects on cloned amylase production of co-overexpression of prlF, a gene that appears to interact with the sec protein export machinery in Escherichia coli, was investigated by comparing three expression systems: (i) a high copy number plasmid with the Bacillus stearothermophilus alpha-amylase gene (amyS) cloned with its promoter downstream of the lac promoter; (ii) a pBR322-based vector with amyS under control of the indigenous Bacillus promoter; and (iii) a temperature-inducible vector with runaway replicon and lambda pL promoter-controlled gene expression. In addition, protease mutants (lon-) of E. coli C600 were used to evaluate the influence of the Lon protease on net enzyme formation and activity degradation during batch fermentations. Our results show that alpha-amylase synthesis occurred during exponential growth and ceased in the stationary phase. While strong promoters on high copy number plasmids severely impaired cell viability, resulting in culture lysis at mid-log phase, co-overexpression of prlF greatly improved cell viability, as well as the yield and specific production of alpha-amylase for the expression constructs considered. lon deficiency slightly increased amylase stability during the late stationary phase. However, the specific productivity of lon- strains was only about 40-60% that of the isogenic E. coli C600 equivalent.

Improvement of cloned α‐amylase gene expression in fed‐batch culture of recombinant Saccharomyces cerevisiae by regulating both glucose and ethanol concentrations using a fuzzy controller

The effect of ethanol concentration on cloned gene expression in recombinant Saccharomyces cerevisiae strain 20B-12 containing one of two plasmids, pNA3 and pNA7, was investigated in batch cultures. Plasmids pNA3 and pNA7 contain the alpha-amylase gene under the control of the SUC2 or PGK promoter, respectively. When the ethanol concentration was controlled at 2 to 5 g/L, the gene expressions were two times higher than those at 20 g/L ethanol. The increase the gene expression by maintaining both the ethanol and glucose concentrations at low levels, a fuzzy controller was developed. The concentrations of glucose and ethanol were controlled simultaneously at 0.15 and 2 g/L, respectively, in the production phase using the fuzzy controller in fed-batch culture. The synthesis of alpha-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory alpha-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory alpha-amylase activities of cells harboring plasmids pNA3 and pNA7 in fed-batch culture were 175 and 395 U/mL, and their maximal specific activities 7.7 and 12.4 U/mg dry cells, respectively. These values are two to three times higher in activity and three to four times higher in specific activity than those obtained when glucose only was controlled.

Characterization of a new sporulation factor in Bacillus subtilis.

We report the existence and partial purification of sporulation factor, which stimulates sporulation of Bacillus subtilis at low cell density. Proline or arginine is required for stimulation under the conditions of our assay. Sporulation factor is a small heat-stable substance produced by the cells during exponential growth phase. It is required in small amounts and is resistant to various proteolytic agents. Several spo mutants were tested for the ability to produce functional sporulation factor. All of these mutants produce factor and do not sporulate in the presence of factor from wild-type cells. Sporulation factor is not involved in the induction of alpha-amylase synthesis at the initiation of sporulation.

Catabolite repression of beta-glucanase synthesis in Bacillus subtilis.

beta-Glucanase synthesis in Bacillus subtilis was repressed by glucose and other substrates of glycolysis. Experiments with different pts mutants showed that the phosphoenolpyruvate: sugar phosphotransferase system is not involved in carbon catabolite repression of beta-glucanase synthesis. Carbon catabolite repression of beta-glucanase synthesis was completely abolished in a ccpA mutant. An operator structure similar to those upstream of amyE and the xyl operon was found and was shown by site-directed mutagenesis to be the target for carbon catabolite repression of beta-glucanase synthesis. The presence of this operator on a multi-copy plasmid resulted in a reduced repression of both beta-glucanase and alpha-amylase synthesis. It seems likely that the gene encoding these enzymes are part of one regulon with respect to catabolite repression.

Comparison of three different promoter systems for secretory α‐amylase production in fed‐batch cultures of recombinant Saccharomyces cerevisiae

Cloned gene expression in recombinant Saccharomyces cerevisiae 20B-12 containing three different plasmids was compared in batch and fed-batch cultures. The plasmids pNA3, pNA7, and pNA9 contain the alpha-amylase gene under the control of SUC2, PGK, and GAL7 Promoters, respectively. The synthesis of alpha-amylase was therefore induced by low glucose concentration for the SUC2 and PGK promoters, and by galactose for GAL7 promoter. The specific cell growth rates were similar among cells harboring the three different plasmids; they decreased from 0.35 to 0.38 h(-1) during the cell growth phase to 0.03 to 0.06 h(-1) during the production phase. The secretory alpha-amylase activity of cells harboring plasmid pNA7 was 129 U/mL in fed-batch culture, which was 1.4 and 2 times as high as the activities of cells harboring plasmids pNA3 and pNA9, respectively. The secretion ratios (amount of extracellular alpha-amylase activity/amounts of total alpha-amylase activity) of cells harboring plasmids pNA3, pNA7, and pNA9 were 91.4%, 94.5%, and 95.3%, respectively.

Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley aleurone protoplasts.

The effects of gibberellic acid (GA3) and abscisic acid (ABA) on the temporal and spatial dynamics of cytosolic calcium concentration ([Ca2+]i) in aleurone protoplasts of barley (Hordeum vulgare L. cv. Himalaya) were measured by using fluorescence ratio analysis and confocal microscopy. After 4-6 h of treatment, GA3 induced a sustained increase in [Ca2+]i from 50 to 150 nM in aleurone protoplasts. The increase in [Ca2+]i preceded the GA3-induced increase in alpha-amylase synthesis and secretion by 4 h. The elevation of [Ca2+]i was highest in the peripheral cytoplasm and may play a role in coordinating the secretory events there. Reducing Ca2+ levels in the incubation medium to below 500 microM inhibited the increase in [Ca2+]i and the GA3-induced stimulation of alpha-amylase synthesis and secretion. These data suggest that GA3 may increase [Ca2+]i by increasing the influx of Ca2+ at the plasma membrane. ABA reversed the effect of GA3 on [Ca2+]i within 3 h, 2 h before its effect on alpha-amylase production could be detected. Thus changes in [Ca2+]i may play a role in mediating the effects of GA3 and ABA on the synthesis and secretion of alpha-amylase in the aleurone cell.

Small cytoplasmic RNA of Bacillus subtilis: functional relationship with human signal recognition particle 7S RNA and Escherichia coli 4.5S RNA.

Small cytoplasmic RNA (scRNA; 271 nucleotides) is an abundant and stable RNA of the gram-positive bacterium Bacillus subtilis. To investigate the function of scRNA in B. subtilis cells, we developed a strain that is dependent on isopropyl-beta-D-thiogalactopyranoside for scRNA synthesis by fusing the chromosomal scr locus with the spac-1 promoter by homologous recombination. Depletion of the inducer leads to a loss of scRNA synthesis, defects in protein synthesis and production of alpha-amylase and beta-lactamase, and eventual cell death. The loss of the scRNA gene in B. subtilis can be complemented by the introduction of human signal recognition particle 7S RNA, which is considered to be involved in protein transport, or Escherichia coli 4.5S RNA. These results provide further evidence for a functional relationship between B. subtilis scRNA, human signal recognition particle 7S RNA, and E. coli 4.5S RNA.

Cell growth and alpha-amylase production characteristics of Bacillus subtilis.

Growth, differential rate of alpha-amylase synthesis and production characteristics of Bacillus subtilis DP 1 (isolate from starch materials) in comparison with 10 Bacillus strains were examined in batch fermentation. The effect of the carbon and nitrogen source was evaluated with regard to cell growth and enzyme production. The pH optimum of enzyme activity was 6.5 and temperature optimum of 60 degrees C.

Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A

Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.

Metabolic derepression of alpha-amylase gene expression in suspension-cultured cells of rice.

We present evidence to show that the alpha-amylase gene family in rice is under two different modes of regulation: 1) hormonal regulation in germinating seeds, and 2) metabolic repression in cultured cells by available carbohydrate nutrients. Expression of alpha-amylase genes in deembryoed rice seeds is known to be induced by exogenous gibberellic acid. On the other hand, expression of alpha-amylase genes in suspension-cultured cells is induced by the deprivation of carbohydrate nutrient. A lag period of 2-4 h is required for the induction of alpha-amylase mRNA in sucrose-depleted medium. The induction of alpha-amylase expression is extraordinarily high and levels of alpha-amylase mRNA can be increased 8-20-folds after 24 h of sucrose starvation. The synthesis and secretion of alpha-amylase is also dependent upon the level of carbon source. The derepression or repression of alpha-amylase synthesis can be readily reversed by the deprivation or replenishment of sucrose in the medium, respectively. Glucose and fructose exert a repression on the alpha-amylase synthesis similar to that of sucrose. A hypothesis that explains the induction of alpha-amylase synthesis by carbohydrate starvation is proposed. Our data have suggested a hitherto undiscovered, potentially important control mechanism of carbohydrate metabolism in higher plants.

Simple structured model for α‐amylase synthesis by Bacillus amyloliquefaciens

A predictive, simple, structured model describing the synthesis of alpha-amylase by Bacillus amyloliquefaciens was formulated. Three key intracellular processes were identified (i.e, translation, and excretion) along with two key intracellular components (i.e., mRNA and the intracellular form of the alpha-amylase enzyme). Nearly all the model parameters were estimated by means of performing independent experiments, primarily fed-batch experiments. The model was shown to predict transient system behavior in batch and in fed-batch operation with some limitation and minor model parameter revisions. Since a principal objective was to demonstrate that independent experimental parameter determination can be used to construct the predictive model, further fine-tuning of the parameters may be necessary before application for optimization and control purposes.

Contribution of aleurone layer and scutellum to alpha-amylase synthesis and secretion in wheat and rice grains

Contribution of aleurone layer and scutellum to alpha-amylase synthesis and secretion in wheat and rice grains

Barley aleurone layer cell protoplasts as a transient expression system

Protoplasts were prepared from barley aleurone layers using 'Onozuka' cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of alpha-amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley alpha-amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormone-responsive elements in hydrolase genes.

Effects of spermidine on the synthesis of alpha-amylase in cotyledons of mung bean seedlings

Effects of spermidine on the synthesis of alpha-amylase in cotyledons of mung bean seedlings