Influence of carbon source on α-amylase production by Aspergillus oryzae

The influence of the carbon source on alpha-amylase production by Aspergillus oryzae was quantified in carbon-limited chemostat cultures. The following carbon sources were investigated: maltose, maltodextrin (different chain lengths), glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate. A. oryzae did not grow on galactose as the sole carbon source, but galactose was co-metabolized together with glucose. Relative to that on low glucose concentration (below 10 mg/l), productivity was found to be higher during growth on maltose and maltodextrins, whereas it was lower during growth on sucrose, fructose, glycerol, mannitol and acetate. During growth on acetate there was no production of alpha-amylase, whereas addition of small amounts of glucose resulted in alpha-amylase production. A possible induction by alpha-methyl-D-glucoside during growth on glucose was also investigated, but this compound was not found to be a better inducer of a-amylase production than glucose. The results strongly indicate that besides acting as a repressor via the CreA protein, glucose acts as an inducer.     

Induction and regulation of alpha-amylase synthesis in a cellulolytic thermophilic fungus Myceliophthora thermophila D14 (ATCC 48104).

The alpha-amylase enzyme synthesis was higher when M. thermophila D-14 (ATCC 48104) was grown in culture medium incorporated with starch or other carbohydrates containing maltose units. Maximum enzyme production was attained with 1% starch followed by a gradual decrease with increasing concentration. Marked decrease in alpha-amylase synthesis occurred with the addition of glucose to the culture medium and this decreasing activity was proportional to the concentration of glucose. The enzyme synthesis was resumed as soon as the glucose concentration fell below a critical level. The addition of cAMP did not eliminate the repressive activity of glucose. The findings suggest that extracellular alpha-amylase synthesis in M. thermophila D-14 was inducible and subject to catabolite repression.     

Production and Characteristics of Raw-Potato-Starch-Digesting alpha-Amylase from Bacillus subtilis 65

A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting alpha-amylase. The electrophoretically homogeneous preparation of enzyme (molecular weight, 68,000) digested and solubilized raw corn starch to glucose and maltose with small amounts of maltooligosaccharides ranging from maltotriose to maltoheptaose. This enzyme was different from other amylases and could digest raw potato starch almost as fast as it could corn starch, but it showed no adsorbability onto any kind of raw starch at any pH. The mixed preparation with Endomycopsis glucoamylase synergistically digested raw potato starch to glucose at 30 degrees C. The raw-potato-starch-digesting alpha-amylase showed strong digestibility to small substrates, which hydrolyzed maltotriose to maltose and glucose, and hydrolyzed p-nitrophenyl maltoside to p-nitrophenol and maltose, which is different from the capability of bacterial liquefying alpha-amylase.     

Production of alpha-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

alpha-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55 degrees C and 5.5, respectively, and the apparent K(m) for starch was 0.8 mg ml. The products of alpha-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of alpha-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial alpha-amylase. Activities of alpha-amylase up to 4.4 U ml (1 U represents 1 mumol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml), alpha-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed alpha-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of alpha-amylase (initial concentration, 2.5 mg ml), they were found to be less effective than starch, but maltose was almost as effective. The fungal alpha-amylase was found to be stable at 60 degrees C in the presence of low concentrations of starch (相似文献   

Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A. oryzae alpha-amylase.

The physiology of three strains of Aspergillus nidulans was examined--a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations and the biomass formation and alpha-amylase production was characterised. Overexpression of the creA gene resulted in a lower maximum specific growth rate and a slightly higher repression of the alpha-amylase production during conditions with high glucose concentration. No expression of creA also resulted in a decreased maximum specific growth rate, but also in drastic changes in morphology. Furthermore, the expression of alpha-amylase was completely derepressed and creA thus seems to be the only regulatory protein responsible for glucose repression of alpha-amylase expression. The effect of different carbon sources on the alpha-amylase production in the creA deletion strain was investigated and it was found that starch was the best inducer. The degree of induction by starch increased almost linearly with the concentration of starch in starch/glucose mixtures. High-density batch cultivation was performed with the creA deletion strain and a final titre of 6.0 g l(-1) of alpha-amylase was reached after 162 h of cultivation.     

Kinetics of α-amylase production in a continuous culture ofBacillus licheniformis

As found during continuous cultivation ofBacillus licheniformis on a semisynthetic medium (glucose or maltose as C source), the specific rate of α-amylase production is proportional to growth rate but is repressed by higher substrate concentrations. Besides glucose or maltose, peptone was also used as an alternative carbon source during cultivation. The specific rate of production of the enzyme on maltose is half that found with glucose.     

Mode of alpha-amylase production by the shochu koji mold Aspergillus kawachii

Aspergillus kawachii produces two kinds of alpha-amylase, one is an acid-unstable alpha-amylase and the other is an acid-stable alpha-amylase. Because the quality of the shochu depends strongly on the activities of the alpha-amylases, the culture conditions under which these alpha-amylases are produced were examined. In liquid culture, acid-unstable alpha-amylase was produced abundantly, but, acid-stable alpha-amylase was not produced. The acid-unstable alpha-amylase was produced significantly when glycerol or glucose was used as a carbon source, similarly to the use of inducers such as starch or maltose. In liquid culture, A. kawachii assimilated starch at pH 3.0, but no alpha-amylase activity was recognized in the medium. Instead, the alpha-amylase was found to be trapped in the cell wall. The trapped form was identified as acid-unstable alpha-amylase. Usually, acid-unstable alpha-amylase is unstable at pH 3.0, so its stability appeared to be due to its immobilization in the cell wall. In solid-state culture, both kinds of alpha-amylase were produced. The production of acid-stable alpha-amylase seems to be solid-state culture-specific and was affected by the moisture content in the solid medium.     

Statistical optimization of a high maltose-forming,hyperthermostable and Ca2+-independent alpha-amylase production by an extreme thermophile Geobacillus thermoleovorans using response surface methodology

AIM: Statistical optimization for maximum production of a hyperthermostable, Ca2+-independent and high maltose-forming alpha-amylase by Geobacillus thermoleovorans. METHODS AND RESULTS: G. thermoleovorans was cultivated in 250 ml flasks containing 50 ml of chemically defined glucose-arginine medium (g l(-1): glucose 20; arginine 1.2; riboflavin 150 microg ml(-1); MgSO4. 7H2O 0.2; NaCl 1.0; pH 7.0). The medium was inoculated with 5 h-old bacterial inoculum (1.8x10(8) CFU ml(-1)), and incubated in an incubator shaker at 70 degrees C for 12 h at 200 rev min(-1). The fermentation variables optimized by 'one variable at a time' approach were further optimized by response surface methodology (RSM). The statistical model was obtained using central composite design (CCD) with three variables: glucose, riboflavin and inoculum density. An over all 24 and 70% increase in enzyme production was attained in shake flasks and fermenter because of optimization by RSM, respectively. A good coverage of interactions could also be explained by RSM. The end products of the action of alpha-amylase on starch were maltose (62%), maltotriose (31%) and malto-oligosaccharides (7%). CONCLUSIONS: RSM allowed optimization of medium components and cultural parameters for attaining high yields of alpha-amylase, and further, a good coverage of interactions could be explained. The yield of maltose was higher than maltotriose and malto-oligosaccharides in the starch hydrolysate. SIGNIFICANCE AND IMPACT OF THE STUDY: By applying RSM, critical fermentation variables were optimized rapidly. The starch hydrolysate contained a high proportion of maltose, and therefore, the enzyme can find application in starch saccharification process for the manufacture of high maltose syrups. The use of this enzyme in starch saccharification eliminates the addition of Ca2+.     

Cell growth and alpha-amylase production characteristics of Bacillus amyloliquefaciens

Growth and alpha-amylase production characteristics of Bacillus amyloliquefaciens strain F (ATCC 23350) in batch cultures are examined using glucose or maltose as the carbon source. While the cell growth is rapid when glucose is used as the carbon source, higher cell mass, higher total and specific enzyme activities, and higher enzyme production rates are obtained when maltose is used as the carbon source. The overall specific enzyme activity decreases with an increase in the initial concentration of carbon source. The oxygen requirement and carbon dioxide generation vary linearly with the maximum amount of cell mass produced. For experiments conducted using glucose as the carbon source, the kinetics of cell growth and glucose consumption are described using a special form of the Vavilin equation. For a given amount of initial carbon source, the enzyme synthesis capability is retained by the microorganism, although at a substantially reduced level, under severe oxygen limitation.     

Maltose adaptive mutant of heterotrophic marine bacterium, Alteromonas espejiana Bal-31: changes in the growth and the induction of extracellular alpha-amylase

Growth of the heterotrophic marine bacterium, Alteromonas espejiana Bal-31 was inhibited in the presence of sucrose, maltose and even glucose, but not with starch. Extracellular alpha-amylase was induced with a lag phase of 2 h in the presence of starch. In contrast, cell growth of the S2a mutant was not affected by the addition of maltose, and starch was ineffective in the induction of extracellular alpha-amylase in this mutant. Activity of extracellular alpha-amylase was induced from the S2a mutant with a 4-h lag phase in the presence of maltose, and the high level of enzyme activity was maintained for at least 24 h. Activity of alpha-amylase induced by both wild type starch and S2a mutant maltose cultures were mainly observed in extracellular locations. This activity could be stopped by tetracycline treatment, indicating that enzyme induction was dependant on gene expression and not on enzyme protein secretory mechanisms. Our results showed that the mutation in S2a changed the growth and the modulation of the specific alpha-amylase in response to carbon nutrients.     

Regulation of a thermostable α-amylase of Streptomyces thermoviolaceus CUB74: maltotriose is the smallest inducer

We have examined induction and repression by various sugars and carbon sources of the synthesis of a thermostable alpha-amylase in its natural host, S thermoviolaceus CUB74. The smallest molecule capable of inducing synthesis of the enzyme was maltotriose whereas maltose had no effect which might suggest a different control system from that found in other streptomycete amylases. Addition of mannitol to the growth medium impeded the alpha-amylase induction whereas glucose had no effect. After cloning of its gene into a new streptomycete host, S lividans TK24, the S thermoviolaceus alpha-amylase could not be induced by any of the sugars tested.     

Purification, biochemical characterization, and gene cloning of a new extracellular thermotolerant and glucose tolerant maltooligosaccharide-forming alpha-amylase from an endophytic ascomycete Fusicoccum sp. BCC4124

An endophytic fungus, Fusicoccum sp. BCC4124, showed strong amylolytic activity when cultivated on multi-enzyme induction enriched medium and agro-industry substrates. alpha-Amylase and alpha-glucosidase activities were highly induced in the presence of maltose and starch. The purified target alpha-amylase, Amy-FC1, showed strong hydrolytic activity on soluble starch (kcat/Km=6.47 x 10(3) min(-1)(ml/mg)) and selective activity on gamma- and beta-cyclodextrins, but not on alpha-cyclodextrin. The enzyme worked optimally at 70 degrees C in a neutral pH range with t(1/2) of 240 min in the presence of Ca(2+) and starch. Maltose, matotriose, and maltotetraose were the major products from starch hydrolysis but prolonged reaction led to the production of glucose, maltose, and maltotriose from starch, cyclodextrins, and maltooligosaccharides (G3-G7). The amylase showed remarkable glucose tolerance up to 1 M, but was more sensitive to inhibition by maltose. The deduced protein primary structure from the putative gene revealed that the enzyme shared moderate homology between alpha-amylases from Aspergilli and Lipomyces sp. This thermotolerant, glucose tolerant maltooligosaccharide-forming alpha-amylase is potent for biotechnological application.     

Alpha-amylase production from catabolite derepressed Bacillus subtilis KCC103 utilizing sugarcane bagasse hydrolysate

A catabolite derepressed Bacillus subtilis strain KCC103 was used to produce alpha-amylase in medium containing sugarcane bagasse hydrolysate (SBH). Addition of SBH (1% reducing sugar (w/v)) to the nutrient medium supported maximum alpha-amylase production of 67.4 Um l(-1). HPLC analysis of SBH showed the presence of glucose, xylose and arabinose in the ratio of 0.9:1.0:0.16 (w/w/w). In SBH-medium glucose and xylose were consumed completely while arabinose remained unutilized. Uptake rate of glucose was 2-folds higher than xylose but rate of alpha-amylase production with xylose was 1.5-folds higher than glucose. Arabinose had no effect on growth and alpha-amylase synthesis. Further, alpha-amylase production in SBH-medium was enhanced to 144.5 Um l(-1) (2.2-fold) by response surface methodology where the levels of SBH, and other media components were varied. The modified medium consisted of (in gl(-1)) SBH: 24; peptone: 17.43; yeast extract: 1.32 and beef extract: 1.82. High level of SBH showed no significant inhibition of alpha-amylase synthesis. The derepressed strain KCC103 is useful to produce alpha-amylase economically in short time (30-36 h).     

Process design for recombinant protein production based on the promoter,P<Subscript><Emphasis Type="Italic">malK</Emphasis></Subscript>

P_malK is induced through activation of MalT, by the formation of maltotriose and cyclic adenosine monophosphate (cAMP). The possibility to influence endogenous inducer levels is used to vary the production rates in specifically designed production protocols. Induction based on a batch process protocol on maltose gives low production rates, as the result of a lack of cAMP, which is shown to be of major importance to fully induce this promoter. Two mechanisms are thus used to influence the levels of maltotriose and/or cAMP formation: (1) catabolite derepression achieved from low glucose concentration and (2) catabolite derepression/inducer exclusion from diauxic growth on glucose/maltose. Fed-batch processes based on limited amounts of glucose result in product accumulation of up to 10% of the total protein. Depending on the feed of limiting glucose, different production profiles are developed. The initial increase in the production rate is due to maltotriose formation from endogenous glycogen degradation while, later in the process, production can be further supported by elevated levels of cAMP, provided the feed rate is sufficiently low. The introduction of maltose after a preceding fed-batch process on glucose can be efficiently used to produce maltotriose in combination with cAMP formation in the event of catabolite derepression. This leads to higher production rates and a further increase in product accumulation of up to 30% of the total protein. The diauxic growth phase resulting from the shift in carbon source can be shortened and even avoided by the design of the preceding feed-rate of glucose. It is postulated that proper design of the inoculum and initial phases of production can reduce basal levels of product formation. With this promoter, the production rate can be as high as 65 units mg^–1 h^–1 and the time to reach a maximal production rate can be designed to take up to 8 h. Furthermore, the duration of the production rate can be as long as 7 h.     

Production and partial characterization of high molecular weight extracellular alpha-amylase from Thermoactinomyces vulgaris isolated from Egyptian soil

Optimizing production of alpha-amylase production by Thermoactinomyces vulgaris isolated from Egyptian soil was studied. The optimum incubation period, temperature and initial pH of medium for organism growth and enzyme yield were around 24 h, 55 degrees C and 7.0, respectively. Maximum alpha-amylase activity was observed in a medium containing starch as carbon source. The other tested carbohydrates (cellulose, glucose, galactose, xylose, arabinose, lactose and maltose) inhibited the enzyme production. Adding tryptone as a nitrogen source exhibited a maximum activity of alpha-amylase. Bactopeptone and yeast extract gave also high activity comparing to the other nitrogen sources (NH4CI, NH4NO3, NaNO3, KNO3, CH3CO2NH4). Electrophoresis profile of the produced two alpha-amylase isozymes indicated that the same pattern at about 135-145 kDa under different conditions. The optimum pH and temperature of the enzyme activity were 8.0 and 60 degrees C, respectively and enzyme was stable at 50 degrees C over 6 hours. The enzyme was significantly inhibited by the addition of metal ions (Na+, Co2+ and Ca2+) whereas CI- seemed to act as activator. The enzyme was not affected by 0.1 mM EDTA while higher concentration (10 mM EDTA) totally inactivated the enzyme.     

Fed-batch fermentation for the production of alpha-amylase by Bacillus amyloliquefaciens

Fed-batch cultures were performed to maximize the alpha-amylase activity in a bioreactor. Kinetic equations containing a catabolite repression effect were used to model the enzyme formation from Bacillus amyloliquefaciens. Fed-batch culture experiments were performed using maltose to implement the optimal feeding strategy. Optimal fed-batch culture based on sequential parameter estimation was performed successfully using off-line analysis while the fermentation was in progress. The enzyme activity from the fed-batch culture employing maltose was higher than that of the batch culture by 60%. Enzyme production using starch showed similar trends to those obtained using maltose.     

Enhanced production of alpha-amylase in fed-batch cultures of Bacillus subtilis TN106[pAT5

Growth of Bacillus subtilis TN106[pAT5] and synthesis of plasmid-encoded protein (alpha-amylase) are investigated in batch, continuous, and fed-batch cultures using a defined medium containing glucose and/or starch as the carbohydrate source. The batch culture studies reveal that reduced availability of arginine hampers growth of recombinant cells (which lack an arginine synthesis gene) but promotes production of alpha-amylase and substitution of glucose by starch as the carbohydrate source leads to slower growth of recombinant cells and increased production of alpha-amylase per unit cell mass. Retention of recombinant cells over prolonged periods in continuous cultures is not possible without continuous application of antibiotic selection pressure owing to segregational plasmid instability. Fed-batch experiments with constant volumetric feed rate demonstrate that alpha-amylase production is enhanced at lower feed concentration of starch (sole carbohydrate source) and lower volumetric feed rate. Such slow addition of starch is however not conducive for growth of recombinant cells. The expression of the thermostable alpha-amylase gene carried on the recombinant plasmid pAT5 (derived from a plasmid isolated from a thermophilic bacterium) is promoted at higher temperatures, while growth of recombinant cells is depressed. In all batch and fed-batch experiments, production of alpha-amylase is observed to be inversely related to growth of recombinant cells. The efficacy of two-stage bioreactor operations, with growth of recombinant cells being promoted in the first stage and alpha-amylase production in the second stage, in attaining increased bulk alpha-amylase activity is demonstrated. (c) 1993 John Wiley & Sons, Inc.     

A biosensor for the determination of amylase activity

A new biosensing flow injection method for the determination of alpha-amylase activity has been introduced. The method is based on the analysis of maltose produced during the hydrolysis of starch in the presence of alpha-amylase. Maltose determination in the flow system was allowed by the application of peroxide electrode equipped with an enzyme membrane. The membrane was obtained by immobilisation of glucose oxidase, alpha-glucosidase and optionally mutarotase on a cellophane, co-crosslinked by gelatin-glutaraldehyde together with bovine serum albumine. alpha-Glucosidase hydrolyses maltose to alpha-D-glucose, which is converted to beta-D-glucose by mutarotase. beta-D-Glucose is then determined via glucose oxidase. The new biosensor has the limit of detection of 50 nmol l(-1) maltose, which means 2 nkat ml(-1) in alpha-amylase activity units, when the reaction time of amylase was 5 min (determined with respect to a signal-to-noise ratio 3:1). When the reaction time of alpha-amylase was 30 min, the limit of detection was 0.5 nkat ml(-1). A linear range of current response was 0.1-3 mmol l(-1) maltose, with a response time of 35s. The biosensor was stable at least two months and retained 70% of its original activity (with mutarotase the stability is decreased to 3 weeks). When the enzyme membrane was stored in a dry state at 4 degrees C in a refrigerator, the lifetime was approximately 6 months (with mutarotase only 3 months).     

Growth defects of Escherichia coli cells which contain the gene of an alpha-amylase from Bacillus coagulans on a multicopy plasmid

An alpha-amylase gene from Bacillus coagulans has previously been cloned in Escherichia coli and shown to direct the synthesis of an enzymically active protein of 60,000 Dal (Cornelis et al., 1982). In one particular E. coli host, strain HB101, amylase was found to accumulate in the periplasmic space. To study the processing and the location of the amylase, plasmid pAMY2 was introduced into E. coli 188 which is a strain constitutive for alkaline phosphatase, a periplasmic marker, and for beta-galactosidase, a cytoplasmic marker. Abnormally large amounts of both alpha-amylase and beta-galactosidase were found in the culture fluid of cells grown in rich medium. Furthermore a severe growth defect was found when cells containing pAMY2 were grown in maltose and glycerol media, while the ability to grow on glucose remained normal. This defect could be reversed by two types of spontaneous mutations. Mutations in the first class are located on the plasmid and correspond to the insertional inactivation of the amylase gene by IS1. Mutations in the second class are located on the host chromosome. These results suggest that the synthesis and export of B. coagulans alpha-amylase is deleterious to E. coli, especially in media containing maltose or glycerol as sole carbon source.     

Production of surfactant and detergent-stable,halophilic, and alkalitolerant alpha-amylase by a moderately halophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. Strain <Emphasis Type="Italic">TSCVKK</Emphasis>

A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl₂ at pH 8.0 at 30 °C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 °C. The amylase that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose, and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase.