Action pattern of alpha-amylase from Aspergillus oryzae in concentrated media

The influence of concentrated maltotetraose solutions on fungal alpha-amylase activity and specificity has been determined. It has been found that the enzyme is not inhibited by 500 g/L substrate concentration and that transglycosylation reactions are increased with rising substrate concentration. The amount of oligosaccharides of polymerization degree higher than maltotetraose reaches 20% (w/w). Moreover, the effect of polyhydric alcohols, known as water activity depressors has been analyzed: the presence of polyols does not modify the amount of transglycosylation products, but changes the hydrolysis pattern by favoring the formation of low polymerization degree oligosaccharides.     

Statistical medium optimization and production of a hyperthermostable lipase from Burkholderia cepacia in a bioreactor

AIM: Statistical medium optimization for maximum production of a hyperthermostable lipase from Burkholderia cepacia and its validation in a bioreactor. METHODS AND RESULTS: Burkholderia cepacia was grown in shake flasks containing 1% glucose, 0.1% KH2PO4, 0.5% NH4Cl, 0.24% (NH4)2HPO4, 0.01% MgSO4.7H2O and 1% emulsified palm oil, at 45 degrees C and pH 7.0, agitated at 250 rev min(-1) with 6-h-old inoculum (2% v/v) for 20 h. A fourfold enhancement in lipase production (50 U ml(-1)) and an approximately three fold increase in specific activity (160 U mg(-1)) by B. cepacia was obtained in a 14 litre bioreactor within 15 h after statistical optimization following shake flask culture. The statistical model was obtained using face centred central composite design (FCCCD) with five variables: glucose, palm oil, incubation time, inoculum density and agitation. The model suggested no interactive effect of the five factors, although incubation period, inoculum and carbon concentration were the important variables. CONCLUSIONS: The maximum lipase production was 50 U ml(-1), with specific activity 160 U mg(-1) protein, in a 14 litre bioreactor after 15 h in a medium obtained after statistical optimization in shake flasks. Further, the model predicted reduction in time for lipase production with reduction in total carbon supply. SIGNIFICANCE AND IMPACT OF THE STUDY: Statistical optimization allows quick optimization of a large number of variables. It also provides a deep insight into the regulatory role of various parameters involved in enzyme production.     

Kinetics of alpha-amylase secretion in Aspergillus oryzae.

Pulse and pulse-chase experiments have been performed to study L-[(35)S] methionine incorporation and protein secretion kinetics in Aspergillus oryzae. Pulse experiments confirmed the mechanism of methionine uptake reported previously for Penicillium chrysogenum (Benko et al., 1967). Pulse-chase experiments were carried out to investigate the alpha-amylase secretion kinetics in A. oryzae. No unglycosylated alpha-amylase was detected neither intracellularly nor extracellularly demonstrating that glycosylation was not the rate controlling step in the secretory pathway. The pulse chase experiments indicated that there are two pools of intracellular alpha-amylase: a fast secreted and a slow secreted. The secretion of those two pools were described with a kinetic model, which was fitted to the pulse chase experiments.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Statistical media optimization for growth and PHB production from methanol by a methylotrophic bacterium

Media components were optimized by statistical design for cell growth and PHB production of Methylobacterium extorquens DSMZ 1340. Four important components of growth media were optimized by central composite design. The growth increased from an OD = 1.35 for Choi medium as control to an OD = 2.15 for optimal medium. Then media components for PHB production were optimized. Optimization of five important factors was conducted by response surface method. The optimal composition of PHB production medium was found to be at 7.8 (g/L) Na₂HPO₄ · 12H₂O, and surprisingly at zero concentration of (NH₄)₂SO₄, KH₂PO₄, MgSO₄ and MnSO₄. The PHB production was found to be 2.95 (g/L) at this medium. RSM results indicated that a deficiency of nitrogen and magnesium is crucial for PHB accumulation in this microorganism. Also, PHB production was carried out in a 5 L fermentor at the optimum condition which resulted in 9.5 g/L PHB and 15.4 g/L cell dry weight with 62.3% polymer content.     

Lipase production of Aspergillus oryzae

Aspergillus oryzae produced a small amount of lipase (0.05–0.8 U/wet-g of solid medium) in solid cultures, in contrast to the larger amount (0.46 U/ml) in a shake-flask culture in a modified GYP medium containing 2% glucose, 1% yeast extract and 2% Polypepton. Optimum conditions of lipase production in the submerged culture of A. oryzae were determined in terms of pH, composition of medium, and temperature. In a shake-flask culture at 28°C, the maximum amount of lipase increased to 0.78 U/ml upon the addition of 3% soybean oil to the modified GYP medium. In a jar fermentor culture, 30 U/ml lipase activity was obtained after 72 h at 28°C under appropriate conditions. Lipase production was greatly influenced by the culture temperature, and the optimum temperature for lipase production was about 24°C with a narrow temperature range, which was 10 degrees lower than that for the growth. In the submerged cultures, two kinds of lipase at least exhibiting different substrate specificities were also suggested.     

Lipid production by Aspergillus oryzae from starch substrates

Summary Reports that starch is a poor substrate for lipid production are attributed to the low available C:N ratio which occurs because starch is not directly available to the microbial cell. If cultural conditions were established which gave rapid and extensive amylase activity, sufficient starch hydrolysis would occur to give a high ratio of available C:N, conditions favourable to lipid accumulation. This hypothesis was tested experimentally with Aspergillus oryzae and mycelium with 37% lipid content was obtained with starch as sole carbon source in defined culture media.     

Effect of low oxygen concentrations on growth and alpha-amylase production of Aspergillus oryzae in model solid-state fermentation systems

Oxygen transfer in the fungal mat is a major concern in solid-state fermentation (SSF). Oxygen supply into the mycelial layers is hampered by diffusion limitation. For aerobic fungi, like Aspergillus oryzae, this oxygen depletion can be a severely limiting factor for growth and metabolite production. This paper describes the effects of a low oxygen concentration on growth at the levels of individual hyphae, colonies and overcultures, and on alpha-amylase production in overcultures. PDA medium was used to study the effect of a low oxygen concentration on hyphal elongation rate and branching frequency of hyphae, and radial extension rate of colonies of A. oryzae. We found similar saturation constants (K(O2)) of 0.1% (v/v in the gas phase) for oxygen concentration described with Monod kinetics, for branching frequency of hyphae and colony extension rate. When A. oryzae was grown as an over-culture on wheat-flour model substrate at 0.25% (v/v) oxygen concentration, the reduction in growth was more pronounced than as individual hyphae and a colony on PDA medium. Experimental results also showed that the specific alpha-amylase production rate under the condition of 0.25% (v/v) oxygen was reduced. Because the value of K(O2) is relatively low, it is reasonable to simplify the kinetics of growth of A. oryzae to zero-order kinetics in coupled diffusion/reaction models.     

Aspergillus oryzae alpha-amylase partition in potassium phosphate-polyethylene glycol aqueous two-phase systems

The aim of this work is to study the partitioning of alpha-amylase from Aspergillus oryzae in polyethylene glycol-potassium phosphate systems formed by polymers of different molecular masses with different total concentrations, several NaCl concentrations and different volume ratio between the phases and at different temperatures. The enzyme was partitioned towards the top phase in the 2000-molecular-mass polyethylene glycol systems and towards the bottom phase in the other systems analyzed with higher molecular mass. The protein-medium interaction parameter (A) was determined; it increased in the same way as PEG molecular mass. The enthalpic and entropic changes found, in general, were negative and were shown to be associated by an entropic-enthalpic compensation effect suggesting that the ordered water structure in the chain of polyetyleneglycol plays a role in protein partition. The recovery in each of the phases was calculated in order to choose the best systems to be applied to enzyme isolation either from a polymer-rich or a polymer-poor phase.Enzymatic activity, circular dichroism and fluorescence were studied for the protein alone and in the presence of the different phases of the aqueous two-phase systems (ATPSs) in order to understand how they affect the enzymatic structure and the role of the protein-polymer interaction in the partitioning process. Secondary structure is not affected, in general, by the presence of the phases that do affect the enzymatic activity; therefore, there should be a change in the tertiary structure in the enzyme active site. These changes are more important for PEG 8000 than for PEG 2000 systems according to the results of the quenching of the intrinsic fluorescence. In a bio-separation process, the A. oryzae alpha-amylase could be isolated with ATPSs PEG 2000/Pi or PEG 8000/Pi with a high recovery, in the top or bottom phases, respectively.     

Insoluble complex formation between alpha-amylase from Aspergillus oryzae and polyacrylic acid of different molecular weight

The insoluble complex formation between alpha-amylase and the strong anionic polyelectrolyte polyacrylic acid was studied by using turbidimetric and enzymatic activity. The highest molecular weight polyacrylic acid (100,000 Da and 240,000 Da) proved to be suitable precipitating agents. They were insoluble at pH lower than 4–5, with a stoichiometric ratio polymer mol per protein mol of 1:52 and 1:154, respectively. Electrostatic interactions are not the only factor in the formation of insoluble complexes. High percentage of alpha-amylase enzymatic activity maintains throughout time, even in the presence of polyelectrolyte.The application of precipitation conditions found when applying a bovine homogenate showed that it is not suitable for purification even if it proved to be useful methodology for the concentration of the enzyme and can be used as a first step of purification.     

Rapid induction of alpha-amylase by nongrowing mycelia of Aspergillus oryzae.

A rapid induction system for synthesis of alpha-amylase by the funga Aspergillus oryzae M-13 was established. The mycelia were prepared from 20-h cultures grown on a peptone-glycerol medium and starved for 5 h; maltose was the optimum inducer tested. During h 1 of induction, formation of both intra- and extracellular alpha-amylases occurred at an almost identical rate (70 to 80 microgram/g of cells-h) without a detectable lag period. After a 1-h induction period, a remarkable increase in the extracellular concentration of the enzyme occurred, and a maximum rate (330 microgram/g of cells-h) was reached after 1.5 h of induction. During h 2 of induction, no significant change in mycelial weight was observed. Purified samples of intra- and extracellular enzymes formed in the induction system showed identical properties as examined by behavior in diethylaminoethyl-cellulose column chromatography, gel filtration, discontinuous gel electrophoresis, electrofocusing, optimal conditions for the reaction, heat stability, and molecular weight.     

Rapid induction of alpha-amylase by nongrowing mycelia of Aspergillus oryzae.

A rapid induction system for synthesis of alpha-amylase by the funga Aspergillus oryzae M-13 was established. The mycelia were prepared from 20-h cultures grown on a peptone-glycerol medium and starved for 5 h; maltose was the optimum inducer tested. During h 1 of induction, formation of both intra- and extracellular alpha-amylases occurred at an almost identical rate (70 to 80 microgram/g of cells-h) without a detectable lag period. After a 1-h induction period, a remarkable increase in the extracellular concentration of the enzyme occurred, and a maximum rate (330 microgram/g of cells-h) was reached after 1.5 h of induction. During h 2 of induction, no significant change in mycelial weight was observed. Purified samples of intra- and extracellular enzymes formed in the induction system showed identical properties as examined by behavior in diethylaminoethyl-cellulose column chromatography, gel filtration, discontinuous gel electrophoresis, electrofocusing, optimal conditions for the reaction, heat stability, and molecular weight.     

Efficient production and partial characterization of aspartyl aminopeptidase from Aspergillus oryzae

Aims: Aspartyl aminopeptidase (DAP) has a high degree of substrate specificity, degrading only amino-terminal acidic amino acids from peptides. Therefore, attention is focused here on the efficient production of this enzyme by a recombinant Aspergillus oryzae and characterization of its biochemical properties. Methods and Results: The gene encoding DAP was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a Co²⁺-containing medium. The enzyme was extracted from the mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high reactivity towards peptide substrate rather than synthetic substrates. Conclusions: Recombinant A. oryzae DAP was purified to homogeneity with an increased specific activity, when cultivated in a Co²⁺-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help in increasing the specific activity of other metalloproteases and their functional analysis. Significance and Impact of the Study: Recombinant DAP produced using a cobalt ion in culture media of A. oryzae and purification process allow high yield of the enzyme activity.     

Protease and alpha-amylase synthesis by washed cells of Aspergillus oryzae 251-90

Regularities of protease and alpha-amylase production by washed cells of Aspergillus oryzae 251-90 were being studied. The results obtained enabled us to assume a constitutive character of the both enzymes synthesis by the given producer. Sources of carbon, nitrogen and sulphur take part in regulation of the protease production, whereas in the case of the alpha-amylase synthesis only carbon sources that are important. Elimination of phosphorus from the medium affects the synthesis of both enzymes. Celatin stimulates the production of the two enzymes, being a supplier of amino acids.     

Model for carbohydrase action. Aspergillus oryzae alpha-amylase degradation of maltotriose.

Aspergillus oryzae alpha-amylase catalyzes degradation of oligosaccharides by a variety of pathways. We present here a quantitative study of the degradation of maltotriose by this amylase. Our results lead to a scheme involving multiple transglycosylation reactions and shifted binding due to simultaneous binding of two substrate molecules. The scheme is able to account for the diverse body of information collected for the enzyme. The effect of substrate concentration on the products of maltotriose degradation is correctly predicted over a 10(4)-fold concentration range, and the time course of maltotriose degradation is closely approximated by this scheme. The initial velocity data, which show deviation from Michaelis-Menton kinetics, are also consistent with the formulated scheme. The scheme is proposed as a general model of carbohydrase action.     

Application of a statistical design to the optimization of parameters and culture medium for alpha-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product)

The production optimization of alpha-amylase (E.C.3.2.1.1) from Aspergillus oryzae CBS 819.72 fungus, using a by-product of wheat grinding (gruel) as sole carbon source, was performed with statistical methodology based on three experimental designs. The optimisation of temperature, agitation and inoculum size was attempted using a Box-Behnken design under the response surface methodology. The screening of nineteen nutrients for their influence on alpha-amylase production was achieved using a Plackett-Burman design. KH(2)PO(4), urea, glycerol, (NH(4))(2)SO(4), CoCl(2), casein hydrolysate, soybean meal hydrolysate, MgSO(4) were selected based on their positive influence on enzyme formation. The optimized nutrients concentration was obtained using a Taguchi experimental design and the analysis of the data predicts a theoretical increase in the alpha-amylase expression of 73.2% (from 40.1 to 151.1 U/ml). These conditions were validated experimentally and revealed an enhanced alpha-amylase yield of 72.7%.