Optimization and scale-up of α-amylase production by Aspergillus oryzae using solid-state fermentation of edible oil cakes.

BACKGROUND: Amylases produced by fungi during solid-state fermentation are the most widely used commercial enzymes to meet the ever-increasing demands of the global enzyme market. The use of low-cost substrates to curtail the production cost and reuse solid wastes are seen as viable options for the commercial production of many enzymes. Applications of α-amylases in food, feed, and industrial sectors have increased over the years. Additionally, the demand for processed and ready-to-eat food has increased because of the rapid growth of food-processing industries in developing economies. These factors significantly contribute to the global enzyme market. It is estimated that by the end of 2024, the global α-amylase market would reach USD 320.1 million (Grand View Research Inc., 2016). We produced α-amylase using Aspergillus oryzae and low-cost substrates obtained from edible oil cake, such as groundnut oil cake (GOC), coconut oil cake (COC), sesame oil cake (SOC) by solid-state fermentation. We cultivated the fungus using these nutrient-rich substrates to produce the enzyme. The enzyme was extracted, partially purified, and tested for pH and temperature stability. The effect of pH, incubation period and temperature on α-amylase production using A. oryzae was optimized. Box-Behnken design (BBD) of response surface methodology (RSM) was used to optimize and determine the effects of all process parameters on α-amylase production. The overall cost economics of α-amylase production using a pilot-scale fermenter was also studied. RESULTS: The substrate optimization for α-amylase production by the Box-Behnken design of RSM showed GOC as the most suitable substrate for A. oryzae, as evident from its maximum α-amylase production of 9868.12 U/gds. Further optimization of process parameters showed that the initial moisture content of 64%, pH of 4.5, incubation period of 108 h, and temperature of 32.5 °C are optimum conditions for α-amylase production. The production increased by 11.4% (10,994.74 U/gds) by up-scaling and using optimized conditions in a pilot-scale fermenter. The partially purified α-amylase exhibited maximum stability at a pH of 6.0 and a temperature of 55 °C. The overall cost economic studies showed that the partially purified α-amylase could be produced at the rate of Rs. 622/L. CONCLUSIONS: The process parameters for enhanced α-amylase secretion were analyzed using 3D contour plots by RSM, which showed that contour lines were more oriented toward incubation temperature and pH, having a significant effect (p < 0.05) on the α-amylase activity. The optimized parameters were subsequently employed in a 600 L-pilot-scale fermenter for the α-amylase production. The substrates were rich in nutrients, and supplementation of nutrients was not required. Thus, we have suggested an economically viable process of α-amylase production using a pilot-scale fermenter.

How Different Snacks Produce a Distinct Effect in Salivary Protein Composition.

Saliva secretion changes in response to different stimulation. Studies performed in animals and humans suggest that dietary constituents may influence saliva composition, although the dynamics of these changes, and how they are specific for each type of food, are little known. The objective of the present study was to access the short-term effects of different foods in salivation and salivary protein composition. Twelve participants were tested for four snacks (yoghurt, bread, apple and walnuts). Non-stimulated saliva was collected before and at 0', 5' and 30' after each snack intake. Flow rate, total protein, alpha-amylase enzymatic activity and salivary protein profile were analyzed. Yoghurt and apple were the snacks resulting in higher salivary changes, with higher increases in flow rate and alpha-amylase activity immediately after intake. The expression levels of immunoglobulin chains decreased after the intake of all snacks, whereas cystatins and one pink band (proline-rich proteins-PRPs) increased only after yoghurt intake. Walnut's snack was the one resulting in lower changes, probably due to lower amounts eaten. Even so, it resulted in the increase in one PRPs band. In conclusion, changes in saliva composition varies with foods, with variable changes in proteins related to oral food processing and perception.

Leucine Affects α-Amylase Synthesis through PI3K/Akt-mTOR Signaling Pathways in Pancreatic Acinar Cells of Dairy Calves.

Dietary nutrient utilization, particularly starch, is potentially limited by digestion in dairy cow small intestine because of shortage of α-amylase. Leucine acts as an effective signal molecular in the mTOR signaling pathway, which regulates a series of biological processes, especially protein synthesis. It has been reported that leucine could affect α-amylase synthesis and secretion in ruminant pancreas, but mechanisms have not been elaborated. In this study, pancreatic acinar (PA) cells were used as a model to determine the cellular signal of leucine influence on α-amylase synthesis. PA cells were isolated from newborn Holstein dairy bull calves and cultured in Dulbecco's modifed Eagle's medium/nutrient mixture F12 liquid media containing four leucine treatments (0, 0.23, 0.45, and 0.90 mM, respectively), following α-amylase activity, zymogen granule, and signal pathway factor expression detection. Rapamycin, a specific inhibitor of mTOR, was also applied to PA cells. Results showed that leucine increased ( p < 0.05) synthesis of α-amylase as well as phosphorylation of PI3K, Akt, mTOR, and S6K1 while reduced ( p < 0.05) GCN2 expression. Inhibition of mTOR signaling downregulated the α-amylase synthesis. In addition, the extracellular leucine dosage significantly influenced intracellular metabolism of isoleucine ( p < 0.05). Overall, leucine regulates α-amylase synthesis through promoting the PI3K/Akt-mTOR pathway and reducing the GCN2 pathway in PA cells of dairy calves. These pathways form the signaling network that controls the protein synthesis and metabolism. It would be of great interest in future studies to explore the function of leucine in ruminant nutrition.

Expression of the enzymatically active legumain-like cysteine proteinase TvLEGU-1 of Trichomonas vaginalis in Pichia pastoris.

The legumain-like cysteine proteinase TvLEGU-1 from Trichomonas vaginalis plays a major role in trichomonal cytoadherence. However, its structure-function characterization has been limited by the lack of a reliable recombinant expression platform to produce this protein in its native folded conformation. TvLEGU-1 has been expressed in Escherichia coli as inclusion bodies and all efforts to refold it have failed. Here, we describe the expression of the synthetic codon-optimized tvlegu-1 (tvlegu-1-opt) gene in Pichia pastoris strain X-33 (Mut+) under the inducible AOX1 promoter. The active TvLEGU-1 recombinant protein (rTvLEGU-1) was secreted into the medium when tvlegu-1-opt was fused to the Aspergillus niger alpha-amylase signal peptide. The rTvLEGU-1 secretion was influenced by the gene copy number and induction temperature. Data indicate that increasing tvlegu-1-opt gene copy number was detrimental for heterologous expression of the enzymatically active TvLEGU-1. Indeed, expression of TvLEGU-1 had a greater impact on cell viability for those clones with 26 or 29 gene copy number, and cell lysis was observed when the induction was carried out at 30 °C. The enzyme activity in the medium was higher when the induction was carried out at 16 °C and in P. pastoris clones with lower gene copy number. The results presented here suggest that both copy number and induction temperature affect the rTvLEGU-1 expression in its native-like and active conformation.

Utilization of glycerin byproduct derived from soybean oil biodiesel as a carbon source for heterologous protein production in Pichia pastoris.

Crude glycerol, also known as glycerin, is the main byproduct of the biodiesel industry. It has been estimated that up to 40,000 tons of glycerin will be produced each year by 2020. This study evaluated the value-added use of crude glycerol derived from soybean biodiesel preparation as a carbon source for heterologous protein production using the yeast Pichia pastoris. Eleven glycerin samples were obtained by methanolysis of soybean oil using different acids or bases as catalysts. Cell growth experiments showed that crude glycerol containing either potassium or sodium hydroxide resulted in 1.5-2 times higher final cell densities when compared to glycerol P.A. Finally, crude glycerol containing sodium hydroxide was successfully utilized for constitutive heterologous α-amylase production in P. pastoris. This study demonstrated that crude glycerol without any purification steps may be directly used as carbon source for protein production in P. pastoris.

Construction of an artificial secYEG operon allowing high level secretion of α-amylase.

The key components of the major secretion pathway in bacteria, the Sec pathway, are the proteins SecA, an ATPase that generates the energy required for protein translocation, and the heterotrimeric protein complex SecYEG, which functions as the preprotein-conducting channel through the cytoplasmic membrane, named translocon. Overexpression of exoproteins can cause jamming of the membrane, e.g., due to a shortage of translocons. Therefore, we decided to increase the number of translocons by first creating an artificial secYEG operon and then fusing it to an inducible promoter. By Western- and Northern-blot analysis, we could first show that the amount of the SecY protein and the secYEG transcript can be increased after addition of the inducer. Next, we proved by immunoblot experiments that the amount of α-amylase secreted in the presence of increased amounts of SecYEG proteins is enhanced. Therefore, increasing the number of translocons is accompanied by a concomitant increase in the amount exoenzymes. This finding will be of importance for high-level secretion of recombinant proteins.

Influence of media composition on the production of alkaline α-amylase from Bacillus subtilis CB-18.

BACKGROUND: Starch, a homopolysaccharide is an important and an abundant food reserve and energy source. Starches are processed to yield different products which find many industrial applications. Alpha-amylases hydrolyze starch by cleaving α-1,4-glucosidic bonds and have been used in food, textile and pharmaceutical industries [Sun et al. 2010]. Enzymatic conversion of starch with amylase presents an economically superior alternative to the conventional method of starch gelatinization. Alkaline α-amylase has an important position in the global enzyme market as a constituent of detergent. In this paper, we screened soil bacteria and an isolate, alkalophilic Bacillus subtilis CB-18 was found to produce an alkaline α-amylase in different media. MATERIAL AND METHODS. Screening of the isolates for amylolytic activity was carried out by growing bacteria isolated from the soil in starch agar plates and subsequently staining the plates with iodine solution to reveal zones of hydrolysis of starch. The selected isolate, Bacillus subtlis CB-18 was grown in different media at alkaline pH to evaluate the influence of media composition on alkaline α-amylase production. Enzyme assay was carried out by growing the culture in a broth medium and obtaining cell - free culture supernatant after centrifugation at 2515 × g for 15 minutes Amylase activity was determined by incubating 0.5 ml of crude enzyme solution in 0.1M Tris/HCl buffer (pH 8.5) with 0.5 ml of 1% soluble starch solution. The reaction was terminated by the addition of DNS reagent and reducing sugar produced from the amylolytic reaction was determined. RESULTS: Bacillus subtilis CB-18 used for this work was selected because it produced 7 mm zone diameter on starch agar plate. This organism was cultured in different alkaline broth media containing 2% soluble starch as inducer carbohydrate for α-amylase production. Among the carbon sources used for enzyme production, sorbitol was the best to stimulate enzyme production with α-amylase activity of 758 U/mL after 48 h. Peptone was the best nitrogen source for enzyme production with α-amylase activity of 680 U/mL after 48 h. Metal ions including Ca (2+), Mn(2+) and Mg(2+) stimulated enzyme production while Hg(2+) and Ag(+) repressed enzyme production. The best enzyme yields were observed in basal media containing agro-based substrates. CONCLUSION: This work reports the production of alkaline α-amylase by Bacillus subtlis CB-18 in different media. Enzyme production was highest when agro-based media were used to formulate the media.

Synthesis of vesicle cargo determines amplitude of Ca(2+)-sensitive exocytosis.

Here we examine the potential coupling between the synthesis of secretory proteins and the sensitivity of exocytosis to the concentration of free Ca(2+) in the cytosol ([Ca(2+)](i)) in plant cell. We therefore monitor in tobacco protoplasts the excursion of the membrane capacitance in response to an elevation of [Ca(2+)](i) as a measure for exocytotic activity. The data show that a ramp like elevation of [Ca(2+)](i) generates in protoplasts from wild type plants and from transgenic plants, which overexpress the secreted α-amylase, an exocytotic burst with an initial steep and a subsequent slow phase. The largest capacitive burst is obtained in α-amylase producing plants and the amplitude of the [Ca(2+)](i) evoked C(m) excursion is a function of the amylase synthesis of the plants. The data support a model according to which plant cells have at least two serial [Ca(2+)](i) sensitive processes in the final steps of their exocytotic pathway. The overproduction of a secreted cargo does not affect the kinetics of this process but the number of vesicles in pools upstream of the [Ca(2+)](i) sensitive steps.

Changes in selected physical property and enzyme activity of rice and barley koji during fermentation and storage.

UNLABELLED: Koji are solid-state fermentation products made by inoculating steamed grains with the spores of fungi, particularly Aspergillus spp. This research was undertaken to identify the fermentation and storage conditions optimal for the production and maintenance of selected hydrolytic enzymes, such as α-amlyase and protease, in koji. Steamed rice and barley were inoculated with 2 × 10 ¹¹ Aspergillus oryzae spores per kilogram of grains and fermented for 118 h in a growth chamber at 28 to 32 °C with controlled relative humidities. Samples were drawn periodically during fermentation and storage at -20, 4, or 32 °C, and α-amylase and protease activity, mold counts, a(w), moisture contents, and pH of collected samples were determined. It was observed that the a(w), moisture contents, and pH of the koji were influenced by the duration of fermentation and temperature of storage. The α-amylase activity of both koji increased as the populations of A. oryzae increased during the exponential growth phase. The enzyme activity of barley koji was significantly higher than that of rice koji, reaching a peak activity of 211.87 or 116.57 U at 46 and 58 h, respectively, into the fermentation process. The enzyme activity in both products started to decrease once the mold culture entered the stationary growth phase. The protease activities of both koji were low and remained relatively stable during fermentation and storage. These results suggest that rice and barley koji can be used as sources of α-amylase and desired enzyme activity can be achieved by controlling the fermentation and storage conditions. PRACTICAL APPLICATION: Amylases and proteases are 2 important hydrolytic enzymes. In the food industry, these enzymes are used to break down starches and proteins while reducing the viscosity of foods. Although amylases and proteases are found in plants and animals, commercial enzymes are often produced using bacteria or molds through solid state fermentation, which is designed to use natural microbial process to produce enzymes in a controlled environment. A properly produced and maintained koji with a high hydrolytic enzyme activity can serve as an important source of the enzymes for the food industry.

Raw starch fermentation to ethanol by an industrial distiller's yeast strain of Saccharomyces cerevisiae expressing glucoamylase and α-amylase genes.

Industrial strains of a polyploid, distiller's Saccharomyces cerevisiae that produces glucoamylase and α-amylase was used for the direct fermentation of raw starch to ethanol. Strains contained either Aspergillus awamori glucoamylase gene (GA1), Debaryomyces occidentalis glucoamylase gene (GAM1) or D. occidentalis α-amylase gene (AMY), singly or in combination, integrated into their chromosomes. The strain expressing both GA1 and AMY generated 10.3% (v/v) ethanol (80.9 g l(-1)) from 20% (w/v) raw corn starch after 6 days of fermentation, and decreased the raw starch content to 21% of the initial concentration.

Amylase and cyclic amp receptor protein expression in human diabetic parotid glands.

BACKGROUND: Salivary dysfunction and oral disorders have been described in both type 1 and type 2 diabetes mellitus. However, the cellular and molecular consequences of diabetes on oral tissues remain to be ascertained. The purpose of this investigation was to study, by means of electron microscopy, the morphologic and molecular changes that occur in salivary glands during diabetes. METHODS: Biopsy samples of parotid glands were excised from non-diabetic and diabetic (type 1 and type 2) consenting patients and processed by standard methods for routine morphology and electron microscopic immunogold labeling. Specific antibodies were used to determine and quantify the expression of secretory proteins (alphaamylase and the regulatory subunit of type II protein kinase A). RESULTS: Morphologic changes in the diabetic samples included increased numbers of secretory granules, and alterations in internal granule structure. Quantitative analysis of immunogold labeling showed that labeling densities were variable among the parotid gland samples. In type 1 diabetes amylase expression was greater than in non-diabetic glands, whereas in type 2 diabetes it was not significantly changed. Expression of type II regulatory subunits was slightly, although not significantly, increased in acinar secretory granules of type 1 diabetic samples and was unchanged in type 2 diabetic samples. CONCLUSIONS: Our data show that diabetes elicits specific changes in secretory protein expression in human salivary glands, thus contributing to the altered oral environment and oral disease associated with diabetes.

Development of an α-amylase reporter system for efficient screening of clones with highly expressed heterologous protein in Hansenula polymorpha.

To check feasibility and effectiveness of the α-amylase reporter system, two vectors were designed and tested using hepatitis B virus surface antigen (HBsAg) and Homo sapiens granulocyte-macrophage colony stimulating factor 2 (hGM-CSF2) as a model. By integrating the vector containing two independent cassettes into the same genome locus, high-producing clones of HBsAg (or hGM-CSF2) were screened using the α-amylase as a reporter. Results show there was a positive correlation (Correlation coefficient, R (2) > 0.95) between the yield of recombinant proteins and the α-amylase activity of corresponding transformants, which was independent of the gene dosage.

Antioxidant status, alpha-amylase production, growth, and survival of hemoglobin bearing Escherichia coli exposed to hypochlorous acid.

In the present work, two matched strains of E. coli that bear a recombinant R-amylase gene (MK57) or the R-amylase gene and vgb (MK79-hemoglobin expressing strain) were exposed to HOCl. In these cells, glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), alpha-amylase production, growth and lethality were assessed in the presence and absence of HOCl. It was observed that the hemoglobin makes cells highly susceptible to killing by HOCl. The maximum survival for both strains was with stationary phase cells at any concentration of HOCl. Both strains grown in the presence of 0.0125-0.075 mg/liter HOCl showed a substantial increase in SOD activity and GSH level, with MK79 being the most increased strain in this respect, while the level of CAT activity was decreased in a dose depended manner. Growth of MK57 and MK79 strains decreased as HOCl concentration increased. However, HOCl at concentration above zero enhanced alpha-amylase production (about 2-fold) in both MK79 and MK57. Furthermore, total amylase production (at all HOCl concentrations) by MK79 was always greater than that by MK57. The results indicate that except for survival, the hemoglobin helps cells to grow better and produces more recombinant products and activates general defense systems more in response to oxidative stress when compared with the non-hemoglobin-containing counterpart.

Characterization of the seabass pancreatic alpha-amylase gene and promoter.

Seabass (Lates calcarifer) pancreatic alpha-amylase gene was cloned and characterized. The alpha-amylase cDNA has 1620 bp and the deduced polypeptide has 522 amino acids. Southern blot indicated that there are two gene copies in the seabass genome. Sequence analysis showed that except for the loss of an intron in seabass, the coding region and the exon/intron boundaries are highly homologous to those of mammalian amylases. However, the promoter regions are distinctively divergent. To investigate the seabass amylase promoter, a series of deletion mutants was generated and fused to the luciferase reporter gene, followed by studies of their functional activity in rat AR42J cell line. Besides identifying several potential regulatory elements that have been previously identified in the human and mouse pancreatic amylase promoter, we have identified a glucocorticoid response element (GRE). However, while the human and mouse pancreatic amylase promoters are highly homologous between nucleotide -160 and transcription start site where GRE is located, the 5' promoter deletion mutants revealed that the GRE of the seabass amylase promoter was located far upstream -947 to -776 bp of the promoter. Site-directed mutagenesis of the putative GRE and electrophoretic mobility shift assays (EMSA) confirmed that this region was responsible for dexamethasone induction. However, no functional PTF-1 binding site, which is responsible for pancreas-specific transcription in higher vertebrates, was identified in seabass amylase promoter. Instead a Hepatocyte Nuclear Factor 3 binding site was found to modulate the amylase promoter expression. The evolutionary significance of this divergence in promoter regulation between seabass and mammals requires further studies.

Coconut oil cake--a potential raw material for the production of alpha-amylase.

Solid-state fermentation (SSF) was carried out using coconut oil cake (COC) as substrate for the production of alpha-amylase using a fungal culture of Aspergillus oryzae. Raw COC supported the growth of the culture, resulting in the production of 1372 U/gds alpha-amylase in 24 h. Process optimization using a single parameter mode showed enhanced enzyme titre, which was maximum (1827 U/gds) when SSF was carried out at 30 degrees C for 72 h using a substrate with 68% initial moisture. Supplementation with glucose and starch further enhanced enzyme titre, which was maximum (1911 U/gds) with 0.5% starch. However, maltose inhibited the enzyme production. Studies on the effect of addition of external organic and inorganic nitrogenous compounds further showed a positive impact on enzyme synthesis by the culture. Increase of 1.7-fold in the enzyme activity (3388 U/gds) was obtained when peptone at 1% concentration was added to the fermentation medium. The enzyme production was growth-related, the activity being the maximum when the fungal biomass was at its peak at 72 h. Use of COC as raw material for enzyme synthesis could be of great commercial significance. To the best of our knowledge this is the first report on alpha-amylase production using COC in SSF.

Two-dimensional gel electrophoresis analysis of the abundance of virulent exoproteins of group A streptococcus caused by environmental changes.

Group A streptococci regulate the expression of virulence factors in response to environmental change. In order to investigate this mechanism, the growth of group A streptococci and the abundance of virulent exoprotein production in culture supernatant were analyzed by two-dimensional gel electrophoresis (2-D electrophoresis) under several culture conditions. Judging from alterations in their growth, group A streptococci were affected by various environmental stresses. Under high O(2) and low CO(2 )concentrations, streptococcal pyrogenic exotoxin B (SpeB) and streptococcal pyrogenic exotoxin F (SpeF) significantly decreased, and the streptococcal inhibitor of complement (Sic) increased. At 30 degrees C, increases in endo-beta- N-acetylglucosaminidase (EndoS) and alpha-amylase were also detected, while at 41 degrees C EndoS became undetectable and SpeB and SpeF decreased. Sic, SpeF and mitogenic factor 3 (Mf3) decreased when cells were cultured in higher NaCl concentrations, and EndoS disappeared following culture of the cells in high glucose concentration. An increase in acid phosphatase and a decrease in several other proteins were detected when the cells were cultivated in high iron concentrations. These results suggest that group A streptococci have a versatile adaptation system that responds to several environmental stresses by altering the level of exoprotein production.

Gibberellin-mediated proteasome-dependent degradation of the barley DELLA protein SLN1 repressor.

DELLA proteins are nuclear repressors of plant gibberellin (GA) responses. Here, we investigate the properties of SLN1, a DELLA protein from barley that is destabilized by GA treatment. Using specific inhibitors of proteasome function, we show that proteasome-mediated protein degradation is necessary for GA-mediated destabilization of SLN1. We also show that GA responses, such as the aleurone alpha-amylase response and seedling leaf extension growth, require proteasome-dependent GA-mediated SLN1 destabilization. In further experiments with protein kinase and protein phosphatase inhibitors, we identify two additional signaling steps that are necessary for GA response and for GA-mediated destabilization of SLN1. Thus, GA signaling involves protein phosphorylation and dephosphorylation steps and promotes the derepression of GA responses via proteasome-dependent destabilization of DELLA repressors.

Thermostable alpha-amylase production by an extreme thermophile Bacillus thermooleovorans.

AIMS: alpha-Amylase production by a newly isolated thermophile, Bacillus thermooleovorans, was studied under different cultivation conditions. METHODS AND RESULTS: The influence of various carbon and nitrogen sources on alpha-amylase production was quantified in batch fermentation in shake flasks. Starch and tryptone were observed to be the ideal carbon and nitrogen sources, respectively. Cultivation of the organism in a chemically defined medium consisting of glucose, riboflavin, cysteine, MgSO4, K2HPO4 and NaCl led to a near twofold increase in the production of alpha-amylase in comparison with that in the complex medium. The increase in enzyme production was achieved using vitamins and amino acids. When the organism was grown in a laboratory fermenter in the optimized complex medium, the noticeable effects were the near abolition of the lag phase, a 2.2-fold increase in enzyme production and a reduction in optimal production time from 12 to 4-5 h. CONCLUSION: Enhancement of amylase production was achieved under various cultivation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus thermooleovorans produces a calcium-independent and thermostable amylase which can find use in starch saccharification.

Activity of glycogen depolymerizing enzymes in extracts from brain tumor tissue (anaplastic astrocytoma and glioblastoma multiforme).

An approximately threefold increase in glycogenolytic activity of the neutral alpha-1,4-glucosidase and a twofold increase in the same activity of the acid isoform have been found in extracts of anaplastic astrocytoma and glioblastoma multiforme tumors of brain tissue. "Maltase activity" of the respective enzymes increased by 60-80% in both kinds of tumor extracts. However a significant decrease in a-amylase and almost complete disappearance of phosphorylase activities have also been found in both kinds of tumors.

Production of amylases by Aspergillus tamarii

A strain of Aspergillus tamarii, a filamentous fungus isolated from soil, was able to produce both Ó-amylase and glucoamylase activities in mineral media supplemented with 1(per cent) (w/v) starch or maltose as the carbon source. Static cultivation led to significantly higher yields than those obtained using shaking culture. The production of amylases was tolerant to a wide range of initial culture pH values (from 4 to 10) and temperature (from 25 to 42degree C). Two amylases, once Ó-amylase and one glucoamylase, were separated by ion exchange chromatography. Both partially purified enzymes had optimal activities at pH values between 4.5 and 6.0 and were stable under acid conditions (pH 4.0-7.0). The enzymes exhibited optimal activities at temperatures between 50(degree) and 60(degree) C and wete stable for more than ten hours at 55(degree) C.