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.

Production, purification and applications of raw starch degrading and calcium-independent α-amylase from soil rich in extremophile.

Calcium independent, raw starch hydrolyzing, acidic α-amylase (66 kDa) was synthesized by Bacillus subtilis S113 that is an aerobic, rod-shaped and Gram +ve bacteria. Purification of the enzyme was performed by HiTrap Capto Q (Ion-exchange chromatography; 19.28 fold; 22.41% yield). The purified enzyme was found stable at broad acidic pH (4-6.5) and high-temperature range (40-80 °C), that fulfilled the necessary criteria and laid the foundation to be utilized in starch saccharification industry. Kinetic studies of the enzyme revealed that Km and Vmax of the enzyme was 0.22% and 357.14 U/mg respectively. Scanning electron microscopy studies showed that the enzyme was capable of completely hydrolyzing raw wheat and potato starch, further confirming its role in the starch industry. It was found that only 7.93% of the activity was loss at 4 °C when kept for one year.

Enhanced production of α-amylase by Penicillium chrysogenum in liquid culture by modifying the process parameters.

In this paper, we have assessed the role of changing physicochemical parameters and substrate types on the production of α-amylase enzyme from Penicillium chrysogenum, with a view to determining the optimal conditions required for its maximum production. The findings of this research revealed that, at pH 6 using linseed oil cake as substratum, α-amylase enzyme production was maximum (550.0 U/mL), when the fungi was incubated for 6 days at 30 °C in 0.1 M acetate buffer. Further, reasonably good production of the α-amylase enzyme was also observed at pH 9 with all the experimented carbon sources as substrates. Moreover, statistical analysis, using analysis of variance (ANOVA) carried out to study the impact of different studied parameters on the α-amylase enzyme production revealed that incubation period of 6-18 days is highly significant (p = 0.01) factor in amylotic activity of the P. chrysogenum. Under the researched out optimal conditions, P. chrysogenum is an economically viable option for the industrial and biotechnological production of α-amylase enzyme.

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.

Production and characterization of alpha-amylase from mango kernel by Fusarium solani NAIMCC-F-02956 using submerged fermentation.

Microbial production of enzymes using low valued agro industrial wastes is gaining importance globally. Mango is one of the major fruit processed into a variety of products. During processing 40-50% of solid waste is generated in form of peel and stones. After decortications of mango stone, kernel is obtained which is a rich source of starch (upto 60%). It was utilized as a substrate for alpha-amylase production using Fusarium soloni. Maximum alpha-amylase production (0.889 U g(-1)) was recorded using a substrate concentration of 5% (w/v), pH-4 and temperature 30 degrees C on 9th day of incubation. Supplementation of production medium with micronutrients viz., Ca2+, Fe2+ or Mg2+ improved the enzyme production while, Zn2+, B3+ or Mn2+ ions exhibited inhibitory effect. The extracellular protein was precipitated by ammonium sulphate up to 70% saturation, dialyzed and purified (27.84 fold) by gel-exclusion (Sephadex G-75) chromatography. Protein profiling on 12% SDS-PAGE revealed three bands corresponding to 26, 27 and 30 kDa molecular sizes. The optimum amylase activity was achieved at pH 5.0 at 40 degrees C. The Michaelis constant (KM), Vmax and activation energy (-Ea) were found to be 3.7 mg ml(-1), 0.24 U mg(-1) and 42.39 kJ mole(-1), respectively.

Dose- and tissue-specific interaction of monoterpenes with the gibberellin-mediated release of potato tuber bud dormancy, sprout growth and induction of α-amylases and ß-amylases.

Gibberellins (GA) are involved in bud dormancy release in several species. We show here that GA-treatment released bud dormancy, initiated bud sprouting and promoted sprout growth of excised potato tuber bud discs ('eyes'). Monoterpenes from peppermint oil (PMO) and S-(+)-carvone (CAR) interact with the GA-mediated bud dormancy release in a hormesis-type response: low monoterpene concentrations enhance dormancy release and the initiation of bud sprouting, whereas high concentrations inhibit it. PMO and CAR did, however, not affect sprout growth rate after its onset. We further show that GA-induced dormancy release is associated with tissue-specific regulation of α- and ß-amylases. Molecular phylogenetic analysis shows that potato α-amylases cluster into two distinct groups: α-AMY1 and α-AMY2. GA-treatment induced transcript accumulation of members of both α-amylase groups, as well as α- and ß-amylase enzyme activity in sprout and 'sub-eye' tissues. In sprouts, CAR interacts with the GA-mediated accumulation of α-amylase transcripts in an α-AMY2-specific and dose-dependent manner. Low CAR concentrations enhance the accumulation of α-AMY2-type α-amylase transcripts, but do not affect the α-AMY1-type transcripts. Low CAR concentrations also enhance the accumulation of α- and ß-amylase enzyme activity in sprouts, but not in 'sub-eye' tissues. In contrast, high CAR concentrations have no appreciable effect in sprouts on the enzyme activities and the α-amylase transcript abundances of either group. The dose-dependent effects on the enzyme activities and the α-AMY2-type α-amylase transcripts in sprouts are specific for CAR but not for PMO. Different monoterpenes therefore may have specific targets for their interaction with hormone signalling pathways.

Strain improvement and genetic characterization of indigenous Aspergillus flavus for amylolytic potential.

Aspergillus flavus FCBP-231, a filamentous fungus, was genetically modified for its ability to reveal extra cellular alpha-amylase activity. For strain improvement, the selected strains were subjected to UV irradiation (5-40 min exposure) and EMS treatment (50-300 microg mL(-1)) for hyper activity of an alpha-amylase enzyme. The mutants were quantitatively compared with the parental strain. UV and chemical mutagenesis brought about a dramatic enhancement in enzymatic activity. The mutant strains Af-UV-5.3 and Af-Ch-5.7 exhibited 79 and 110% more enzyme activity than the native strain A. flavus FCBP-231. This improvement in enzyme activity of the mutants suggests that they are suitable strains to be used in biotechnology. RAPD-PCR analysis revealed different patterns of amplicons of native as well as mutant derivatives, which suggested that the mutation imparted changes in the genetic make up of the mutants probably involved enzyme production control.

Production and characterization of alpha-amylase from Aspergillus niger JGI 24 isolated in Bangalore.

Five fungal isolates were screened for the production of alpha-amylase using both solid-state and submerged fermentations. The best amylase producer among them, Aspergillus niger JGI 24, was selected for enzyme production by solid-state fermentation (SSF) on wheat bran. Different carbon and nitrogen supplements were used to enhance enzyme production and maximum amount of enzyme was obtained when SSF was carried out with soluble starch and beef extract (1% each) as supplements. Further attempts to enhance enzyme production by UV induced mutagenesis were carried out. Survival rate decreased with increase in duration of UV exposure. Partial purification of the enzyme using ammonium sulphate fractionation resulted in 1.49 fold increase in the enzyme activity. The enzyme showed a molecular weight of 43 kDa by SDS-PAGE. Metal ions Ca2+ and Co2+ increased the enzyme activity. The enzyme was optimally active at 30 degrees C and pH 9.5.

Affinity purification of trypsin inhibitor with anti-Aspergillus flavus activity from cultivated and wild soybean.

Trypsin inhibitors (TI) from wild-type soybean (Glycine soya) (WBTI) and domesticated soybean (Glycine max) (SBTI) were purified using prepared chitosan resin-trypsin as filler on the affinity chromatography column. The SBTI/WBTI purification fold by affinity chromatography was 718- and 279-fold, with the activity recovery of 62% and 59%, respectively. It was found that SBTI and WBTI exerted a strong inhibition of Aspergillus. flavus growth, with IC(50) of 1.6 and 1.0 micromol/l. This growth inhibition was possibly the result of the inhibition on alpha-amylase activity of A. flavus by both the SBTI and WBTI. This was further supported by the fact that in the presence of SBTI and WBTI at 9.0 and 6.0 microg/g (peanut) on peanuts inhibited the germination and growth of A. flavus. Accordingly, characterization of the mode of action of SBTI and WBTI could constitute a first step leading to resistance to A. flavus invasion.

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.

Production and characterization of a thermostable alpha-amylase from Nocardiopsis sp. endophyte of yam bean.

Thermostable amylolytic enzymes have been currently investigated to improve industrial processes of starch degradation. Studies on production of alpha-amylase by Nocardiopsis sp., an endophytic actinomycete isolated from yam bean (Pachyrhizus erosus L. Urban), showed that higher enzyme levels were obtained at the end of the logarithmic growth phase after incubation for 72 h at pH 8.6. Maximum activity of alpha-amylase was obtained at pH 5.0 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C, and 50% of residual activity at 90 degrees C for 10 min. Extracellular enzyme from Nocardiopsis sp. was purified by fractional precipitation with ammonium sulphate. After 60% saturation produced 1130 U mg-1 protein and yield was 28% with purification 2.7-fold. The enzyme produced by Nocardiopsis sp. has potential for industrial applications.

Possible involvement of phosphoinositide-Ca2+ signaling in the regulation of alpha-amylase expression and germination of rice seed (Oryza sativa L.).

We have studied the effects of neomycin, a potent inhibitor of inositol phospholipid-specific phospholipase C (PLC), on the germination of rice seed and the gibberellin-induced expression of alpha-amylase in the aleurone layer and the scutellar tissues. It was shown that, in the absence of exogenous Ca2+, neomycin markedly reduced the germination speed and seedling growth of rice seeds and inhibited the gibberellin-induced expression of alpha-amylase in both secretory tissues. In addition, neomycin decreased the formation of inositol 1,4,5-trisphosphate (IP3) in the gibberellin-treated aleurone layer and the scutellar tissues. However, the inhibitory effects on the germination speed and the expression of alpha-amylase activity were overcome by supplementation of Ca2+. In addition, gibberellin elevated the level of IP3, and ABA prevented the gibberellin-induced formation of IP3, although ABA alone did not alter the IP3 level. The expression of a membrane-bound PLC molecule in rice aleurone layer was shown to be induced by gibberellin, and the gibberellin-induced expression of PLC was markedly delayed by treatment with ABA. These results strongly suggest that the phosphoinositide-Ca2+ signal transduction pathway may play an important role in the gibberellin-induced expression of alpha-amylase molecules closely related to the germination processes of rice seed.

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.

Cloning of a Ca(2+)-ATPase gene and the role of cytosolic Ca2+ in the gibberellin-dependent signaling pathway in aleurone cells.

The ultimate goal of this investigation was to identify intermediary steps in the gibberellin (GA)-dependent signaling pathway in rice aleurone cells. By using a differential display approach, a number of putative GA-responsive genes were isolated. One of them, a GA-responsive Ca(2+)-ATPase gene, was identified and partially characterized. A genomic clone and a cDNA clone were isolated and sequenced. The deduced amino acid sequence showed that this protein resembles an endoplasmic reticulum membrane Ca(2+)-ATPase. In a transient assay in rice aleurone cells, expression of the introduced Ca(2+)-ATPase cDNA bypassed the GA requirement for stimulating the expression of a major target gene, the alpha-amylase c gene (Osamy-c). This result suggests that GA-dependent expression of this Ca(2+)-ATPase gene (OsCa-atpase) plays an important role in the GA-dependent signal-transduction pathway. To investigate the possible involvement of other proteins and genes that may affect the intracellular Ca2+ level, compounds which can block different putative steps in the signal-transduction pathway were introduced into rice aleurone cells, and then the level of the OsCa-atpase transcript or the Osamy-c transcript was monitored. In the presence of GA, the rice Ca(2+)-ATPase and the Ca2+ channels appeared to co-regulate the local concentration of cytosolic Ca2+. The release of Ca2+ from the internal stores to the cytoplasm was presumably initiated by inositol-1,4,5-triphosphate which reached a peak level within 25 min after GA induction. As a second messenger, Ca2+ binds to calmodulin (CaM), and the Ca2+/CaM complex regulates the cytosolic Ca2+ by affecting expression of the OsCa-atpase. Finally, a working model is proposed for the GA-dependent signaling pathway in aleurone cells.

Structure and expression of alpha-amylase gene from Vigna mungo.

A single copy of the alpha-amylase gene, composed of three introns and four exons, was found in Vigna mungo. Examination of levels of alpha-amylase and its mRNA in detached cotyledons indicated that attachment of the embryonic axis is not required for expression of the gene in cotyledons of germinating seeds.