Efficient hydrolysis of starch by α-amylase immobilized on cloisite 30B and modified forms of cloisite 30B by adsorption and covalent methods

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. 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. 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.

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

A novel high maltose-forming α-amylase from Rhizomucor miehei and its application in the food industry

Starch is an inexpensive and renewable raw material for numerous industrial applications. However, most starch-based products are not cost-efficient due to high-energy input needed in traditional enzymatic starch conversion processes. Therefore, α-amylase with high efficiency to directly hydrolyze high concentration raw starches at a relatively lower temperature will have a profound impact on the efficient application of starch. A novel raw starch digesting α-amylase (named AmyZ1) was screened and cloned from a deep-sea bacterium Pontibacillus sp. ZY. Phylogenetic analysis showed that AmyZ1 was a member of subfamily 5 of glycoside hydrolase family 13. When expressed in Escherichia coli, the recombinant AmyZ1 showed high activity at pH 6.0–7.5 and 25–50 °C. Its optimal pH and temperature were 7.0 and 35 °C, respectively. Similar to most α-amylases, AmyZ1 activity was enhanced (2.4-fold) by 1.0 mM Ca2+. Its half-life time at 35 °C was also extended from about 10 min to 100 min. In comparison, AmyZ1 showed a broad substrate specificity toward raw starches, including those derived from rice, corn, and wheat. The specific activity of AmyZ1 towards raw rice starch was 12,621 ± 196 U/mg, much higher than other reported raw starch hydrolases. When used in raw starch hydrolyzing process, AmyZ1 hydrolyzed 52%, 47% and 38% of 30% (w/v) rice, corn, and wheat starch after 4 h incubation. It can also hydrolyze marine raw starch derived from Chlorella pyrenoidosa, resulting in 50.9 mg/g DW (dry weight of the biomass) of reducing sugars after 4 h incubation at 35 °C. Furthermore, when hydrolyzing raw corn starch using the combination of AmyZ1 and commercial glucoamylase, the hydrolysis rate reached 75% after 4.5 h reaction, notably higher than that obtained in existing starch-processing industries. As a novel raw starch-digesting α-amylase with high specific activity, AmyZ1 efficiently hydrolyzed raw starches derived from both terrestrial and marine environments at near ambient temperature, suggesting its application potential in starch-based industrial processes.

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AmyZ1: a novel α-amylase from marine bacterium Pontibacillus sp. ZY with high activity toward raw starches

High-level expression of a novel α-amylase from Thermomyces dupontii in Pichia pastoris and its application in maltose syrup production.

The invention relates to an efficient preparation method and an application of thermomyces dupontii [alpha]-amylase. Thermomyces dupontii [alpha]-amylase gene is cloned and undergoes efficient expression in pichia pastoris; and by conducting high-density fermentation for 168h, the enzymatic activity of supernatant liquid can reach 38314.2U/mL, and a protein content can reach 28.7mg/mL. The optimumpH value and the optimum temperature of enzyme are respectively 6.5 and 60 DEG C; and the enzymatic activity is kept stable within a pH value ranging from 4.5 to 10.0 and at a temperature under 55 DEG C. The enzyme is free from dependency on Ca2+ and the enzyme has a certain trans-glucoside activity. The enzyme is applicable to the production of malt syrup in which a malt content is 51.8%; and the enzyme, when applied to steamed buns and breads, can increase specific volume and reduce hardness. The enzyme has a great application potential in food industry.

Efficient preparation method and application of thermomyces dupontii [alpha]-amylase

The invention relates to rhizomucor miehei alpha-amylase, and an encoding gene and an application of the rhizomucor miehei alpha-amylase. A protein has alpha-amylase activity; the gene of the proteinis efficiently expressed in pichia pastoris; the maximum enzyme activity after high density fermentation of the pichia pastoris is 29794.2U/mL; the specific enzyme activity after purification is 3502.1U/mg; an optimum reaction pH (potential of hydrogen) is 6.0; the protein keeps stable when the pH is 4.5-8.0; an optimum reaction temperature is 75 DEG C; and the protein keeps the higher enzymic activity below 65 DEG C and has better thermal resistance. The amylase can increase a specific volume of steamed bread by 7.7% and retard aging of the steamed bread when the amylase is applied to the steamed bread; and a maltose content can reach 54.1% when the amylase is applied to maltose production. The amylase has good utilization potentiality in the food industry.

Yu-chuan Wang

Papers

A novel high maltose-forming α-amylase from Rhizomucor miehei and its application in the food industry

High-level expression of a novel α-amylase from Thermomyces dupontii in Pichia pastoris and its application in maltose syrup production.

Efficient preparation method and application of thermomyces dupontii [alpha]-amylase

Rhizomucor miehei alpha-amylase, and encoding gene and application of rhizomucor miehei alpha-amylase