The Effect of Aluminum Oxide Nanoparticles on the Activity of Amylases of the Enzyme Preparation of Microbial Origin

На основании анализа литературных данных показано, что изучение влияния наночастиц, в том числе оксида алюминия, на активность целевых ферментов микробных ферментных препаратов, применяемых в пивоваренном производстве, целесообразно. Приведены данные о влиянии наночастиц Al2O3 на процесс гидролиза крахмала в модельных средах под действием амилаз ферментного препарата «АПСубтилин П». Показано, что присутствие наночастиц в реакционной среде приводит к снижению амилолитической активности и, как следствие, снижению концентрации редуцирующих веществ в гидролизате на 12-15% (при содержании наночастиц 0,1 мг/см3) и на 34% (при содержании наночастиц 1,0 мг/см3) по сравнению с контролем. Установлено, что увеличение продолжительности ферментативного гидролиза с 60 до 120 мин приводит к ослаблению ингибирующего воздействия наночастиц Al2O3 на амилолитическую активность ферментного препарата. При этом присутствие наноразмерных объектов указанного типа не оказало негативного воздействия на результаты лабораторного затирания, проводимого по настойному способу с тремя паузами: основные показатели первого сусла опытных вариантов не уступали аналогичным характеристикам контрольного образца. На основании приведенных данных сделано заключение о нежелательности присутствия наночастиц Al2O3 в реакционных средах амилаз микробного происхождения в концентрациях, превышающих 0,1 мг/см3. The expediency of studying the effect of nanoparticles, in particular, aluminum oxide, on the activity of target enzymes of enzyme preparations used in brewing is substantiated based on the analysis of the literature data. Data on the effect of Al2O3 nanoparticles on the process of starch hydrolysis in model media under the action of amylases of the enzyme preparation «APSubtilin P» are presented. It has been shown that the presence of nanoparticles in the reaction medium leads to a decrease in amylolytic activity and, as a consequence, a decrease in the concentration of reducing substances in the hydrolyzate by 12-15% (at nanoparticle content of 0.1 mg/cm3) and by 34% (at nanoparticle content of 1, 0 mg/cm3) compared to the control. It was found that the increase in the duration of enzymatic hydrolysis from 60 to 120 min leads to a weakening of the inhibitory effect of Al2O3 nanoparticles on the amylolytic activity of the enzyme preparation. At the same time, the presence of nanoscale objects of this type did not have a negative impact on the results of laboratory mashing, carried out according to the infusion method with three pauses: the main indicators of the first wort of the experimental variants were not inferior to those of the control sample. Based on the data presented, it was concluded that the presence of Al2O3 nanoparticles in the reaction media of amylases of microbial origin in concentrations exceeding 0.1 mg/cm3 is undesirable.

RESEARCH OF AMYLOLYTIC ACTIVITY OF CULTURAL LIQUIDS IN BIOTECHNOLOGY OF SYMBIOTIC CROPS МЕDUSOMYCES GISEVII AND ORYZAMYCES INDICI

Based on the literature review found that the natural symbionts Medusomyces gisevii and Oryzamyces indici biotechnology are a valuable objects. It is urgent to find ways to impact on it in order to obtain these or other products of its life activity. At present, it is urgent to search for microorganisms producing enzymes, including amylase. One of the most promising in terms of biological objects is a natural microbial symbiont Medusomyces Gisevii (tea fungus) and Oryzamyces Indici, which, thanks to the not identical, microbiological composition and different growing conditions may have a different composition of metabolites. Studies of the amylolytic activity of the culture liquid Medusomyces Gisevii and Oryzamyces indici with different cultivation periods have been carried out. Cultivation of the fungus was carried out in the laboratory according to the classic method. The optimal concentration of sucrose for Medusomyces Gisevii and Oryzamyces Indici biomass growth was set at 5%. Sucrose concentration of 15% and above is not recommended for use due to inhibition of biomass growth with increasing concentration of carbohydrates in the culture medium. It is established that these symbionts start to show the amylolytic activity already on the 10th day of cultivation cultivation in standard nutrient medium, medium supplemented with 10 % milk and serum-based medium and stores it in the course of the experiment (30 days). Over time the amylolytic activity increases. However, the intensity of metabolism of the microorganisms, the criterion of which is the ratio of the total and exogenous amylase is most pronounced in the early stages of cultivation. It was found that the culture medium of polycultures shows high amylolytic activity. This fact allows us to consider the Medusomyces Gisevii and Oryzamyces Indici inoculum as a promising biotechnological raw material source of amylase enzyme.

Microbial Interactions between Amylolytic and Non-Amylolytic Lactic Acid Bacteria Strains Isolated during the Fermentation of Pozol

Pozol is a Mexican beverage prepared from fermented nixtamalized maize dough. To contribute to understanding its complex microbial ecology, the effect of inoculating on MRS-starch pure and mixed cultures of amylolytic Sii-25124 and non-amylolytic W. confusa 17, isolated from pozol, were studied on their interactions and fermentation parameters. These were compared with L. plantarum A6, an amylolytic strain isolated from cassava. Microbial growth, kinetic parameters, amylolytic activity, lactic acid production, and hydrolysis products from starch fermentation were measured. The population dynamics were followed by qPCR. L. plantarum A6 showed higher enzymatic activity, lactic acid, biomass production, and kinetic parameters than pozol LAB in pure cultures. Mixed culture of each pozol LAB with L. plantarum A6 showed a significant decrease in amylolytic activity, lactic acid yield, specific growth rate, and specific rate of amylase production. The interaction between Sii-25124 and W. confusa 17 increased the global maximum specific growth rate (µ), the lactic acid yield from starch (Ylac/s), lactic acid yield from biomass (Ylac/x), and specific rate of lactic acid production (qlac) by 15, 30, 30, and 40%, respectively compared with the pure culture of Sii-25124. Interactions between the two strains are essential for this fermentation.

Production, Partial Purification and Characterization of Two α-Amylase Isoforms from Saccharomyces cerevisiae strain YOP 1/2-2 Isolated from Tchapalo (Côte d’Ivoire)

Amylases play an important role in biotechnology and find applications in several industrial fields such as pharmaceutical, food, paper, cosmetics and detergents. Thus, it appears necessary to identify new sources of amylase, especially from microbial origin, due to the low energy consumption, cost-effective, high metabolic diversity, rapid cell growth, non-toxic and eco-friendly characteristics. In the present report, we carried out the production and partial purification of α-amylase by Saccharomyces cerevisiae strains isolated from Tchapalo, a traditional alcoholic beverage of Côte d'Ivoire. Five fungal isolates were screened initially for α-amylase production by using method of wells on Yeast Extract Peptone Dextrose Agar medium, a complete medium for yeast growth. One step DEAE-Sepharose Fast Flow was achieved for partial purification of α-amylase obtained. Among yeasts, isolate S. cerevisiae YOP 1/2-2 was able to provoke starch hydrolysis halo of 15.067±0.12 mm on starch agar plate after 48 h of incubation at 30°C. The partial purification of resulting enzyme showed two protein peaks, designated α-amylase 1 (AMY1) and α-amylase 2 (AMY2) with amylolytic activity and specific activities of 1.57-1.58 U/mg protein. Both isoforms (AMY1 and AMY2) were thermostable with optimal activity at 50 and 55°C, respectively, and at pH ranged from 4.5 to 5.3 in 0.1 M sodium acetate buffer. EDTA and Cd2+ strongly inhibited α-amylase activity by 72-75% and 64-65%, respectively, whereas cations Ca2+ and Mn2+ showed 85-99% and 71% increased amylolytic activity, respectively. All these properties show the potential uses of α-amylases from S. cerevisiae in the industrial transformation of starch.

The Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) Has Digestive Capacity to Degrade Complex Substrates: Functional Characterization and Heterologous Expression of an α-Amylase

Dendroctonus-bark beetles are natural agents contributing to vital processes in coniferous forests, such as regeneration, succession, and material recycling, as they colonize and kill damaged, stressed, or old pine trees. These beetles spend most of their life cycle under stem and roots bark where they breed, develop, and feed on phloem. This tissue is rich in essential nutrients and complex molecules such as starch, cellulose, hemicellulose, and lignin, which apparently are not available for these beetles. We evaluated the digestive capacity of Dendroctonus rhizophagus to hydrolyze starch. Our aim was to identify α-amylases and characterize them both molecularly and biochemically. The findings showed that D. rhizophagus has an α-amylase gene (AmyDr) with a single isoform, and ORF of 1452 bp encoding a 483-amino acid protein (53.15 kDa) with a predicted signal peptide of 16 amino acids. AmyDr has a mutation in the chlorine-binding site, present in other phytophagous insects and in a marine bacterium. Docking analysis showed that AmyDr presents a higher binding affinity to amylopectin compared to amylose, and an affinity binding equally stable to calcium, chlorine, and nitrate ions. AmyDr native protein showed amylolytic activity in the head-pronotum and gut, and its recombinant protein, a polypeptide of ~53 kDa, showed conformational stability, and its activity is maintained both in the presence and absence of chlorine and nitrate ions. The AmyDr gene showed a differential expression significantly higher in the gut than the head-pronotum, indicating that starch hydrolysis occurs mainly in the midgut. An overview of the AmyDr gene expression suggests that the amylolytic activity is regulated through the developmental stages of this bark beetle and associated with starch availability in the host tree.

Effect of Different Steeping and Malting Regimen on the Amylolytic Activities of Some Improved Nigerian Sorghum Grain Varieties

Three Nigerian improved sorghum varieties were evaluated to ascertain how different steeping and malting regimen affect their amylolytic enzyme development. Steeping incorporated air rest and continuous steep regime for 72 h. Samples were withdrawn every 12 h. Germination was then carried out for four days before kilning at 50°C for 24 h. Grain and malt parameters were examined. Results obtained showed variations in the response of sorghum root length to steep regimen and time. CSR-02 gave maximum root length (3.32 cm) after 72 h of air rested steeping. CSR-02, Samsorg 44 and Samsorg 14 had germinative energies of 92.00 ± 4.24, 94.00 ± 1.41 and 96.00 ± 1.41%; germinative capacities of 91.00 ± 1.41, 75.50 ± 2.12 and 88.00 ± 2.83; water sensitivities of 6.50 ± 2.12, 13.50 ± 1.44 and 1.00 ± 0.41 respectively. TKW results were 29.73 ± 0.32, 33.85 ± 1.54 and 33.51 ± 0.41 kg for CSR-02, Samsorg 44 and Samsorg 14 respectively. Variations in the response of the sorghum varieties to various conditions of steep regime and steep period were also observed. Steeping for 48 h seems to be the optimum time for the development of amylolytic activity in all the sorghum varieties at both steeping regimens. Samsorg 14 gave the highest total amylase activity (355.44 µg glucose equivalents), followed by Samsorg 44 (278.08 µg glucose equivalents). Samsorg 14 also showed the highest α-amylase development (276.93 µg glucose equivalents). Air rest was found to be show greater effect on β-amylase development in all the sorghum varieties.

Enzymatic hydrolysis of high gravity extruded corn starch media

Increase of solubles concentration in processable liquid media is one of the trends of technological development of starchy materials bioconversion. It promotes the reduction in operating costs, in heat and energy consumption and increases the efficiency of using capacitive equipment. The use of thermoplastic extrusion in the bioconversion processes as a pretreatment stage is perspective. Extrusion provides intensive gelatinization of starch with a moisture content of 15-30 % that has become a prerequisite for the development of extrusion-hydrolytic technology for obtaining of concentrated hydrolysates from starch-containing raw materials. As a part of the technology development, the effect of the key factors of biocatalysis on the formation of hydrolysis products and the rheological properties of highly concentrated hydrolysates of corn starch has been studied. The dosage of thermostable α-amylase and the concentration of the medium were taken as independent variables. The ranges of variation of the factors were set in the range of 5-13 units of amylolytic activity per 1 g of starch and 40- 60 % soluble concentration in accordance with the central orthogonal second-order design of the experiment. The value of dextrose equivalent in the area of the studied factor space varied from 23 to 40. Dynamic viscosity values were in the range from 89 to 2219 mPa·s. The analysis of the results and the mathematical model showed that an increase in the dextrose equivalent in the hydrolysis products was facilitated by a decrease of the concentration of the medium and an increase in the dosage of α-amylase. The growth dynamics of the dextrose equivalent value decreased with an excess of the dosage of the enzyme preparation of 9 units of amylolytic activity per 1 g of starch. Rheological studies have shown that a dosage of α-amylase of 1- 13 units of amylolytic activity per gram of starch at 40 % concentration of the medium provided dynamic viscosity values in the range 89-780 mPa·s, which is sufficient for the subsequent stages of hydrolyzate processing. Increasing the concentration to 50-60 % requires the introduction of α-amylase at a dosage of more than 5 units of amylolytic activity to ensure a rheologically safe process.

Synthesis of Some Novel Fluorinated/Nonfluorinated α-Amino Acids, Bearing 3-Thioxo-5-oxo-1,2,4-triazin-6-yl and Steroidal Moieties, and Evaluation of Their Amylolytic Effects against Some Fungi, Part-II

Some new fluorinated/nonfluorinated α-amino acids bearing 3-thioxo-5-oxo-1,2,4-triazin-6-yl and steroidal moieties have been obtained from condensation of the corresponding amino-triazinones with the steroid (Epiandrosterone). This was followed by the addition of HCN and, finally, acidic hydrolysis. The structure of the targets was established from their elemental analysis and spectral data. The amylolytic activity of the new products was evaluated against some fungi.