Determination of Amylolytic and Proteolytic Enzyme Activity Characteristics of Thermophilic Actinobacteria Isolated from Amasya-Terziköy Thermal Spring

Significant differences in pH dependence of proteolytic and amylolytic enzyme activities in the gut microbiota between crucian carps from different habitats (the Dniester River and Kuchurgan Reservoir) have been revealed for the first time. The proteolytic activity in fish from these water bodies reaches a peak at pH values varying from 7 to 10, while the peak of amylolytic activity is consistently observed at pH 7.0. Both proteolytic and amylolytic activities are higher in fish from the Kuchurgan Reservoir (characterized by increased trophicity and high abundance of bacterioplankton) than in fish from the Dniester. For comparison, the effect of pH on proteinase and glycosidase activities in the gut mucosa and chyme of the same fish has been studied.

Strains of the ruminal fungi Neocallimastix patriciarum, Orpinomyces joyonii, and Piromyces communis were grown on cellobiose and on cereal grains and then examined for proteolytic and amylolytic activities. On cellobiose all three fungi displayed similar activities, with the exception of little amylolytic activity in the cell-associated fraction of N. patriciarum. Growth on the cereal grains barley, corn, and wheat showed differences in proteolytic and amylolytic activities amongst the ruminal fungi and between the cereal grains. The data suggest that while these fungi are capable of fermenting the cereal grains the mode of enzymatic attack varies both with the particular fungus and the type of cereal grain.

Background: Utilization of endophytic bacteria in order to increase productivity may be viable alternative to organic fertilizers. The goal is to reduce soil pollution and to preserve environment in the spirit of ecological agriculture. Any organism that at some part of its life cycle, colonize the internal plant tissues without causing any type of harm to the host plant are endophytes. Endophytes are known to promote plant growth and nutrient gain. They may improve the plant’s ability to tolerate various types of abiotic and biotic stresses, and enhance the resistance of plants to insects and pests. This study was carried out to isolate beneficial endophytic bacteria from various sources and to screen and identify plant growth promoting activities of isolated bacteria. Further to study field effects of best found endophytic bacterial isolates and its commercial application. Sources were collected from different regions of Maharashtra such as Geolife Research Farm Pune, Mangrove leaves from Mumbai, Khopoli ghat and examined for their bacterial diversity. Isolated bacteria subjected for various tests such as Indole Acetic Acid production, Ammonia production, Hydrogen Cyanide production, Antibiotic resistance and sensitivity assay, Proteolytic and Amylolytic activities, etc. Results: In total 34 bacterial isolates were obtained from five different source. Some isolates were found to exhibit multiple beneficial characteristics to promote plant growth. Among them isolate number E14 has shown maximum production of IAA, same isolate also found positive for ammonia production and HCN production. Whereas isolate number E23 obtained from mangrove leaves is showing maximum nitrogen 12.6mg, positive for Ammonia & HCN production and also showing positive results for Amylolytic and Proteolytic activities. Conclusion: According to field results, the interaction between plant and endophytes can promote plant immunity and play a significant role in sustainable agriculture. Endophytes displaying beneficial traits that can be exploited in agriculture biotechnology

Hot water temperature can be a place for the development of thermophilic bacteria. Thermophilic bacteria produce thermostable enzymes which are needed in various industrial fields such as agriculture, food, detergent, pharmacology, and bioremediation agents. This study aims to isolate and characterize potential proteolytic and amylolytic bacteria from the Bayanan hot spring. Samples were collected from two distinct locations within the geothermal environment: the hot spring source itself and the associated water storage pools. The collected samples underwent a dilution and isolation process to obtain pure bacterial cultures. Subsequently, these isolates were inoculated onto selective media, including skim milk agar (SMA) and nutrient agar (NA), to assess their proteolytic and amylolytic activities, respectively. The presence and extent of these enzymatic activities were determined by measuring the diameter of the clear zone surrounding each bacterial colony. The analysis revealed the presence of eight proteolytic bacterial isolates and twenty amylolytic isolates. Notably, a hydrolysis index threshold of ≥2.5 was implemented to identify isolates with high potential for proteolytic and amylolytic activity. Based on this criterion, two proteolytic bacterial isolates (codes B2-12-C3 and B1-12-B3) exhibited a proteolytic index exceeding 2.5. Furthermore, eight amylolytic isolates displayed an index above 2.5 (codes B1-10-A1, B1-10-A3, B2-8-A1, B2-8-A2, B2-10-A1, B2-10-A2, B2-12-C1, and B2-12-C2). These isolates, particularly those with high hydrolysis index values, has potential as bioremediation agents due to their demonstrated ability to efficiently hydrolyze proteins and starches.

El objetivo de este estudio fue evaluar el efecto de la temperatura y el pH sobre la actividad de las enzimas hidrolíticas de la región cecal de cuyes (Cavia porcellus). Este es el primer reporte sobre la actividad enzimática en el ciego de cuyes, evidenciando que existe actividad amilolítica, celulolítica, proteolítica y lipolítica. Los valores de las actividades evaluadas estuvieron en los siguiente rangos: actividad amilolítica (0.150 - 0.499 U/mg proteína), celulolítica (0.013 - 0.022 U/mg proteína), proteolítica (0.104 - 0.290 U/mg proteína) y lipolítica (2.227 - 4.049 U/mg proteína). Tanto en la actividad celulolítica, lipolítica y proteolítica hubo interacción entre temperatura y pH. La actividad amilolítica solo se vio afectada por la temperatura, evidenciándose mayor actividad enzimática a mayor temperatura (40°C). La actividad amilolítica es estable a cualquiera de los tres pH probados en este estudio. Además, la actividad amilolítica fue 10 veces más alta que la actividad celulolítica.

The chronic effects that polychlorinated biphenyls (PCBs) have on the activities of proteinases and carbohydrates in intestinal mucosa and chyme in juvenile roach Rutilus rutilus (L.) have been studied for the first time. Upon consuming food with PCB 50.8 ng/g wet weight for 218 days, the exposition of fingerlings in aquariums with dirt bottoms (contents PCB 425.6 ng/g dry weight) reduces the proteolytic activity of mucosa to a greater degree than amylolytic activity; the activity of sucrase changes in different directions. In 2-year-old fish receiving PCB only with food (50.8 ng/g wet weight bottom), proteolytic and amylolytic activities of the mucosa are reduced 18 and 35% in comparison with the control (52 days); the sucrose activity is reduced 13% (52 days) and 22% (169 days). In most cases the relation of amylolytic activity to proteolytic activity in fish of the experimental groups is less than in the control. The enzyme activities in chyme change in different directions, depending on the terms and conditions of the experiment.

The digestive tract of fish is colonized by a great number of heterotrophic bacteria. Reported numbers of viable aerobic and anaerobic bacteria range between 10 and 10 and 6.6 ¥ 10-1.6 ¥ 10 CFU g-1, respectively. Consequently, it is widely accepted that fish intestines provide favorable ecologic niches for various taxa of microflora. Resident intestinal bacteria, which could be important for fish digestion, may produce extracellular enzymes supporting food decomposition. If this is the case, intestinal bacteria should exhibit enzymatic activity in relation its food composition. This study examined in vitro proteolytic and amylolytic activities of microflora from the intestinal tract of adult roach Rutilus rutilus L. (widespread in Lithuanian lakes fish, which feed mainly on mollusks and macrophytes). A total of five fish specimens of roach from Dringis Lake, located in the Aukstaitija National Park, Lithuania, were collected by fishing and transported on ice to the laboratory. Intestinal tracts were aseptically removed, divided and separated into three regions (foregut, midgut, hindgut). The roach is a stomachless fish, its intestinal tract comprised from a relatively straight tube with several loops. The foregut comprised the segment from the esophagus until the first loop; midgut was intestine and the rectum was hindgut. The gut content was squeezed from each region and homogenized in a nine-fold volume of a diluent of phosphate buffer (pH 7.0). From five fish specimens were made three integrated intestinal contents samples from foregut, midgut and hindgut. Each sample was serially diluted and 0.05 mL portions of diluents were spread on meat-peptone agar (Oxoid). A total of 60 aerobic and facultative anaerobic bacteria (20 from each part of intestine) were isolated at random for further analysis. Isolates were identified to the genus/family using methods and schemes described by Sakata and Ringo. Each isolate was incubated in a rotary shaker (100 r.p.m.) at 22°C in a mineral salts medium, which contained (l-1): NH4Cl, 9.0 g; K2HPO4, 0.5 g; MgSO4 7H2O, 0.5 g; CaCO3, 3.0 g; glucose, 20.0 g; and tap water (pH 7.0) enriched 10 g of peptone for detection of proteolytic activity (PA), and 10 g of soluble starch for detection of amylolytic activity (AA). Extracellular PA was measured every 24 h for 3 days, AA for 4 days. PA and AA were determined using methods described in ‘Enzyme preparation’. PA was determined by a modified method of Anson. This method is based on the protein (casein) hydrolysis under the action of the protease in the investigated solution, inactivation of protease and precipitation of unhydrolyzed protein with trichloroacetic acid. Neutral protease was tested because intestinal contents pH in carp fish fluctuated between 6.8 and 7.2. A PA unit, used for activity assessment, is the enzyme amount that in 1 min at 30°C converts the amount of casein corresponding to 1 mmol of tyrosine to a form non-precipitable by thrichloroacetic acid. PA was calculated in unit mL-1 (U mL-1) using formula:

Composting is an alternative for recycling organic waste. Microorganisms that can degrade the components of organic waste are an essential part of composting. Isolation and selection of bacteria with the ability to degrade the elements of organic waste are the first steps to obtain an organic waste degrading agent. This study aimed to determine the characteristics and the enzymatic potential (cellulolytic, amylolytic, and proteolytic) of bacteria isolated from soil, cow dung and kitchen waste as candidates for organic waste degradation agents. The research begins with sampling and isolation of bacteria. The isolates obtained were tested qualitatively for their amylolytic, cellulolytic and proteolytic activities. Furthermore, bacterial isolates were characterized morphologically and biochemically. A total of 30 bacterial isolates were isolated from soil samples, cow dung and kitchen waste. The screening result showed that 6 isolates had amylolytic activity, 7 isolates had cellulolytic activity and 3 isolates had proteolytic activity. These bacterial isolates showed various morphological and biochemical characteristics. In general, soil samples, kitchen waste and cow dung contain potential bacteria as organic waste degrading agents

It is known that ionophoric antibiotics regulate ruminal fermentation, improve the utilization of feed protein, and prevent the occurrence of ketosis and steatosis in ruminants. Ionophoric antibiotics and β-acids of hops have a similar spectrum of biological activity, that is, they inhibit the vital activity of most gram-positive microorganisms of the rumen. Bacteria, like other living organisms, need vitamin E as an active antioxidant for cell membranes. The toxicity of tocopherol is very low, so adding it to the diet of ruminants in larger quantities can stimulate celluloselytic rumen bacteria and compensate for the negative effect of ionophores on fiber breakdown. Since rumen bacteria break down a significant part of dietary choline, methionine and carnitine, ruminants must receive them in a protected form, so their influence on rumen fermentation is insignificant. Three groups of cows of the Ukrainian dairy black-spotted breed with milk yields of 5 or more thousand kg during the previous lactation were formed: with signs of clinical ketosis — 4 animals; with subclinical ketosis — 5 animals and clinically healthy — 5 animals. For a month, cows with ketosis were given a treatment supplement containing crushed granules of hop cones (20 g), vitamin E (3 g), and rumen protected choline (50 g), methionine (20 g) and carnitine (1 g). Clinically healthy cows were used as control. In the blood of cows with subclinical ketosis, the additive increased the concentration of glucose and decreased the concentration of β-hydroxybutyrate, these indicators were within the normal range. In cows with symptoms of clinical ketosis, using of the feed additive also reduced the concentration of β-hydroxybutyrate (P<0.01), but it was still higher than normal. In sick cows, amylolytic and lipolytic activity was lower than in healthy cows (P<0.05–0.01). Celluloselytic activity was lower only in cows with clinical ketosis. The proteolytic activity of rumen content changed in the opposite way; it was higher in sick cows (P<0.05–0.01). This is a consequence of the increase in the number and activity of hyper producing ammonia bacteria in the rumen, what is characteristic for ketosis. After treatment of cows with subclinical ketosis, the celluloselytic and amylolytic activities in the rumen fluid were equal to the corresponding indicators of healthy cows, and the proteolytic activity was even slightly lower (P<0.05) than in the control group. Treatment of cows with clinical form of ketosis was not as effective, although the general trends remained. During subclinical and clinical ketosis, a greater amount of ammonia was found in the rumen fluid (P<0.05–0.01), because of higher proteolytic activity. In both forms of ketosis, the concentration of volatile fatty acids in the rumen decreased, and the concentration of lactate increased (P<0.05–0.01). After the treatment, these indicators in cows with subclinical ketosis approached the healthy animals, while the condition of cows with clinical ketosis improved, but the concentration of ammonia continued to differ from healthy animals.

Wheat breadmaking properties in dependance on wheat enzymes status and climate conditions

One of the promising directions in the development of modern feed production is the creation of functional feed additives based on hydrolytic enzyme preparations, which increase the efficiency of nutrient assimilation and enhance overall productivity. At the same time, in aquaculture, due to the likelihood of rapid leaching of additives from the feed into the water, there is a need to ensure greater stability and prolonged action of such enzyme preparations. This paper presents the results of an experimental and model analysis of the efficiency of immobilizing the enzyme preparation Protosubtilin on a natural inorganic carrier — basalt tuff — with the aim of developing functional feed additives for use in fish farming technologies. The immobilization process was carried out by adsorbing an enzyme solution onto ground basalt tuff from the “Polytske-2” deposit, which is characterized by high porosity, significant mechanical strength, and excellent adsorption capacity. The study analyzed the effect of the type of immobilization (static or dynamic), exposure duration, and temperature factor on the amount of enzyme preparation adsorbed on the carrier, as well as on the retention of its proteolytic and amylolytic activities. It was shown that dynamic immobilization conditions (mixing) do not contribute to increased efficiency of the process and even reduce it with prolonged exposure (3 hours), which is probably due to the disturbance of stable enzyme binding on the carrier surface. Under static conditions, an increase in the amount of adsorbed enzyme preparation was observed with an increase in temperature from +20 °C to +30 °C; however, a significant decrease in enzyme activity, especially proteolytic activity, was also recorded, which limits the feasibility of using higher temperatures. The optimal immobilization mode was determined to be static adsorption for 1–3 hours at +20 °C, which ensures the best combination of sufficient enzyme binding and retention of catalytic activities. The obtained results confirm the potential of using basalt tuff as an effective, affordable, and environmentally safe carrier for creating enzyme-based functional feed additives, which will contribute to the development of modern technologies in feed production and aquaculture. Keywords: enzyme immobilization, basalt tuff, Protosubtilin, feed additives, proteolytic activity, amylases, aquaculture.

The use of detergents with enzymatic activity is very popular if we consider their real economic advantages - reducing energy and water consumption but also reducing the environmental impact by using more biodegradable components related to organic and inorganic components. The purpose of our study was to evaluate the impact of environmental factors such as the variability of water hardness in the public water supply network and the temperature applied to the amylolytic, lipolytic and proteolytic enzyme activity of 6 detergents available on the market. The determinations were performed by modified methods from the European Pharmacopoeia 8th Edition in determination the enzymatic activity of the pancreas powder. The determinations show a significant influence of increased water hardness on lipase and protease activity in detergent (p [0.01 *) between extreme hardness values, while amylolytic activity does not vary significantly with increasing water hardness.

Recently, a paternally expressed quantitative trait nucleotide (QTN) in the regulatory sequence of the IGF-II gene with effects on muscle growth and fat deposition was discovered in the pig. This QTN is also known as the IGF-II intron3 G3072A mutation. The aim of the current study was to determine the effects of age, muscle type, and IGF-II genotype (Apat, mutant allele vs. Gpat, wild-type allele) on muscle proteolytic and lipolytic enzyme activities. At approximately 4, 8, 16, and 26 wk of age, boars (n = 6 to 15 per genotype x age group) were slaughtered and mu- and m-calpain (CALP), calpastatin (CAST), cathepsins (CATH) B+L and H, acid lipase, and phospholipase activities were measured in Longissimus thoracis et lumborum, Semimembranosus, and Triceps brachii muscle samples taken soon after slaughter. Activities of CATH B+L and H, mu- and m-CALP, and acid lipase were not affected by the IGF-II genotype. Activity of CAST was greater (P < 0.005) and m-CALP:CAST was less (P < 0.05) in Apat animals. Because CAST activity and m-CALP:CAST are known to be related to protein degradation, satellite cell fusion, or both, it is likely that differences in proteolytic enzyme activities are involved in the greater percentage of muscle mass in Apat animals. Age and muscle type influenced proteolytic and lipolytic enzyme activities (P < 0.05), except for mu- and m-CALP (no effect of muscle) and acid lipase (no effect of age). The same pattern in mu-CALP, CAST, and m-CALP:CAST with age was found during growth for the 3 muscles, although clear differences (P < 0.05) between muscles existed. In general, and in agreement with previous reports, greater enzyme activities were found in the more oxidative Triceps brachii muscle compared with the other 2 muscles. A remarkable increase (P < 0.05) from 16 to 26 wk of age in mu-CALP, CAST, mu-CALP:CAST, and CATH H and a large decrease (P < 0.05) in acid phospholipase and m-CALP:CAST was found. For m-CALP and CATH B+L, a gradual decrease (P < 0.05) was found with age. Although age effects on enzyme activities could only partly be interpreted biologically in relation to the muscle growth rate, this study showed that proteolytic and lipolytic enzyme activities change during growth.

Lactic acid bacteria (LAB) play a central role in the food industry due to their ability to produce beneficial metabolites and enhance the technological and sensory qualities of fermented products. Additionally, they contribute to human health by supporting immune function and maintaining gut microbiota balance through probiotic effects. This study aimed to isolate and characterize LAB from dromedary camel milk (DCM) collected in semi-arid regions of Algeria, evaluating their technological functionalities and antimicrobial activities. A total of 31 LAB strains were isolated from raw DCM samples. Strains were identified using MALDI-TOF MS and characterized for acidification kinetics, lipolytic, proteolytic, and amylolytic activities, exopolysaccharide (EPS) and acetoin production, and antimicrobial properties against common foodborne pathogens. Four species were identified, with Enterococcus italicus reported for the first time in this environment. Significant inter-strain variability (p < 0.0001) was observed in all tested properties. Three strains (BLC9, BLC12, BLC14) acidified milk rapidly to pH 4.6 within 12 h. Proteolytic activity was detected in 87.10% of strains, while EPS and acetoin were produced by 29.03 and 48.39%, respectively. Lipolytic and amylolytic activities were generally weak. Notably, 74.19% of the strains exhibited antimicrobial activity, inhibiting at least one pathogen, with inhibition zones varying significantly (p < 0.0001). Dromedary camel milk from Algerian semi-arid regions represents a rich source of LAB strains with promising technological and antimicrobial potential. These native isolates could be further developed for use in additive-free fermented foods and natural biopreservation systems, supporting sustainable and functional food innovation.

The enzymatic reactions of microbial communities are rapid and adequate to changes in the composition of organic matter and environmental factors; therefore, they can be used as descriptors of biological responses in freshwater systems. The objective of this work was to determine the proteolytic enzymatic activity in the Almendares and San Juan rivers (western Cuba) and their relationship with physicochemical and microbiological indicators. The Almendares River (Havana, Cuba) presented water quality index between very poor and unusable for consumption, which makes impossible for recreational uses. In contrast, the waters of the San Juan River (Artemisa, Cuba) are classified as excellent and good in most of the sampling stations during the study period. The greatest proteolytic activities were detected in the San Juan River compared to the Almendares River, which indicates that in more contaminated ecosystems, the proteolytic enzyme activity decreases compared to another less impacted system. In addition, the annual variation in the proteolytic activity of both rivers was evidenced. In the San Juan River, a correlation was observed among proteolytic activity, the concentration of proteolytic bacteria and the physicochemical indicators of water quality, suggesting that proteolytic activity is related to naturally occurring contaminants. In contrast, in the Almendares River, this relationship was not observed, which suggests that the proteolytic activity is not directly affected by the indicators measured in this study.