A Modified Brewing Procedure Informed by the Enzymatic Profiles of Gluten-Free Malts Significantly Improves Fermentable Sugar Generation in Gluten-Free Brewing

The mashing step underpins the brewing process, during which the endogenous amylolytic enzymes in the malt, chiefly β-amylase, α-amylase, and limit dextrinase, act concurrently to rapidly hydrolyze malt starch to fermentable sugars. With barley malts, the mashing step is relatively straightforward, due in part to malted barley’s high enzyme activity, enzyme thermostabilities, and gelatinization properties. However, barley beers also contain gluten and individuals with celiac disease or other gluten intolerances should avoid consuming these beers. Producing gluten-free beer from gluten-free malts is difficult, generally because gluten-free malts have lower enzyme activities. Strategies to produce gluten-free beers commonly rely on exogenous enzymes to perform the hydrolysis. In this study, it was determined that the pH optima of the enzymes from gluten-free malts correspond to regions already typically targeted for barley mashes, but that a lower mashing temperature was required as the enzymes exhibited low thermostability at common mashing temperatures. The ExGM decoction mashing procedure was developed to retain enzyme activity, but ensure starch gelatinization, and demonstrates a modified brewing procedure using gluten-free malts, or a combination of malts with sub-optimal enzyme profiles, that produces high fermentable sugar concentrations. This study demonstrates that gluten-free malts can produce high fermentable sugar concentrations without requiring enzyme supplementation.

Process Improvisation Strategies for the Enhancement of Lipase Activity

In this present study, lipase-producing bacteria were isolated and screened from an indigenous soil sample and were used for lipase production with high enzyme activity. In the production medium, different production media were screened and lipase production was induced by olive oil, 14 mL/L. It was observed from Luedeking and Piret model that the lipase production was mixed growth associated with maximum activity at 37°C and at pH 7. Statistical optimization using Response Surface Methodology was performed to understand the interaction of different parameters and the standardized conditions obtained were as follows: Peptone 10 g/L, yeast extract 7.5 g/L and olive oil 14 mL/L. The predicted data were validated and the model predicted was significant with a maximum specific activity of 1.1 µmole/min/mg proteins. The lipasespecific activity was enhanced by 10% and 23% after a single parameter and statistical optimization.

Preparation of enzymatically highly active pegylated-D-amino acid oxidase and its application to antitumor therapy

Background: D-amino acid oxidase (DAO) is an H2O2-generating enzyme, and tumor growth suppression by selective delivery of porcine DAO in tumors via the cytotoxic action of H2O2 has been reported. DAO isolated from Fusarium spp. (fDAO) shows much higher enzyme activity than porcine DAO, although the application of fDAO for antitumor treatment has not yet been determined. Objective: The purpose of this study was to prepare enzymatically highly active pegylated-fDAO, and to determine whether it accumulates in tumors and exerts a potent antitumor effect in tumor-bearing mice. Methods: Polyethylene glycol (PEG; Mw. 2000) was conjugated to fDAO to form PEGylated fDAO (PEG-fDAO). PEGfDAO was intravenously administered into S180 tumor-bearing mice, and the body distribution and antitumor activity of PEG-fDAO was determined. Results: The enzyme activity of PEG-fDAO was 26.1 U/mg, which was comparable to that of fDAO. Intravenously administered PEG-fDAO accumulated in tumors with less distribution in normal tissue except in the plasma. Enzyme activity in the tumor was 60–120 mU/g-tissue over 7–20 h after i.v. injection of 0.1 mg of PEG-fDAO. To generate the H2O2 in the tumor tissue, PEG-fDAO was intravenously administered, and then, D-phenylalanine was intraperitoneally administered after a lag time. No remarkable tumor suppression effect was observed under conditions used in this study, compared to the non-treated group. Conclusion: The results suggest that PEG-fDAO maintained high enzymatic activity after pegylation. Treatment with PEGfDAO conferred high enzyme activity on tumor tissue; 3–6 fold higher than that of previously reported pDAO; however, high enzyme activity in the plasma limited repeated treatment owing to lethal toxicity, which seemingly led to poor therapeutic outcome. Overall, the use of PEG-fDAO is promising for antitumor therapy, although the suppression of DAO activity in the plasma would also be required rather than only the increase in DAO activity in the tumor for an antitumor effect.

Mechanism of the feedback-inhibition resistance in aspartate kinase of Corynebacterium pekinense: from experiment to MD simulations

Corynebacterium pekinense AK was successfully modified and two mutants A380C and T379N/A380C with high enzyme activity were constructed. The mechanism of their feedback-inhibition resistance was thoroughly studied from experiment to MD simulations.

Change of Endoglucanase Activity and Rumen Microbial Community During Biodegradation of Cellulose Using Rumen Microbiota

Treatment with rumen microorganisms improves the methane fermentation of undegradable lignocellulosic biomass; however, the role of endoglucanase in lignocellulose digestion remains unclear. This study was conducted to investigate endoglucanases contributing to cellulose degradation during treatment with rumen microorganisms, using carboxymethyl cellulose (CMC) as a substrate. The rate of CMC degradation increased for the first 24 h of treatment. Zymogram analysis revealed that endoglucanases of 52 and 53 kDa exhibited high enzyme activity for the first 12 h, whereas endoglucanases of 42, 50, and 101 kDa exhibited high enzyme activities from 12 to 24 h. This indicates that the activities of these five endoglucanases shifted and contributed to efficient CMC degradation. Metagenomic analysis revealed that the relative abundances of Selenomonas, Eudiplodinium, and Metadinium decreased after 12 h, which was positively correlated with the 52- and 53-kDa endoglucanases. Additionally, the relative abundances of Porphyromonas, Didinium, unclassified Bacteroidetes, Clostridiales family XI, Lachnospiraceae and Sphingobacteriaceae increased for the first 24 h, which was positively correlated with endoglucanases of 42, 50, and 101 kDa. This study suggests that uncharacterized and non-dominant microorganisms produce and/or contribute to activity of 40, 50, 52, 53, and 101 kDa endoglucanases, enhancing CMC degradation during treatment with rumen microorganisms.

Statistical Optimization of Parameters for the Enhancement of Lipase Activity

In this present study, lipase producing bacteria were isolated and screened from an indigenous soil sample and was used for lipase production with high enzyme activity. Different production media were screened and lipase production was found to be induced by olive oil, 14 mL/L, in the production medium. It was observed from Luedeking and Piret model that the lipase production was mixed growth associated with its maximum activity at 37 °C and pH 7. To understand the interaction of different parameters, statistical optimization using Response Surface Methodology was performed and the standardized conditions obtained were as follows: Peptone 10 g/L, yeast extract 7.5 g/L and olive oil 14 mL/L. The predicted data were validated and the model predicted was significant with maximum specific activity of 1.1 µmole/min/mg proteins. The lipase specific activity was found to be enhanced by 10% and 23% after a single parameter and statistical optimization respectively.

INVESTIGATION OF ALKALINE PHOSPHATASE EXPRESSION IN THE SMALL INTESTINES OF THE BRONZE TURKEY (MELEAGRIS GALLOPAVO)

The aim of our investigation was to study the expression of the enzyme Alkaline phosphatase in the small intestines of the bronze turkey in the age aspect. Forty clinically healthy bronze turkeys (twenty males and twenty females) were studied. The groups of the birds were at age 1, 7, 14, 28, 35, 49, 56, 90, 120, and 240 days. Each group consisted of five males and five female birds. The enzyme’s expression was investigated by Gomori staining. The most significant expression of tissue alkaline phosphatase was observed in the epithelial cells of the duodenum. It was weaker in the jejunum and weakest in the ileum. In the three intestinal segments, high enzyme activity was observed during the first weeks of hatching (from the 1st to the 14th day in the duodenum and ileum, and from the 1st to the 28th day in the jejunum).

Активність та вміст ізозимів аспартатамінотрансферази в репродуктивних органах корів

Activity and isozyme content of aspartate transaminase in cow reproductive organs at different physiological states and ovarian hypofunction were studied. It was registered, that activity of aspartate transaminase (AST) in ovarian tissue and endometrium at corpus luteum state is 132.0 ± 8.23 and 172.9 ± 8.70 μm/min×mg of protein, lower on 26.3 and 5.5% at follicle growth and at early corpus luteum is 66.5 ± 2.10 and 147.1 ± 13.26 μm/min×mg of protein. Lowest AST activity is registered at hypofunction state (61.8 ± 4.57 і 146.1 ± 9.33 μm/min×mg of protein). In cow reproductive organ tissues and antral fluid from ovarian follicles was registered two bands of proteins, that have AST activity. In reproductive organs main part is mitochondrial isozyme (АSТ2; 78.8–97.7%) in antral fluid – cytosole (АSТ1; 72.8–88.2%). In ovarian and uterus tissues was established high enzyme activity at late corpus luteum, that characterizes intensive aspartate conversion to oxaloacetate and possible utilization of substeates to maintain energetic and sytnthetic needs in cow reproductive organs. Antral fluid of big follicles from ovarian at follicle growth and at state of late corpus luteum characterizes by high activity and content of AST, that points on follicle cell growth – granulose and oocyte. In ovarian tissue and endometrium at hypofunction lower activity and isozyme content point on substrate deficiency for transamination and violation of physiological functions.