Purification and Enzymatic Properties of a New Thermostable Endoglucanase From Aspergillus Oryzae HML366

Aspergillus oryzae HML366 is a newly screened cellulase-producing strain. The endoglucanase HML ED1 from A. oryzae HML366 was quickly purified by two-step method ammonium sulfate precipitation and strong anion exchange column. SDS-PAGE electrophoresis indicated that the molecular weight of the enzyme was 68 kDa. The optimum temperature of the purified endoglucanase was 60 ℃ and the enzyme activity was stable below 70 ℃. The optimum pH was 6.5, and the enzyme activity was stable at pH between 4.5 to 9.0. The analysis indicated that additional Na+, K+, Ca2+, and Zn2+ reduced the catalytic ability of enzyme to the substrate, but Mn2+ enhanced its catalytic ability to the substrate.The Km and Vmax of the purified endoglucanase was 8.75 mg/mL and 60.24 μg/min·mL, respectively. Our study demonstrated that A. oryzae HML366 can produce a heat-resistant and wide pH tolerance endoglucanase HML ED1, which has potential industrial application value in bioethanol, paper, food, textile, detergent and pharmaceutical industries.

Production and Identification of Polypeptide from Silks by Hydrothermal Method

There are two kinds of proteins in silk, sericin and silk fibroin. Polypeptide compounds from silk sericin and silk fibroin were prepared by hydrothermal method. The process of silk dissolution was investigated under different solid-liquid ratio, reaction temperature and reaction time. By controlling the operating parameters of hydrothermal method, the temperature, material ratio and time were further optimized, and the best experimental results were obtained, the expected decomposition of silks occurred when the ratio of silks to waters was selected as 1 to 10, at 140 degree in 30 min. The molecular weight of polypeptide was detected by SDS-PAGE electrophoresis and analysed by MALDI-TOF-MS. The results showed that the molecular weight of the polypeptide obtained from silks was about 6000-8000Da. After literature research, the polypeptide with such molecular weight could have better performance for some functional additives.

POLYMORPHISM OF TAGLGAP MICROSATELITE LOCUS AND ITS CONNECTION WITH ALLELELIC VARIETIES OF GLIADINS OF BREAD WHEAT

Introduction. Gliadins are monomeric and highly polymorphic storage proteins of wheat endosperm, which together with glutenins form a gluten complex that determines the breadmaking properties of wheat. Allelic variants of gliadins are an important feature in the selection of material for breeding, but their determination by electrophoresis in acid PAGE is quite difficult. Aim. The aim of this study was to investigate the polymorphism of the Taglgap microsatellite locus and to analyze its correspondence to the polymorphism of allelic variants of gliadins that have been revealed by acid PAGE electrophoresis. Methods. 140 cultivars and lines of bread wheat of Ukrainian and foreign selection were analyzed. Electrophoresis of storage proteins was performed in an acid PAGE according to the method of F. O. Poperellia (1989), allelic variants were designated according to the international nomenclature (Metakovsky et al., 2018). DNA was isolated by CTAB method and PCR was performed with primers to the Taglgap microsatellite (Devos et al., 1995). PCR products were fractionated in 7% PAGE and stained with silver staining method. Nucleotide sequences were searched by BLAST and aligned by MAFT methods. The main results. 19 allelic variants of gliadins and 11 alleles of the Taglgap locus were identified. In the collection of Ukrainian varieties there were Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1l and Gli-B1o allelic variants and alleles of Taglgap 216 bp, 237 bp, 246 bp, 248 bp, 252 bp, 267 bp, 270 bp and null. In the foreign collection of varieties − Gli-B1a, Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1i, Gli-B1j, Gli-B1k, Gli -B1l, Gli-B1m, Gli-B1n, Gli-B1o, Gli-B1p, Gli-B1q, Gli-B1r, Gli-B1s and 213 bp, 216 bp, 237 bp, 246 bp, 248 bp, 250 bp, 252 bp, 270 bp, 285 bp and null. Nucleotide sequence analysis in the NCBI database showed the presence of a number of other alleles of the Taglgap microsatellite not only in bread wheat but also in some species of the Triticum L. and Aegilops L. genus. Conclusions. The detected polymorphism correlates with the polymorphism of allelic variants of gliadins of Gli-B1 locus and makes it possible to identify Gli-B1a, Gli-B1d, Gli-B1h and Gli-B1l allelic variants, and for Ukrainian varieties with high probability also Gli-B1b allelic variant. However, this marker does not allow identifying Gli-B1c, which is important for selection.

Proteolytic Analysis of Different Tuna (Thunnus albacares) Viscera for Obtaining Protein Hydrolysates

This project aims to obtain and characterize protein hydrolysates from the solid residues (dark muscle) and effluents (cooking water) generated from the processing of yellowfin tuna (Thunnus albacares) and evaluate their potential to be applied as organic fertilizer in farming. From the studied viscera (stomach, pyloric blind, liver, pancreas, and intestine), it was found that the pyloric blind of the tuna digestive system represents an adequate source of alkaline proteases that act on the dark muscle of tuna and cooking water, at pH optimum of 10.5 and temperature of 50 °C. The results of SDS-PAGE electrophoresis and enzymatic inhibition indicate that the proteolytic activity exhibited by the enzymatic extract of the pyloric blind of tuna is mainly due to serine protease enzymes, especially trypsin type, and it showed a proteolytic activity similar to that of a commercial protease coming from Bacillus sp. It is possible to use these enzymes in processes that require pH values between 8 and 11 and mild temperatures conditions (30-50°C). The cooking water showed a low yield and high ash content, which may decrease the protein hydrolysates' nutritional quality and functional properties.

Soybean and Lupine Addition in Hen Nutrition—Influence on Egg Immunoreactivity

Modifying hen fodder is a common way of changing eggs composition today. However, there is no information on the effect of the source of protein in the fodder replacement on egg allergenicity. This research aimed to detect potential differences in the immunoreactivity and protein composition of eggs from hens fed with fodder containing legume. The aim of the first step of the study was to select the proper solvent for extracting allergenic proteins from hen eggs. Two of them (containing Tween 20 and Triton 100) were selected, based on protein profile and concentration analysis. Egg-white- and egg-yolk-proteins extracts prepared with them were checked for potential differences, using SDS-PAGE electrophoresis, and then the Western-blot method, using sera from children allergic to eggs and soy. Preliminary studies on the influence of fodder composition on the composition of egg proteins suggest that the addition of soy and lupine to fodder modifies the expression of egg proteins. The observed differences in the immunoreactivity of proteins contained in hen egg-white samples do not seem to be as significant as the appearance of protein with a molecular weight of ~13 kDa in the yolk of eggs obtained from soybean-fed hens. This protein may increase the immunoreactivity of eggs for children allergic solely to soy.

Effects of Pasteurization and High-Pressure Processing of Camel and Bovine Cheese Quality, and Proteolysis Contribution to Camel Cheese Softness

The effects of high-pressure processing (HPP) compared to thermal treatments on the quality of camel vs. bovine cheeses were studied. The study showed that camel milk has a lower microbial load compared to bovine milk, which is maintained during 7 days' storage of the processed milk. The effect of three HPP treatments (350, 450, and 550 MPa for 5 min at 4°C) and two pasteurization treatments (65°C for 30 min and 75°C for 30 s) on the quality of soft unripened camel and bovine milk cheeses were accessed. The cheeses were evaluated for pH, yield, proximate composition, textural and rheological properties, microstructure, and protein profile by SDS-PAGE electrophoresis. The effects of the treatments on cheese's hardness were different between the camel and bovine cheeses; while heat treatment at 65°C for 30 min gave the hardest bovine milk cheese (1,253 ± 20), HPP treatment at 350 MPa for 5 min gave the highest value for camel milk cheese (519 ± 5) (p < 0.05). The hardness of the cheeses was associated with low yield and moisture content. SDS-PAGE electrophoresis revealed that extensive proteolysis might have contributed to the softness of camel cheeses compared to bovine and suggested the involvement of some residual enzyme activities.

Restriction site-associated DNA from Python-implemented Digestion Simulations (RApyDS): a companion tool for RAD sequencing experimental design

Reduced representation sequencing is a practical approach for obtaining genetic variations from a random subsample of the genome. RADseq (Restriction Site-Associated DNA Sequencing), as one of the more popular reduced representation approaches, is currently being used in a wide array of applications including marker development, phylogenetics, and population genomics. A crucial step in designing a RADseq experiment is the selection of one or a pair of restriction enzymes (RE) that will result in sufficient density of loci to meet the objectives of the study, which is not straightforward because of difficulties in obtaining a standard set of REs that can generally be applied to RADseq experimental designs. Here we present RApyDS, a simulation tool that provides users with evaluation metrics to aid in choosing suitable REs based on their target RADseq design. RApyDS can perform simulations for single- or double-digest RADseq, preferably with a supplied reference genome. The tool outputs an overview page, electrophoresis visualization, mapping of restriction cut sites, and RAD loci density across the genome. If supplied with an annotation file, the program can also output evaluation metrics for a specified genomic feature. The tool is currently available at https://github.com/pgcbioinfo/rapyds.

External morphological stages and protein variations along with the embryonic development of the longarm river prawn Macrobrachium tenellum (Smith, 1871)

A good understanding of a given species' embryology is important to settle the larval rearing bases when juveniles are required for culture purposes or conservation programs. Changes in embryonic morphology, protein concentration, and protein type occurring in prawn eggs were analyzed in the present work. Berried females of Macrobrachium tenellum were collected in the Colotepec River, Oaxaca, Mexico. The eggs were taken from the ovigerous mass and embryonic stages classified by their color. Morphological changes in the embryos allowed identifying six embryonic stages based on color, egg size, and morphological features. Determinations of the protein extract were executed in SDS-PAGE (electrophoresis in polyacrylamide gels) and, subsequently, proteomic analyses were also performed. Protein bands along embryonic development and their molecular weights are presented and commented.

Meat tenderization using bromelain enzyme extracted from pineapple waste

Tenderization is an important method to improve the quality and palatability of meat. In comparison to conventional chemical and physical methods of meat tenderization, enzymatic treatment is more economical and healthy. In the present study proteolytic enzyme bromelain was used for this purpose. Bromelain enzyme was extracted from pineapple, quantified and optimized concentration was used for treating the muscle fibre of meat. Tenderness was measured. pH and cooking loss were not significantly (P>0.05) decreased, but water-holding capacity and protein content were significantly (P<0.05) increased with the concentration and ageing. Duration of marination was optimized and SDS- PAGE electrophoresis was conducted to estimate protein. The results proved that bromelain enzyme is an effective method of tenderization used to improve the quality of meat.