Property Improvement of α-Amylase from Bacillus stearothermophilus by Deletion of Amino Acid Residues Arginine 179 and Glycine 180

Proizveden je mutant AmySΔR179-G180 delecijom arginina (Arg179) i glicina (Gly180) u α-amilazi iz bakterije Bacillus stearothermophilus (AmyS) pomoću ciljane mutageneze, a radi poboljšanja njezinih svojstava. Amilaze AmyS i AmySΔR179-G180 eksprimirane su u bakteriji Bacillus subtilis i pročišćene taloženjem pomoću amonijeva sulfata, te su im opisana i uspoređena svojstva. Delecijom aminokiselina Arg179 i Gly180 poboljšala se termostabilnost α-amilaze AmySΔR179-G180, a vrijeme poluraspada pri 100 °C bitno se produljilo s 24 na 33 min. Osim toga, ova je amilaza otpornija na djelovanje kiselina i treba manje kalcija za održavanje aktivnosti. Sekrecijska svojstva rekombinantnog soja ispitana su šaržnom fermentacijom u fermentoru od 7,5 L, te je dobivena velika aktivnost α-amilaze. Postignuta je najveća aktivnost od 3300 U/mL i produktivnost od 45,8 U/(mL•h).To improve the properties of α-amylase from Bacillus stearothermophilus (AmyS), a deletion mutant AmyS∆R179-G180 was constructed by deleting arginine (Arg179) and glycine (Gly180) using site-directed mutagenesis. AmyS and AmyS∆R179-G180 were expressed in Bacillus subtilis and purified by ammonium sulfate precipitation, after which the enzymatic properties were characterized and compared. By deleting amino acids Arg179 and Gly180, the thermostability of α-amylase AmyS∆R179-G180 was enhanced and the half-life at 100 °C significantly increased from 24 to 33 min. In addition, AmyS∆R179-G180 exhibited greater acid resistance and lower calcium requirements to maintain α-amylase activity. The secretory capacity of the recombinant strain was evaluated by fed-batch fermentation in a 7.5-litre fermentor in which high α-amylase activity was obtained. The highest activity reached 3300 U/mL with a high productivity of 45.8 U/(mL•h)

Zinc-Chelating Mechanism of Sea Cucumber (Stichopus japonicus)-Derived Synthetic Peptides

In this study, three synthetic zinc-chelating peptides (ZCPs) derived from sea cucumber hydrolysates with limited or none of the common metal-chelating amino-acid residues were analyzed by flame atomic absorption spectroscopy, circular dichroism spectroscopy, size exclusion chromatography, zeta-potential, Fourier transform infrared spectroscopy, Raman spectroscopy and nuclear magnetic resonance spectroscopy. The amount of zinc bound to the ZCPs reached maximum values with ZCP:zinc at 1:1, and it was not further increased by additional zinc presence. The secondary structures of ZCPs were slightly altered, whereas no formation of multimers was observed. Furthermore, zinc increased the zeta-potential value by neutralizing the negatively charged residues. Only free carboxyl in C-terminus of ZCPs was identified as the primary binding site of zinc. These results provide the theoretical foundation to understand the mechanism of zinc chelation by peptides

Influence of Refrigerated Storage on Water Status, Protein Oxidation, Microstructure, and Physicochemical Qualities of Atlantic Mackerel (<i>Scomber scombrus</i>)

Moisture migration, protein oxidation, microstructure, and the physicochemical qualities of Atlantic mackerel during storage at 4 °C and 0 °C were explored in this study. Three proton components were observed in mackerel muscle using low-field nuclear magnetic resonance relaxation, which were characterized as bound water, immobilized water, and lipid. The relaxation peak of immobilized water shifted to a shorter relaxation time and its intensity decreased with the proceeding of the storage process. T1 and T2 weighted images obtained by magnetic resonance imaging showed a slightly continuous decrease in the intensity of water. The significant decrease in sulfhydryl (SH) content and the increase in carbonyl group (CP) content, disulfide bond content, and hydrophobicity revealed the oxidation of protein during storage. The contents of α-helixes in proteins decreased while that of random coils increased during storage, which suggested changes in the secondary structure of mackerel protein. The storage process also caused the contraction and fracture of myofibrils, and the granulation of endolysin protein. In addition, the drip loss, total volatile basic nitrogen (TVB-N), thiobarbituric acid-reactive substances (TBARS) value, and b* value increased significantly with the storage time

Influence of Refrigerated Storage on Water Status, Protein Oxidation, Microstructure, and Physicochemical Qualities of Atlantic Mackerel (Scomber scombrus)

Moisture migration, protein oxidation, microstructure, and the physicochemical qualities of Atlantic mackerel during storage at 4 &deg;C and 0 &deg;C were explored in this study. Three proton components were observed in mackerel muscle using low-field nuclear magnetic resonance relaxation, which were characterized as bound water, immobilized water, and lipid. The relaxation peak of immobilized water shifted to a shorter relaxation time and its intensity decreased with the proceeding of the storage process. T1 and T2 weighted images obtained by magnetic resonance imaging showed a slightly continuous decrease in the intensity of water. The significant decrease in sulfhydryl (SH) content and the increase in carbonyl group (CP) content, disulfide bond content, and hydrophobicity revealed the oxidation of protein during storage. The contents of &alpha;-helixes in proteins decreased while that of random coils increased during storage, which suggested changes in the secondary structure of mackerel protein. The storage process also caused the contraction and fracture of myofibrils, and the granulation of endolysin protein. In addition, the drip loss, total volatile basic nitrogen (TVB-N), thiobarbituric acid-reactive substances (TBARS) value, and b* value increased significantly with the storage time

RNA Sequencing Analysis to Capture the Transcriptome Landscape during Tenderization in Sea Cucumber Apostichopus japonicus

Sea cucumber (Apostichopus japonicus) is an economically significant species in China having great commercial value. It is challenging to maintain the textural properties during thermal processing due to the distinctive physiochemical structure of the A. japonicus body wall (AJBW). In this study, the gene expression profiles associated with tenderization in AJBW were determined at 0 h (CON), 1 h (T_1h), and 3 h (T_3h) after treatment at 37 &deg;C using Illumina HiSeq&trade; 4000 platform. Seven-hundred-and-twenty-one and 806 differentially expressed genes (DEGs) were identified in comparisons of T_1h vs. CON and T_3h vs. CON, respectively. Among these DEGs, we found that two endogenous proteases&mdash;72 kDa type IV collagenase and matrix metalloproteinase 16 precursor&mdash;were significantly upregulated that could directly affect the tenderness of AJBW. In addition, 92 genes controlled four types of physiological and biochemical processes such as oxidative stress response (3), immune system process (55), apoptosis (4), and reorganization of the cytoskeleton and extracellular matrix (30). Further, the RT-qPCR results confirmed the accuracy of RNA-sequencing analysis. Our results showed the dynamic changes in global gene expression during tenderization and provided a series of candidate genes that contributed to tenderization in AJBW. This can help further studies on the genetics/molecular mechanisms associated with tenderization

Modulation of miR-382-5p reduces apoptosis of myocardial cells after acute myocardial infarction

Background Acute myocardial infarction (AMI) is a severe cardiovascular condition. Blocking the apoptosis of myocardial cells may mitigate AMI. Excessive expression of Stanniocalcin-1 (STC1) plays a protective role in the heart by inhibiting myocardial cell apoptosis. Here, we looked at the mechanism by which miR-382-5p regulates STC1 and affects myocardial cell apoptosis after AMI. Methods An AMI mouse model with a descending anterior ligament coronary artery and an HL-1 cell model with reproducible hypoxia/reoxygenation (H/R) were established. For pathological changes in myocardial tissues, terminal deoxynucleotidyl transferase dUTP nick end labelling staining and haematoxylin and eosin staining were performed. STC1 mRNA and miR-382-5p levels were measured using quantitative real-time PCR. Protein levels of STC1 and apoptosis-related proteins were measured by western blotting. The 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide assay was used to detect cell viability, and a dual-luciferase reporter assay was carried out to verify potential targets of miR-382-5p. Results The level of miR-382-5p was raised in myocardial tissues of AMI mice and H/R-induced HL-1 cells. Compared with the control group, the myocardial tissue cells in the AMI group were disordered, with evident necrosis of myocardial cells, apoptosis and inflammatory infiltration. Interference with miR-382-5p inhibited myocardial cell apoptosis after H/R, as well as inferior lactate dehydrogenase. Also, miR-382-5p adversely regulated STC1 and the expression of STC1 was increased after transfection with miR-382-5p antagomir. Furthermore, interference with miR-382-5p reduced myocardial cell apoptosis after H/R by increasing the expression level of STC1. Conclusion To summarise, our study showed an increase in miR-382-5p in myocardial tissues in the AMI mouse model. Interference with miR-382-5p reduced apoptosis of myocardial cells after AMI and the effect was achieved by increasing STC1 expression