Purifikacija i svojstva kolagenolitičke proteaze proizvedene s pomoću Bacillus cereus MBL13

A novel collagenase-producing bacterium has been isolated and identified as Bacillus cereus MBL13. From the culture supernatant of B. cereus MBL13 grown on bone collagen as the sole carbon and nitrogen source, an extracellular protease with novel property of hydrolyzing waste animal bones was purified. The molecular mass of the purified collagenolytic protease was estimated to be (38.0±1.5) kDa. As determined by amino acid analysis, it had high contents of asparagine, lysine and serine. The optimum temperature and pH for the collagenase activity were 40 °C and pH=8.0, respectively. The results of the effects of some metal ions, inhibitors and protein substrates suggested that the purified collagenolytic protease is a member of the metalloproteases. Type I collagen (the typical collagen in animal bone) was used as the substrate for determination of Michaelis-Menten kinetics. The obtained Km value was (1.31±0.05) g/L and the corresponding vmax value was (12.54±2.5) μmol/min. The study assumes that the collagenolytic protease purified from B. cereus MBL13 strain could be applied in the hydrolysis of waste animal bones.Izolirana je nova bakterija koja proizvodi kolagenazu, identificirana kao Bacillus cereus MBL13. Iz supernatanta kulture B. cereus MBL13, uzgojene na koštanom kolagenu kao izvoru ugljika i dušika, izdvojena je ekstracelularna proteaza sa sposobnošću razgradnje životinjskih kostiju. Procijenjena je molekularna masa pročišćene kolagenolitičke proteaze od (38,0±1,5) kDa. Analiziran je aminokiselinski sastav i utvrđeno da enzim sadržava veliku količinu asparagina, lizina i serina. Optimalna temperatura za aktivnost kolagenaze bila je 40 °C, a optimalna pH-vrijednost 8,0. Prema utjecaju iona metala, inhibitora i proteinskih supstrata na aktivnost enzima zaključeno je da enzim pripada u grupu metaloproteaza. Kolagen tipa I (tipični sastojak životinjskih kostiju) upotrijebljen je kao supstrat pri određivanju parametara Michaelis-Mentenove kinetike. Km vrijednost bila je (1,31±0,05) g/L, a vmax (12,54±2,5) μmol/min. Zaključeno je da se kolagenolitička proteaza izdvojena iz B. cereus MBL13 može upotrijebiti za razgradnju životinjskih kostiju

Purifikacija i svojstva kolagenolitičke proteaze proizvedene s pomoću Bacillus cereus MBL13

A novel collagenase-producing bacterium has been isolated and identified as Bacillus cereus MBL13. From the culture supernatant of B. cereus MBL13 grown on bone collagen as the sole carbon and nitrogen source, an extracellular protease with novel property of hydrolyzing waste animal bones was purified. The molecular mass of the purified collagenolytic protease was estimated to be (38.0±1.5) kDa. As determined by amino acid analysis, it had high contents of asparagine, lysine and serine. The optimum temperature and pH for the collagenase activity were 40 °C and pH=8.0, respectively. The results of the effects of some metal ions, inhibitors and protein substrates suggested that the purified collagenolytic protease is a member of the metalloproteases. Type I collagen (the typical collagen in animal bone) was used as the substrate for determination of Michaelis-Menten kinetics. The obtained Km value was (1.31±0.05) g/L and the corresponding vmax value was (12.54±2.5) μmol/min. The study assumes that the collagenolytic protease purified from B. cereus MBL13 strain could be applied in the hydrolysis of waste animal bones.Izolirana je nova bakterija koja proizvodi kolagenazu, identificirana kao Bacillus cereus MBL13. Iz supernatanta kulture B. cereus MBL13, uzgojene na koštanom kolagenu kao izvoru ugljika i dušika, izdvojena je ekstracelularna proteaza sa sposobnošću razgradnje životinjskih kostiju. Procijenjena je molekularna masa pročišćene kolagenolitičke proteaze od (38,0±1,5) kDa. Analiziran je aminokiselinski sastav i utvrđeno da enzim sadržava veliku količinu asparagina, lizina i serina. Optimalna temperatura za aktivnost kolagenaze bila je 40 °C, a optimalna pH-vrijednost 8,0. Prema utjecaju iona metala, inhibitora i proteinskih supstrata na aktivnost enzima zaključeno je da enzim pripada u grupu metaloproteaza. Kolagen tipa I (tipični sastojak životinjskih kostiju) upotrijebljen je kao supstrat pri određivanju parametara Michaelis-Mentenove kinetike. Km vrijednost bila je (1,31±0,05) g/L, a vmax (12,54±2,5) μmol/min. Zaključeno je da se kolagenolitička proteaza izdvojena iz B. cereus MBL13 može upotrijebiti za razgradnju životinjskih kostiju

Characteristics and Mechanism of Crayfish Myofibril Protein Gel Deterioration Induced by Autoclaving

Crayfish myofibril protein (CMP) gel deterioration induced by autoclaving was investigated. A series of CMP gels were obtained through treating CMP solutions at different autoclaving conditions from 100 °C/0.1 MPa to 121 °C/0.21 MPa, and then characteristics and the mechanism of gel texture deterioration along with the intensification of autoclaving were explored through determining appearance, texture, protein composition, cross-linking forces, degree of hydrolysis, water state, microstructure of the gels, and average particle size of aggregates. When autoclaving was at above 105 °C/0.103 MPa, texture of CMP gel showed a tendency to severely weaken with the intensification of autoclaving (p < 0.05), hydrophobic interaction and aggregation between proteins weakened gradually (p < 0.05), and moderately bound water in the gel decreased and T22 relaxation time significantly increased (p < 0.05). After heating for 30 min at above 105 °C/0.103 MPa, pores in the microstructure of CMP gel enlarged obviously, and myosin heavy chain (MHC) degraded. It can be concluded that CMP gel deterioration induced by autoclaving was associated with the degradation of MHC and 105 °C might be the critical temperature to ensure good texture of crayfish products

Purification and Properties of a Collagenolytic Protease Produced by Bacillus cereus MBL13 Strain

A novel collagenase-producing bacterium has been isolated and identified as Bacillus cereus MBL13. From the culture supernatant of B. cereus MBL13 grown on bone collagen as the sole carbon and nitrogen source, an extracellular protease with novel property of hydrolyzing waste animal bones was purified. The molecular mass of the purified collagenolytic protease was estimated to be (38.0±1.5) kDa. As determined by amino acid analysis, it had high contents of asparagine, lysine and serine. The optimum temperature and pH for the collagenase activity were 40 °C and pH=8.0, respectively. The results of the effects of some metal ions, inhibitors and protein substrates suggested that the purified collagenolytic protease is a member of the metalloproteases. Type I collagen (the typical collagen in animal bone) was used as the substrate for determination of Michaelis-Menten kinetics. The obtained Km value was (1.31±0.05) g/L and the corresponding vmax value was (12.54±2.5) μmol/min. The study assumes that the collagenolytic protease purified from B. cereus MBL13 strain could be applied in the hydrolysis of waste animal bones

Preparation and characterization of novel eggshell membrane-chitosan blend films for potential wound-care dressing: From waste to medicinal products

A series of different eggshell membrane (ESM) and chitosan (CS) blend films (ESM/CS) were prepared for wound-care dressing. The appearance, transparency and microstructure of the films were characterized. Several wound care-related properties such as the film integrity in solution, pH, protein (BSA) and wound fluid absorption capacity as well as the antibacterial property of ESM/CS films were evaluated. The blend films were more stable than CS film after 95 hours of incubation in solution. The integrity of the blend films improved significantly at the cost of a small insignificant decrease in wound fluid absorption capacity. Besides, the blend films provided an acidic environment (pH = 5.86) for wound healing. The swelling properties of ESM contributed significantly to the increase of BSA absorption capacity of the blend films (from 46.57 mg/g of CS film to 61.07 mg/g of blend film) and helped absorb more nutrients to promote the proliferation and migration of fibroblasts. Addition of CS to ESM also enhanced the antibacterial activity of the films significantly. The results indicated that the EMS/CS blend films with 0.01 g ESM/mL CS solution showed the highest high potential to be used as a wound-care dressing for humans as well as animals.This is a manuscript of an article published as Li, Xiaoyun, Meihu Ma, Dong Uk Ahn, and Xi Huang. "Preparation and characterization of novel eggshell membrane-chitosan blend films for potential wound-care dressing: From waste to medicinal products." International journal of biological macromolecules 123 (2019): 477-484. doi:10.1016/j.ijbiomac.2018.10.215. Posted with permission.</p

Succinylation Modified Ovalbumin: Structural, Interfacial, and Functional Properties

In this study, ovalbumin (OVA) was succinylated with the addition of different levels of succinic anhydride, and the structural and functional properties of succinylated OVA (SOVA) were investigated. SDS−PAGE and FTIR spectrum confirmed the covalent attachment of the succinyl group to OVA. Thermal stability and the absolute value of zeta potential (pH 6.0) of SOVA were enhanced by 14.90% and 76.77% higher than that of the native OVA (NOVA), respectively. Circular dichroism (CD) spectra demonstrated that the succinylation decreased the α−helix and increased β−sheet content to 21.31% and 43.28%, respectively. The content of free sulfhydryl groups increased and intrinsic fluorescence spectra suggested the SOVA became more unfolded and flexible as the degree of succinylation enhanced. Furthermore, succinylation effectively enhanced the solubility and decreased the interface tension (oil−water and air−water interface) of OVA. Compared to NOVA, the emulsifying activity and stability of SOVA were increased by 1.6 times and 1.2 times, respectively, and foaming capacity and stability were enhanced by 2.7 times and 1.5 times, respectively

Effects of Peroxyl Radicals on the Structural Characteristics and Fatty Acid Composition of High-Density Lipoprotein from Duck Egg Yolk

In this study, high-density lipoprotein (HDL) from duck egg yolk was subjected to oxidation with a system based on 2,2&prime;-azobis (2-amidinopropane) dihydrochloride (AAPH)-derived peroxyl radicals. The effects of peroxyl radicals on the protein carbonyl, free sulfhydryl, secondary/tertiary structure, surface hydrophobicity, solubility, particle size distribution, zeta potential and fatty acid composition of HDL were investigated by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier-transform infrared spectroscopy (FTIR), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering and ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The results indicated that the content of protein carbonyl was significantly increased, that of free sulfhydryl was obviously reduced, and the ordered secondary structure was also decreased with increasing AAPH concentration. In addition, the surface hydrophobicity and solubility of HDL showed apparent increases due to the exposure of hydrophobic groups and aggregation of protein caused by oxidation. The fatty acid composition of HDL exhibited pronounced changes due to the disrupted protein&ndash;lipid interaction and lipid oxidation by AAPH-derived peroxyl radicals. These results may help to elucidate the molecular mechanism for the effect of lipid oxidation products on the oxidation of duck yolk proteins

Functional Properties and Extraction Techniques of Chicken Egg White Proteins

Chicken egg whites contain hundreds of proteins, and are widely used in the food, biological and pharmaceutical industries. It is highly significant to study the separation and purification of egg white proteins. This review first describes the structures and functional properties of several major active proteins in egg whites, including ovalbumin, ovotransferrin, ovomucoid, lysozyme, ovomucin, ovomacroglobulin and avidin. Then, the common techniques (including precipitation, chromatography and membrane separation) and some novel approaches (including electrophoresis, membrane chromatography, aqueous two-phase system and molecular imprinting technology) for the separation and purification of egg white proteins broadly reported in the current research are introduced. In addition, several co-purification methods for simultaneous separation of multiple proteins from egg whites have been developed to improve raw material utilization and reduce costs. In this paper, the reported techniques in the last decade for the separation and purification of chicken egg white proteins are reviewed, discussed and prospected, aiming to provide a reference for further research on egg proteins in the future