Effect of degree of deacetylation on solubility of low‐molecular‐weight chitosan produced via enzymatic breakdown of chitosan

Chitosan has emerged as a unique biomaterial, possessing scope in diverse applications in the biomedical, food and chemical industries. However, its high molecular weight is a concern when handling the polymer. Various techniques have been explored for depolymerization of this polymer, wherein enzymes have emerged as the most economic method having minimum degrading effect on the polymer and resulting in formation of side products. Chitosan can be depolymerized using a broad range of enzymes. In this study, various enzymes like α‐amylase, papain, pepsin and bromelain were employed to depolymerize chitosan and convert it into its lower molecular weight counterpart. Further, attempts were made to elucidate the process of depolymerization of chitosan, primarily by determining the change in its viscosity and hence its molecular weight. The process of depolymerization was optimized using a one‐factor‐at‐a‐time approach. The molecular weight of the resultant chitosan was estimated using gel permeation chromatography and infrared spectroscopy. These studies revealed a considerable decrease in molecular weights of chitosan depolymerized by pepsin, papain, bromelain and α‐amylase, resulting in recovery of the low‐molecular‐weight chitosan of 76.09 ± 5, 74.18 ± 5, 55.75 ± 5 and 49.18 ± 5%, respectively. Maximum yield and depolymerization were obtained using pepsin and papain due to their enzymatic recognition pattern, which was also validated using studies involving molecular dynamics. © 2019 Society of Chemical Industry     

LC FT-ICR MS精确分析卵清蛋白在真空干燥过程中的糖基化修饰

研究了卵清蛋白与葡萄糖混合物在真空干燥的过程中,卵清蛋白的糖基化修饰情况。首次采用胃蛋白酶和液相-傅立叶变换离子回旋共振(LC-FT-ICR)精确质谱分析技术对卵清蛋白的糖基化修饰位点进行研究,其蛋白质覆盖率可以达到100%,研究发现在真空干燥过程中,卵清蛋白中的赖氨酸会与还原糖发生美拉德初级反应,形成糖基化修饰,主要发生在卵清蛋白的K47,56,62,182,190,323和370的赖氨酸氨基上,而卵清蛋白中的精氨酸和氮末端氨基未发现被糖基化修饰;同时,采用离子碰撞诱导解离三级质谱CID-MS3验证m/z7863+的离子峰为m/z7323+的离子峰上添加一个162Da。本论文的研究为精确分析蛋白质修饰的一级结构变化提供了重要的研究方法,为真空干燥在实验分析中的应用提供重要参考。     

聚乙二醇/硫酸铵双水相萃取猪胃蛋白酶工艺研究

采用聚乙二醇/硫酸铵[PEG/(NH4)2SO4]建立稳定的双水相体系以分离猪胃蛋白酶。通过上下相体积比(VR)、比活力(SA)、纯化因子(PF)、酶活回收率(η)和SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析测定萃取效果。结果表明,萃取猪胃蛋白酶的最佳配比体系为25%PEG1000:18%(NH4)2SO4,所萃取的猪胃蛋白酶酶活回收率为91.5%,SDS-PAGE结果显示,萃取的蛋白质和猪胃蛋白酶对照品分子量大小一致。     

腐败希瓦氏菌卵黄抗体活性片段的制备及抑菌效果的分析

为探讨卵黄抗体活性片段的制备方法,利用胃蛋白酶对腐败希瓦氏菌卵黄抗体进行酶解,通过高效液相色谱法动态分析酶解过程,建立了一套有效的纯化方法,并对活性片段抑菌效果进行鉴定分析.结果表明,经胃蛋白酶作用6h后,卵黄抗体被酶解为43000u左右及其他更小分子量的蛋白片段,且组分不再随时间发生变化.酶解混合液经Sephacryl S-100层析纯化后,即得纯度大于90％的活性蛋白片段.此蛋白片段保留了较高的抗原结合能力,且抑菌效果明显.研究为制备高纯度的卵黄抗体活性片段提供有效方法,为进一步扩大腐败希瓦氏菌卵黄抗体应用范围奠定理论基础.     

酶法提取胶原的研究

在4~35℃温度范围内,采用酶法成功地从制革固体废弃物中提取胶原,探讨了试验原理及方法,确定了最佳工艺条件:Ca(OH)2浓度的质量分数4.6%、酶浓度的质量分数2‰、酶解温度30℃、反应时间72h,并对所提取的胶原进行了电泳、红外光谱的表征。     

Effect of proteolytic enzymes in gastrointestinal fluids on drug release from polyelectrolyte complex microspheres based on partially phosphorylated chitosan

This study investigated the effect of proteolytic enzymes on in vitro release of ibuprofen from phosphorylated chitosan (PCS) microspheres in simulated gastric fluid (SGF) (pH 1.4) and simulated intestinal fluid (SIF) (pH 7.4). To reduce the enzymatic degradability and to enhance the sustained release property, polyelectrolyte complex microspheres based on PCS were developed and characterized. The ibuprofen release from PCS microspheres was found to be sustained more effectively than that from CS microspheres in the medium containing proteolytic enzymes. It was concluded that the PCS microspheres can be used more successfully as sustained oral drug‐delivery vehicles than CS microspheres due to their lesser enzymatic degradability. Copyright © 2004 Society of Chemical Industry     

Stability of a mineral membrane ultrafiltration reactor for peptide hydrolysis of hemoglobin

Factors involved in the activity decay of an ultrafiltration reactor during the hydrolysis of hemoglobin by pepsin were studied. Biochemical and hydrodynamic parameters were assessed. The product of hydrolysis showed no inhibitory effect. Autolysis and mechanical damage of pepsin were limited by the presence of hemoglobin. Activity decay was mainly a result of the leakage of pepsin through the membrane. Electrostatic charges carried by either hemoglobin or pepsin were involved in the permeation process.     

Peptic and tryptic hydrolysis of native and heated whey protein to reduce its antigenicity

This study examined the effects of enzymes on the production and antigenicity of native and heated whey protein concentrate (WPC) hydrolysates. Native and heated (10 min at 100°C) WPC (2% protein solution) were incubated at 50°C for 30, 60, 90, and 120 min with 0.1, 0.5, and 1% pepsin and then with 0.1, 0.5, and 1% trypsin on a protein-equivalent basis. A greater degree of hydrolysis was achieved and greater nonprotein nitrogen concentrations were obtained in heated WPC than in native WPC at all incubation times. Hydrolysis of WPC was increased with an increasing level of enzymes and higher incubation times. The highest hydrolysis (25.23%) was observed in heated WPC incubated with 1% pepsin and then with 1% trypsin for 120 min. High molecular weight bands, such as BSA, were completely eliminated from sodium dodecyl sulfate-PAGE of both native and heated WPC hydrolysates produced with pepsin for the 30-min incubation. The α-lactalbumin in native WPC was slightly degraded when incubated with 0.1% pepsin and then with 0.1% trypsin; however, it was almost completely hydrolyzed within 60 min of incubation with 0.5% pepsin and then with 0.5% trypsin. Incubation of native WPC with 1% pepsin and then with 1% trypsin for 30 min completely removed the BSA and α-lactalbumin. The β-lactoglobulin in native WPC was not affected by the pepsin and trypsin treatments. The β-lactoglobulin in heated WPC was partially hydrolyzed by the 0.1 and 0.5% pepsin and trypsin treatments and was completely degraded by the 1% pepsin and trypsin treatment. Antigenicity reversibly mimicked the hydrolysis of WPC and the removal of β-lactoglobulin from hydrolysates. Antigenicity in heated and native WPC was reduced with an increasing level of enzymes. A low antigenic response was observed in heated WPC compared with native WPC. The lowest antigenicity was observed when heated WPC was incubated with 1% pepsin and then with 1% trypsin. These results suggested that incubation of heated WPC with 1% pepsin and then with 1% trypsin was the most effective for producing low-antigenic hydrolysates by WPC hydrolysis and obtaining low molecular weight small peptides. Further research is warranted to identify the low molecular weight small peptides in the WPC hydrolysates produced by pepsin and trypsin, which may enhance the use of whey.     

Influence of chemical and biological pretreatments on the degradation of lignocellulosic material by biological systems

A highly lignified lignocellulosic substrate, barley straw, was modified by treatment with a variety of chemical and biological reagents. These reagents had specific effects on different components of the cell wall. The residual materials after the treatments were analysed for their composition and degradability by two biological pretreatments, rumen liquor and pepsin/cellulase. The biological pretreatments (cellulase and hemi-cellulase) gave residues with decreased degradability. Reagents which primarily attack the hemicellulose component gave residues with slightly increased degradabilities. When the treatments primarily hydrolysed the phenolic acid/acetic acid ester bonds, the degradabilities were also slightly increased, but only reagents which primarily attack the core lignin component of the cell wall gave residues with greatly increased degradabililies. Similar results were obtained with both biological systems, but the rumen liquor system was always more efficient than the pepsin/cellulose system.