Evaluation of the binding affinity and antioxidant activity of phlorizin to pepsin and trypsin

Phlorizin(PHL)is a natural compound with strong antioxidant properties mainly found in apples.In this paper,the interaction mechanism of PHL with pepsin and trypsin was comparatively evaluated by computer simulation,fluorescence spectra,circular dichroism(CD),and Fourier transform infrared(FT-IR)spectra at a molecular level.Fluorescence spectra showed that PHL quenches the pepsin/trypsin by static quenching.Thermodynamic parameters indicated that PHL binds to pepsin mainly through hydrogen bonds and van der Waals forces,and that of trypsin was electrostatic forces.The ground state complexes PHL and protease have a moderate affinity of 105 L/mol PHL binds more strongly to trypsin than to pepsin.CD and FT-IR spectra results showed that pepsin/trypsin decreased theβ-sheet content and slightly changed its secondary structure upon PHL.These experimental results are mutually verified with the predicted computer-aid simulation results.Upon PHL and trypsin binding,the antioxidant capacity of PHL was elevated.Nevertheless,the antioxidant capacity of PHL was decreased after binding to pepsin.This work elucidates the binding of PHL binding mechanisms to pepsin/trypsin and provides useful information for the digestion of PHL to improve the application of PHL in food processing.

Interaction of hispidin with pepsin:Multi-spectroscopic analyses and docking simulation

Hispidin is a pyranone compound found in edible and medicinal mushrooms of the Phellinus and Inonotus genera.This investigation used fluorescence spectroscopy,UV absorption spectroscopy,and molecular docking to examine the interaction of hispidin with pepsin.The Stern-Volmer method was used to perform the fluorescence quenching measurements at different temperatures(298 K,303 K,and 310 K).According to the findings,hispidin induced a static quenching mechanism in pepsin that resulted in the creation of a hispidin-pepsin complex with binding constants(Ka)ranging from 9.56×10^(4) to 3.45×10^(5) L mol^(-1).The positive values ofΔH(84.6 kJ mol-1)andΔS(337.9 J mol^(-1) K^(-1))demonstrated that hydrophobic forces contributed to forming the hispidin-pepsin complex.The findings of UV-vis absorption,synchronous fluorescence,and 3D fluorescence spectraspectra demonstrated that hispidin altered the conformation and microenvironment of pepsin.According to the analysis of molecular docking,hispidin got into the pepsin's active cavity.The research clarifies the molecular mechanisms by which hispidin binds to pepsin and helps understand its possible biological activity in vivo.

Isolation and Characterization of Acid-soluble Collagen and Pepsin-soluble Collagen from the Skin of Hybrid Sturgeon

By using the wastes fish skin of sturgeon processed as a raw material, a macromolecule biomaterial of collagen was extracted. Acid-soluble collagen(ASC) and pepsin-soluble collagen(PSC) were successfully isolated from the skin of hybrid sturgeon with two extraction methods. The yields of ASC and PSC based on the wet weight of skin were 5.73 ± 0.11% and 10.26 ± 0.39%, respectively. The denaturation and melting points of ASC(26.83 ℃ and 110.49 ℃) and PSC(26.54 ℃ and 102.99 ℃) were assessed by Circular dichroism(CD) and Differential scanning calorimetry(DSC). ASC and PSC appeared to be dense sheet-like film linked by random-coiled filaments under scanning electron microscopy(SEM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Fourier transform infrared spectroscopy(FTIR) confirmed that both the ASC and PSC were Type I collagen and maintained a complete triple helix structure. These results indicated that both ASC and PSC possessed good biological activity and could be widely used in medical biomaterials and other fields.

Studies on Digestive Enzymes in Alimentary Tract of Southern Sheatfish (Silurus meridionalis Chen) Larvae Ⅰ-Trypsin and Pepsin

The pepsin and trypsin activities and some of the properties of the two enzymes of southern sheatfish larvae were studied. The results were as follows: the highest level of trypsin activity is in the foregut in all measured tissues; from foregut to hindgut, trypsin activities decrease; the pH optimum of trypsin activity is pH9.0; the strongest pepsin activity is in the stomach; the proper density of haemoglobin for detecting pepsin activity is 1.0%. These data are useful in solving applied nutritional problems, such as the adequacy of artificial food to the digestive abilities of the fish.

Isolation and Characterization of Pepsin-Solubilized Collagen from the Skin of Black Carp (<i>Mylopharyngdon piceus</i>)

To make more effective use of underutilized resources, pepsin-solubilized collagen (PSC) was successfully extracted from the skin of black carp (Mylopharyngdon piceus) with a yield of 45.7% based on dry weight. The PSC comprising two identical α1-chains and one α2-chain with no disulfide bond was characterized as type I, and it contained 195 imino acid residues vs. 1000 amino acid residues. The collagen showed an absorption edge around 218 nm, which was lower than the maximum absorption wavelength of other PSC. The denaturation temperature of PSC was 25.6°C, which was lower than that of porcine collagen by approximately 11°C. The isoelectric point (pI) was estimated to be 8.23, and the collagen was soluble at an acidic pH as well as below 40 g/L NaCl. It is thought that the high yield and stability of PSC from the skin of black carp warrant its application as a new source of collagen for industrial purposes.

胃蛋白酶Pepsin A性质与结构分析

胃蛋白酶是一种肽链内切酶,它将蛋白质分解成较小的肽(即蛋白酶)。在胃中产生,是人类和许多其他动物消化系统中的主要消化酶之一,在那里它有助于消化食物中的蛋白质。由胃壁中的主要细胞释放的胃蛋白酶原,与胃液的盐酸混合后,胃蛋白酶原激活成为胃蛋白酶。本文对胃蛋白酶的基本特性进行了初步分析,通过分析胃蛋白酶氨基酸组成、疏水性表明胃蛋白酶具有酸性蛋白、胶原蛋白、黏蛋白等特征,整体呈现疏水性。利用MEGA软件进行胃蛋白酶系统发育分析,结果表明人与日本猕猴亲缘关系高度相近,而牛和日本猕猴两者的亲缘关系较远。使用PSORTII分析工具对胃蛋白酶亚细胞定位进行预测,结果表明胃蛋白酶主要分布于细胞外。最后,采用同源建模法构建了胃蛋白酶的三级结构,并对建模质量进行了评估,结果表明三级结构预测总体准确。

锌试剂与Pepsin作用的UV光谱研究

本文采用 UV光谱法研究锌试剂 ( ZCN)与 Pepsin(胃蛋白酶 ,Porcine mucosa)相互作用的基本条件及反应机理。研究了不同条件对该反应体系的影响 ,确定了最佳 p H2 .1 5为该配合物反应的酸度条件。测得其配合物的最大吸收峰值 λmax为 560 nm和 596nm,其标准曲线质量浓度在 ( 0～ 30 0 ) ug/m L范围内呈线性关系 ,服从朗白比耳定律。最低捡出限为 0 .8mg/L。研究了温度、时间对体系的影响 。

Pepsin nanofilm self—assembly on the positively charged poly （ethylene terephthalate）substrate

Pepsin was assembled on the surface of prepared poly(ethylene terephthalate)(PET-NH3^+) substrates.The composition and structure of the pepsin/PET-NH3^+ assembling films in different condition were characterized by X-ray photoelectron spectroscopy(XPS) and atomic force microscopy(AFM).

Characterization and antioxidant activities of marine pepsin soluble collagen from the skin of yellow goosefish Lophius litulon

Characteristics and antioxidant activities of pepsin-soluble collagen(PSC) from yellow goosefish( Lophius litulon) skins were investigated. PSC was characterized as a type I collagen, and its imino acid content was 193 residues/1 000 residues. PSC's denaturation temperature was ~17.56°C and Fourier transform infrared spectra confirmed the presence of triple helices. Solubility analysis showed good solubility at acidic pH(1–6) or low Na Cl concentrations(≤2%). PSC showed scavenging activity against hydroxyl radicals and superoxide anions in a concentration-dependent manner. Furthermore, PSC could protect D-galactose-induced skin aging by significantly controlling malondialdehyde formation and improving the activity of superoxide dismutase, glutathione peroxidase, catalase, glutathione, and hydroxyproline. PSC may be a promising antioxidant in appropriate applications.

Clinical relevance of salivary pepsin detection in diagnosing gastroesophageal reflux disease subtypes

Background:Gastroesophageal reflux disease(GERD)is heterogeneous with a varied symptom spectrum and reflux profiles.Its definite diagnosis often requires invasive tools including endoscopy or reflux monitoring.The aim of this study was to investigate the clinical relevance of salivary pepsin detection as a non-invasive screening tool to diagnose GERD of different subtypes.Methods:A total of 77 patients with suspected GERD symptoms and 12 asymptomatic controls were analysed.All participants performed symptom evaluation,upper endoscopy,esophageal manometry,and 24-hour multichannel intraluminal impedance-dual pH probe monitoring.Saliva was self-collected across three different time points:at early fasting,postprandially,and at symptom occurrence.Salivary pepsin levels were measured via Peptest.The optimal threshold of salivary pepsin for diagnosing distal or proximal reflux was determined according to a receiver-operating characteristic curve.Results:The average salivary pepsin concentration of suspected GERD patients was significantly higher than that of controls(100.63[68.46,141.38]vs 67.90[31.60,115.06]ng/mL,P=0.044),although no difference was found among patients with different symptom spectrums.The distal reflux group had a higher average pepsin concentration than non-reflux patients(170.54[106.31,262.76]vs 91.13[63.35,127.63]ng/mL,P=0.043),while no difference was observed between the distal reflux group and the proximal reflux group.The optimal cut-off value of salivary pepsin concentration for diagnosing pathological distal reflux was 157.10 ng/mL,which was higher than that for diagnosing pathological proximal reflux(122.65 ng/mL).The salivary pepsin concentration was significantly correlated with distal and proximal reflux parameters.Conclusions:Salivary pepsin measurement can help in identifying true GERD with pathological distal reflux or proximal reflux,regardless of different symptom spectrums.A higher threshold should be applied for diagnosing distal reflux than for proximal reflux.

多光谱法和分子对接模拟法研究茶碱和胃蛋白酶的相互作用

在模拟生理条件下,通过紫外可见吸收光谱法、傅里叶红外光谱、荧光光谱、三维荧光光谱、同步荧光光谱、圆二色谱等方法以及分子对接模拟法对茶碱(TPL)与胃蛋白酶(PEP)的结合机理进行了研究,在分子层面探讨TPL与PEP相互作用的机制,有助于对TPL的药理毒性、药效进行深入研究。根据Stern-Volmer方程,计算在298、 303和308 K三种温度下TPL对PEP的动态荧光猝灭速率常数Kq远大于最大动态荧光猝灭常数2.0×10^(10)L·(mol·s)^(-1),证明TPL对PEP的猝灭方式为静态猝灭。随着TPL浓度的不断升高,PEP的动态猝灭常数Ksv呈规律性下降趋势,TPL可以有效对PEP的内源荧光进行猝灭,进一步判定作用机制为静态猝灭。三维荧光图谱分析表明,体系中随着TPL浓度的不断升高,PEP中代表色氨酸残基与酪氨酸残基以及肽链骨架结构的荧光强度峰值明显降低,且峰位发生红移,表明TPL对PEP的二级结构产生影响。同步荧光图谱分析表明TPL与PEP结合时,主要集中在色氨酸的残基上。红外光谱表明TPL引起PEP内官能团伸缩振动,使PEP的二级结构发生改变。紫外吸收光谱表明随着混合体系中TPL浓度增大,吸收峰峰值逐渐增大,表明TPL可以改变PEP的二级结构。分子对接模拟法表明,TPL与PEP中氨基酸残基GLU13、 VAL30、 TRP39、 GLY76、 GLY78、 PHE117的结合作用力为范德华力,与氨基酸残基THR77、 GLY217形成氢键,与氨基酸残基TYR75、 LEU112、 ILE120、PHE111之间存在疏水作用力,证明两者主要通过范德华力与氢键结合,进一步证明TPL改变了PEP的二级结构。圆二色谱分析显示PEP中β-折叠的占比从50.2%下降至48.8%, α-螺旋结构的占比从8.1%上升到8.4%;β-转角的占比从18.3%上升到18.7%;无规则结构的占比从29.1%上升到29.2%,表明TPL改变了PEP的二级结构。实验研究结果有助于了解TPL与PEP的结合作用机制,为TPL的使用及研究提供了数据依据。

血清circRNA、Notch-3联合胃蛋白酶对胃癌的诊断价值及与分期的相关性

目的探究血清环状RNA(circRNA)、神经源位点缺口同源蛋白3(Notch-3)、胃蛋白酶对胃癌的诊断价值及其与胃癌分期的相关性。方法选取2021年3月至2023年3月该院收治的150例胃癌患者和150例胃部良性病变患者分为研究组和良性组。依据胃癌分期将研究组分为Ⅰ期组(36例)、Ⅱ期组(37例)、Ⅲ期组(35例)及Ⅳ期(42例)组。检测并比较研究组和良性组circRNA、Notch-3及胃蛋白酶表达水平;分析不同胃癌分期circRNA、Notch-3及胃蛋白酶表达水平;绘制受试者工作特征(ROC)曲线分析circRNA、Notch-3、胃蛋白酶单独及联合检测对胃癌的诊断价值;采用Spearman相关进行circRNA、Notch-3、胃蛋白酶与胃癌分期的相关性分析。结果研究组circRNA、Notch-3及胃蛋白酶表达水平高于良性组,差异均有统计学意义(P<0.05)。circRNA、Notch-3及胃蛋白酶的表达水平比较,Ⅰ期组<Ⅱ期组<Ⅲ期组<Ⅳ期组,差异均有统计学意义(P<0.05)。ROC曲线分析结果显示,circRNA、Notch-3、胃蛋白酶单独检测胃癌的曲线下面积(AUC)分别为0.797、0.802、0.848,低于3项指标联合检测的0.901(Z=10.799、11.231、14.492,P<0.05)。circRNA、Notch-3、胃蛋白酶均与胃癌分期呈正相关(r=0.585、0.581、0.645,P<0.05)。结论circRNA、Notch-3及胃蛋白酶的表达水平在胃癌中均有所升高,且胃癌分期越高,其表达水平越高;circRNA、Notch-3及胃蛋白酶均对胃癌有一定诊断价值,其联合检测灵敏度及特异度更高,且circRNA、Notch-3及胃蛋白酶与胃癌分期呈正相关。

胃蛋白酶在声带息肉、声带癌中的作用分析

目的探讨胃蛋白酶在声带息肉、声带癌发病中的作用。方法选取2020年5月至2021年12月诊断为声带息肉患者27例(声带息肉组),声带癌患者27例(声带癌组),健康志愿者23例(对照组);采用反流症状指数(reflux symptom index,RSI)、反流体征评分(reflux finding score,RFS)量表进行评分,胃蛋白酶检测试剂盒(Peptest)进行唾液胃蛋白酶检测,并用免疫组织化学方法检测声带息肉、声带癌患者声带组织中胃蛋白酶表达情况,比较三组RSI、RFS评分、胃蛋白酶检测试剂盒结果,比较声带息肉组和声带癌组胃蛋白酶免疫组化染色结果。结果声带息肉组和声带癌组的RSI、RFS、胃蛋白酶检测结果均高于对照组,差异均有统计学意义(P<0.05);声带息肉组的RSI、胃蛋白酶检测结果和胃蛋白酶免疫组化结果与声带癌组比较差异无统计学意义(P>0.05);声带息肉组的RFS与声带癌组比较差异有统计学意义(P<0.05)。结论胃蛋白酶可能是声带息肉、声带癌的致病因素之一,但发病机制可能有所不同。

乌鳢鱼皮中胶原蛋白的提取工艺及特性研究

该研究利用醋酸和胃蛋白酶结合从乌鳢鱼皮中提取胶原蛋白。采用单因素试验和正交试验考察了醋酸浓度、料液比、酶用量、提取时间等条件对胶原蛋白提取率的影响。结果表明,当醋酸的浓度为0.5 mol/L,胃蛋白酶的添加量为鱼皮质量的1%,提取时间48 h,料液比1∶70,胶原蛋白提取率达到51.2%。通过氨基酸组成分析、圆二色谱、聚丙烯酰胺凝胶电泳等方法对胶原蛋白的结构进行测试,发现该法提取的胶原蛋白具有典型Ⅰ型胶原蛋白特征,其三螺旋结构保持完整,分子质量约为340 kDa。成品中蛋白质含量>95%,羟脯氨酸的含量>5%,灰分<1%(均为质量分数),可用于医用材料、化妆品、功能食品等领域。

Characterization of acid-and pepsin-soluble collagens from spines and skulls of skipjack tuna(Katsuwonus pelamis)

Acid-soluble collagen(ASC) and pepsin-soluble collagen(PSC) from the spine(ASC-SP and PSC-SP) and skull(ASC-SK and PSC-SK) of the skipjack tuna, Katsuwonus pelamis, were successfully isolated and characterized. The yields of ASC-SP, PSC-SP, ASC-SK and PSC-SK were(2.47 ± 0.39)%,(5.62 ± 0.82)%,(3.57 ± 0.40)%, and(6.71 ± 0.81)%, respectively, on the basis of dry weight. The four collagens contained Gly(330.2-339.1 residues/1 000 residues) as the major amino acid, and their imino acid contents were between 168.8 and 178.2 residues/1 000 residues. Amino acid composition, SDS-PAGE, and FTIR investigations confirmed that ASC-SP and ASC-SK were mainly composed of type I collagen, and had higher contents of high-molecular weight cross-links than those of PSC-SK and PSC-SP. The FTIR investigation also certified all the collagens had triple helical structure. The denaturation temperatures of ASC-SK, PSC-SK, ASC-SP, and PSC-SP were 17.8, 16.6, 17.6, and 16.5 °C, respectively. All isolated collagens were soluble at acidic pH(1-5) and lost their solubilities when the NaCl concentration was above 2%(W/V). The isolated collagens from the spines and skulls of skipjack tuna could serve as an alternative source of collagens for further application in food, cosmetic, biomedical, and pharmaceutical industries.

Enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction for the synthesis of 1,2-dihydroquinolines using pepsin from porcine gastric mucosa

An unprecedented enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction of 2-aminobenzaldehyde and a,b-unsaturated aldehydes is achieved. Pepsin from porcine gastric mucosa provided mild and efficient access to diverse substituted 1,2-dihydroquinolines in yields of 38%–97% with 6%–24%enantiomeric excess（ee）. This work not only provides a novel method for the synthesis of dihydroquinoline derivatives, but also promotes the development of enzyme catalytic promiscuity.

酶-超声波辅助提取海蜇胶原蛋白及其组成分析

为确定酶-超声波辅助提取海蜇胶原蛋白的最佳工艺,采用单因素实验和响应面实验对工艺进行优化,考察胃蛋白酶添加量、提取时间、超声时间、超声功率对海蜇胶原蛋白得率的影响,并对胶原蛋白的氨基酸组成和结构进行分析。结果表明,酶-超声波辅助提取海蜇胶原蛋白的最佳条件为胃蛋白酶添加量1%、提取时间48 h、超声时间32 min、超声功率318 W,此时海蜇胶原蛋白得率为38.03%。甘氨酸是海蜇胶原蛋白中的主要氨基酸,为24.96%,海蜇胶原蛋白的紫外吸收峰位于235 nm处。在酰胺A、B带以及Ⅰ、Ⅱ、Ⅲ带中,有海蜇胶原蛋白的典型红外光谱特征吸收峰。该研究为海蜇深加工提供了理论基础。

儿童急性化脓性中耳炎中耳脓液中胰蛋白酶和胃蛋白酶检测分析

目的检测儿童急性化脓性中耳炎(ASOM)的中耳脓液中是否存在胃蛋白酶和胰蛋白酶。方法收集徐州市儿童医院71例急性化脓性中耳炎患儿的中耳脓液样本,根据脓液性状分为浆液性(39例)和粘液性(32例),分别进行pH值测试、免疫印迹实验(WB)及酶联免疫吸附剂检测(Elisa)。所有受试者均由家长辅助完成反流症状指数评分量表(RSI)。结果①71例ASOM患儿中,49.29%(35/71)患儿的RSI评分>13分。②71例中耳脓液样本中,胃蛋白酶阳性率为49.29%(35/71),胰蛋白酶阳性率为42.25%(30/71);35例RSI评分阳性患儿中,胃蛋白酶阳性率为100%(35/35),胰蛋白酶阳性率60%(21/35)。浆液性和粘液性中耳脓液的胃蛋白酶和胰蛋白酶阳性率无显著性差异(P>0.05)。③ELISA检测结果示,胃蛋白酶浓度为47.80(39.80,69.30)ng/ml,胰蛋白酶浓度为291.87±20.45 ng/ml,胰蛋白酶浓度显著高于胃蛋白酶(P<0.05)。浆液性和粘液性中耳脓液的胃蛋白酶和胰蛋白酶浓度之间无显著性差异(P>0.05)。④粘液性中耳脓液pH值为7.39±0.28,浆液性中耳脓液pH值为7.36±0.26,粘液性和浆液性中耳脓液pH值之间差异无统计学意义(P>0.05)。结论ASOM合并咽喉反流性疾病患儿中耳脓液中可检出胃蛋白酶和胰蛋白酶,对儿童ASOM合并咽喉反流性疾病有一定的诊断价值。