CNBr-Papain-Sepharose 4B亲和填料的制备及其性质研究

为探求从鱼类废弃组织及漂洗水中回收Cystatin的高效方法,首先自制了溴化氢-木瓜蛋白酶-琼脂糖凝胶(CNBr-Papain-Speharose 4B)亲和填料,确定了适宜的配基Papain偶联密度,之后测定了该亲和填料的热稳定性、酸碱稳定性、贮藏性能及使用的重复性,最后以该制备的填料对鱼糜漂洗水中的CPIs活性进行了初步回收。以对重组鲢鱼Cystatin吸附率为评价指标,确定适宜Papain偶联密度,即CNBr-Speharose 4B和Papain溶液最佳料液比为1∶7(g∶mL)。CNBr-Papain-Speharose 4B的适宜使用温度为4~40℃,且在pH 3.0~12.0之间稳定。CNBr-Papain-Speharose 4B在4℃下,6个月的贮藏期内,表现较好的稳定性。将填料装柱后进行验证实验,证明该填料的使用重复性能稳定,且最终成功高效地回收了鲢鱼鱼糜漂洗水中的CPIs。

Flavourzyme-Papain复合蛋白酶水解龙虾副产物工艺条件的研究

以底物浓度为10%、Flavourzyme与Papain复合酶比为1:1的条件,利用单因素试验和正交试验,研究了pH值、加酶量、温度、酶解时间对龙虾副产物蛋白质水解度的影响,在此基础上进一步确定了合适的底物浓度。结果表明,Flavourzyme-Papain复合蛋白酶水解龙虾副产物的最佳条件为:pH7.0、加酶量1.5%、温度50～55℃、时间5h,在底物浓度25%的条件下水解,最大水解度为26.2%。

An Orthogonal Experiment of Papain-Assisted Extraction of Polysaccharides from Agrocybe aegirit

[Objective] This study aimed to explore optimum conditions for papain-as- sisted extraction of polysaccharides from Agrocybe aegirit. [Method] Because enzyme is capable of destroying organizational structure of cells, papain was selected to cat- alyze cells to let polysaccharides out. An orthogonal experiment was carried out to investigate the effects of hydrolysis temperature, hydrolysis time, enzyme concentra- tion and solid to liquid ratio on extraction of polysaccharides from A. aegirit. [Result] Through the orthogonal experiment, the optimum papain-assisted extraction conditions were obtained： hydrolysis temperature, 45 ℃; hydrolysis time, 2 h; enzyme concen- tration 2%; solid to liquid ratio, 1：80. [Conclusion] This study will provide a reference for extraction of polysaccharides from A. aegirit.

Inhibition of cysteine protease papain by metal ions and polysulfide complexes,especially mercuric ion

Aim Cysteine proteases are closely associated with many human and non-human pathological processes and are potential targets for metal ions especially Hg^2＋ and the related species. In the present work, on the basis of to the general study on the effects of some metal ions on the activity of papain, a well-known representative of cysteine protease family, the inhibitory effects of Hg^2＋ and polysulfide complexes were studied. Results All the metal ions tested （Hg^2＋, Cu^2＋, Ag^＋, Au^3＋, Zn^2＋, Cd^2＋, Fe^3＋, Mn^2＋, Pb^2＋, Yb^3＋） inhibit the activity of papain anda good correlation between the inhibitory potency and softness-and-hardness was observed. Among the metals, Hg^2＋ was shown to be a potent inhibitor of papain with a Kiof 2 × 10^-7 mol·L^-1 among. Excessive amounts of glutathione and cysteine could reactivate the enzyme activity of papain deactivated by Hg^2＋. These evidences supported that Hg^2＋ might bind to the catalytic site of papain. Interestingly, Hg （Ⅱ） polysulfide complexes were for the first time found to inhibit papain with a Ki of 7 × 10^-6 mol·L^-1, whose potency is close to a well known mercury compound, thimerosal （Ki=2.7 × 10^-6）. In addition, Hg （Ⅱ） polysulfide complexes exhibit good permeability （ 1.9 × 10-5 cm· s^-1） to caco-2 monolayer. Conclusion These results suggested that mercury polysulfide complexes might be potential bioactive species in the interaction with cysteine proteases and other- SH-content proteins, providing a new clue to understand the mechanism of the toxicological and pharmacological actions of cinnabar and other insoluble mercury compounds.

Immobilization of Papain in Biosflica Matrix and Its Catalytic Property

A novel method was developed for papain immobilization through a biomimetic silicification process induced by papain. By incubating papain in a silica precursor solution, the papain-silica composite formed rapidly and oanain was encansulated. The encansulation efficiency and the recovery activity were 82.60% and 83.09%, re-spectively. Compared with enzymes and biomolecules immobilized in biosilica matrix in the presence of additaonal silica-precipitating species, this papaln encapsulation process, a biomimetic approach, realized high encapsulation efficiency by its autosilification activity under mild conditions （near-neutral pH and ambient temperature）. Fur-thermore, the encapsulated papain exhibits enhanced thermal, pH, recycling and storage stabilities. Kinetic analysis showed that the biomimetic silica matrix did not significantly hinder the mass transport of substrate or the release of product.

Effects of Papain Hydrolysis on the Pasting Properties of Wheat Flour

As one of the most effective enzymatic modification methods of protein, papain hydrolysis is applied widely in food production, accompanying starch pasting frequently in order to improve industrial quality. Effects of the papain hydrolysis on flour pasting properties were investigated in five papain/flour concentrations and five time-treatments. The structure of starch and protein networks in slurry was investigated under microscope before and after pasting. Results showed that papain hydrolysis influenced the pasting properties of wheat flour significantly through affecting structural characteristics, amylase activity and exotbermic transition, especially during the early stage of hydrolysis. Peak viscosity, trough, final, integral area, and setback significantly decreased along with the increasing concentration of papain. Both hydrolysis time and concentration of papain had obviously effect on the breakdown. Pasting temperature and pasting time increased significantly with the enhancement of papain concentration. Hydrolysis time exerted minor effect on the pasting temperature and pasting time. The average peak time was slightly prolonged by lower concentration of papain, otherwise slightly shortened by higher concentration.

Ag-induced Efficient Immobilization of Papain on Silica Spheres

Complexation and interaction between silver and amino group were applied to induce an efficient immobilization of papain on silica spheres.Tbe silver nanoparticles were deposited on the silica spheres before p apainwas coupled to the silica spheres. The silica spheres with silver nanoparticles were characterized by high resolution transmission electron microscopy （HR-TEM）, Fournier transform infrared spectroscopy （FT-IR）, and UV-Vis scanning spectrometer. FT-IR spectrum was also used to characterize the immobilized and free papain. Effect of some factors on the activities of the immobilized papain was investigated. It was observed that the coupled yield and relative activity of the papain on Ag/SiO2 were 1.17 and 1.86 times of those on the bare SiO2, respectively. At an optimum concentration of silver, theobserved activity of the immobilized papain was 2.1 timesof that on the bare.silica.In addition, the maximum specific activity of papain immobilized on Ag/SiO2 was 819.9 U·mg·^-1, which is slightly lower than that of the free papain, 906.2 U·mg^-1 . Stability of the immobilized papain was also examined. The resuits indicate that the silver nanoparticles successfully induce a fine immobilization of papain.

THERMAL ACTIVATION OF IMMOBILIZED PAPAIN

Papain (Papainase, EC 3.4.22.2) was immobilized on porous silica beads by cross linking with glutaraldehyde. The thermal activation of this immobilized papain in aqueous system was found at, a temperature range from 50 to 90 degrees C. The higher the temperature, the more active the immobilized papain will possess. At the same time, the durability of the immobilized papain on heating was greatly improved. The effect of additives and salts on the activity of the immobilized papain were also studied. The results showed that the additives and some of the salts studied could markedly enhance the activity of the immobilized papain at elevated temperature.

STUDY ON THE IMMOBILIZATION OF PAPAIN WITH A MACROPOROUS BEAD CARRIER OF COPOLYMER CONTAINING MONOMER UNITS OF NAMINOETHYL ACRYLAMIDE AND VINYL ALCOHOI

A kind of macroporous bead carrier of copolymer containing monomer units of N-aminoethyl acrylamide and vinylalcohol was synthesized, i.e. the MR-AA carrier. Papain was immobilized on the carrier using glutaraldehyde as the couplingagent. The enzymatic activity of the immobilized papain was compared with free papain using casein as a substrate, and theeffects of glutaraldehyde concentration, pH, temperature, time and papain amount added on the activity recovery were alsoinvestigated. The results show that the MR-AA carrier contains reactive primary amine groups, hydrophilic amido links andhydroxyl groups, as well as macroporous structures based on its matrix (MR-AV matrix), furthermore, the activity recoveryof papain in the immobilization could reach 48%/～58%. In comparison with free papain, the resulting immobilized papainexhibits a remarkable thermostability and better reusability.

Optimization of Adsorption Conditions of Papain on Cross-Linked Phage-Displayed Heptapeptide Matrix Using Response Surface Methodology

A new cross-linked heptapeptide matrix(CHM)was prepared and the adsorption performance for papain was investigated.Firstly,a phage-displayed heptapeptide(Ile-Gln-SerPro-His-Phe-Phe)with high affinity to papain was found.Secondly,the heptapeptide was cross-linked to a matrix for papain adsorption.Finally,the adsorption conditions were optimized by response surface methodology.The results indicated that the optimum adsorptionconditionsweredeterminedasinitialpH 6.9,temperature 40℃,and the adsorption capacity of CHM for papain was found to be 55.52 mg/g.Moreover,the papain was purified 78-fold in a single step determined by affinity precipitation.More than87%of the adsorbed papain was desorbed using the eluent solventtriethylamine at pH 7.4.The results show that the CHM is a promising adsorbent for papain purification from crude papaya powder.

Papain Activity in Dextran Solution for Keratin Hydrolysis

High concentration of polymer solution in cosmetics has the essential role in keeping water on peel of skin. This study includes the papain activity in the high concentrated dextran solution for keratin hydrolysis. Papain is the industrial and a representative hydrolase. The concentration of dextran was ranged to 100 g/L. In the increasing concentration of dextran, the activity of papain decreased due to the low diffusivity of papain and substrate. Even in concentrated dextran solution, keratin powder was possible to be hydrolyzed at the high efficiency.

Preparation of Dendrimers from Macromolecular Cores and Papain Immobilization

Using amino-poly(ethylene glycol) and amino-propyl silica gel as initiator cores, two dendrimers were prepared by a two-step procedure. The progress of dendrimers during each step was monitored by infrared spectroscopy and elemental analysis. Papain was crosslinked to the solid dendrimer with silica gel core by glutaraldehyde, and the enzymatic activity of the immobilized papain kept almost unchanged after repeated usage for fifteen times.

Synergistic precipitant powered self-assembly of papain for cross-linked enzyme crystals preparation

In this study,we prepared cross-linked enzyme crystals(CLECs)of papain to further broaden the application of the enzyme with high activity in extreme environments.Initially,papain crystals were successfully obtained based on the micro-batch,batch,and expanded batch crystallization experiments.Specifically,ammonium sulfate and polyethylene glycol 6000(PEG6000)were synergistically used as the precipitants,while L-cysteine was applied to enhance the activity of papain.Furthermore,the interaction between L-cysteine and papain was modeled by molecular docking technique.It was found that L-cysteine could form a hydrogen bond with aspartic acid residue(Asp)at site 158,and the electrostatic attraction with lysine residue(Lys)at site 156 was also quite obvious.Then the enzyme crystals were cross-linked by glutaraldehyde at optimized conditions.The papain CLECs were identified by various methods,and it was found that the thermal stability and enzymatic activity both increased compared to the raw enzyme.More importantly,it could be applied at more rigorous conditions,for example,pH of 4.

加味独活寄生合剂对膝骨关节炎小鼠血清IL-6、IL-1β、TNF-α及滑膜炎症的影响

目的探讨加味独活寄生合剂对膝骨关节炎小鼠血清白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)及滑膜炎症的影响,初步判定加味独活寄生合剂治疗膝骨关节炎的最佳剂量。方法将36只C57/BL6j小鼠随机分为空白组、模型组、西药对照组及中药低、中、高剂量组,除空白组外均通过膝关节注射木瓜蛋白酶溶液建立膝骨关节炎模型,空白组、模型组予生理盐水灌胃,西药对照组予双醋瑞因胶囊溶液灌胃,中药低、中、高剂量组分别按照6.28、12.56、25.12 g/kg剂量灌胃加味独活寄生合剂。连续灌胃8周后,各组小鼠进行改良Lequesen MG量表评分,ELISA测定各组小鼠血清中IL-6、IL-1β、TNF-α含量;HE染色及Masson染色观察膝关节滑膜组织结构形态及纤维组织增生情况。结果与空白组相比,模型组改良Lequesen MG量表评分显著升高(P<0.01),血清炎症因子含量明显升高(P<0.01),HE染色可见滑膜组织丰富的血管增生及大量炎性细胞浸润等滑膜炎症表现,Masson染色可见大量胶原纤维增生;西药对照组、中药低剂量组改良Lequesen MG量表评分较模型组显著降低(P<0.01),血清炎症因子含量均明显降低(P<0.01),HE染色见滑膜炎症改善,Masson染色见胶原纤维增生减轻;中药中、高剂量组较西药对照组和中药低剂量组改良Leque⁃sen MG量表评分显著降低(P<0.01),血清炎症因子含量明显降低(P<0.01);HE染色示滑膜炎症明显好转,Masson染色见胶原纤维增生明显减轻。结论加味独活寄生合剂可降低膝骨关节炎小鼠血清IL-6、IL-1β、TNF-α含量,改善膝关节滑膜炎症;12.56 g/kg剂量的加味独活寄生合剂对膝骨关节炎小鼠病情改善最明显。

五味子蛋白酶解前后抗氧化活性和功能特性

以五味子蛋白为原料,评价中性蛋白酶和木瓜蛋白酶对五味子蛋白酶解前后的抗氧化活性和功能特性;根据两种蛋白酶最适酶解条件,对五味子蛋白酶解,通过扫描电子显微镜、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、游离巯基和二硫键含量测定分析其结构,并测定其体外清除羟基自由基、DPPH自由基和ABTS~+自由基的能力,测定亚铁离子螯合能力和铁离子还原能力,并对不同酶解产物的持水性、持油性、乳化性和乳化稳定性、起泡性和泡沫稳定性等功能特性进行评价;结果显示,五味子蛋白经酶解后相对分子量显著下降,中性蛋白酶酶解产物分子量最低,中性蛋白酶酶解产物水解度最高,为26.45%,酶解效果优于木瓜蛋白酶。以五味子蛋白为参照,酶解可以提升其抗氧化能力,中性蛋白酶酶解产物抗氧化能力优于木瓜蛋白酶酶解产物,但木瓜蛋白酶酶解产物具有较好的还原能力。五味子蛋白经酶解后,溶解性、乳化性和乳化稳定性、起泡性和泡沫稳定性均提高,并且中性蛋白酶酶解产物功能特性优于木瓜蛋白酶酶解产物。扫描电子显微镜观察到五味子蛋白经两种不同蛋白酶酶解后,产生不同程度的碎片和条状结构,并且木瓜蛋白酶的酶解产物中存在部分较大的片状结构,中性蛋白酶酶解产物呈现出细小的无规则碎片结构,相对松散。同时与五味子蛋白相比,两种酶解产物游离巯基含量均增加,其中中性蛋白酶酶解产物二硫键含量最小。综上,酶解可以降低五味子蛋白分子量,提升五味子蛋白的抗氧化活性和功能特性。

超声波联合木瓜蛋白酶降解壳聚糖的工艺研究

采用超声波技术联合木瓜蛋白酶对壳聚糖进行降解,以还原糖释放量为指标,通过单因素实验研究了壳聚糖质量浓度、溶液p H、超声功率及超声时间对壳聚糖降解的影响。并利用傅里叶变换红外光谱(FT-IR)及X-射线衍射(XRD)分别对降解前后的壳聚糖进行结构表征分析。结果表明:超声波联合木瓜蛋白酶可以有效降解壳聚糖,在壳聚糖质量浓度为6 g/L、溶液p H为4.5、超声功率为480 W、超声时间为180 min条件下,加入木瓜蛋白酶,降解效果最明显,还原糖释放量达到1.456 g/L;FT-IR结果表明,降解后壳聚糖的结构基本没有变化;XRD结果表明,降解后壳聚糖的晶体结构受到了破坏。

响应面法优化长茎葡萄蕨藻多糖提取工艺及抗氧化活性

为了探究提取长茎葡萄蕨藻多糖的最优工艺及抗氧化活性,对目前已有的多糖提取方法进行筛选,并采用单因素实验和响应面实验的方法,对料液比、提取温度、提取时间、木瓜蛋白酶添加量和提取次数这5个因素进行优化。结果显示,添加木瓜蛋白酶提取长茎葡萄蕨藻多糖的方法最高效便捷,且当料液比1∶40,提取温度50℃,提取时间3 h,提取次数2次,以及木瓜蛋白酶添加量为2.0%时,长茎葡萄蕨藻多糖提取率相对较高,可达到41.24%±0.09%。进一步的实验结果显示,长茎葡萄蕨藻多糖对DPPH和ABTS自由基均具有良好的清除活性,其IC50值分别为2.32和0.67 mg/mL。研究表明,使用优化后的木瓜蛋白酶酶解法能有效提高长茎葡萄蕨藻多糖的提取率,且长茎葡萄蕨藻多糖具有良好的抗氧化活性。本研究可为长茎葡萄蕨藻多糖的开发利用提供理论基础和参考依据。

Bushen Qiangjin capsule(补肾强筋胶囊) inhibits the Wnt/β-catenin pathway to ameliorate papain-induced knee osteoarthritis in rats

OBJECTIVE:To evaluate the molecular mechanism underlying the beneficial effect of Bushen Qiangjin capsule(补肾强筋胶囊,BSQJ),a Traditional Chinese Medicine,on knee osteoarthritis(KOA).METHODS:In the present study,32 female Sprague-Dawley rats were randomly divided into four groups:control,KOA,high-dose BSQJ(H-BSQJ),and low-dose BSQJ(L-BSQJ).After successfully establishing the KOA model by intra-articular injection of papain,H-BSQJ and L-BSQJ groups were intragastrically administered 0.243 and 0.122 g/kg BSQJ,respectively,daily for 6 weeks.At the end of the experiment,knee articular cartilage tissues of rats were collected for evaluation by hematoxylin and eosin staining,Safranin O-Fast Green staining,and terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling assay.Serum interleukin-1βand tumor necrosis factor-αlevels of rats were detected with an enzyme-linked immunosorbent assay method.Gene expression of Wnt-4,β-catenin,Frizzled-2,glycogen synthase kinase-3β(GSK-3β),cysteinyl aspartate-specific proteinases 3 and 9(caspases 3 and 9),collagen typeⅡalpha 1(Col2 a1),and matrix metalloproteinases 1 and 13(MMP-1 and MMP-3)of rat knee articular cartilage was quantified by reverse transcription-quantitative polymerase chain reaction analysis.Wnt-4,β-catenin,Frizzled-2,GSK-3β,cleaved caspase-3,and cleaved caspase-9 protein expression in rat knee articular cartilage was determined by western blot analysis.RESULTS:BSQJ obviously reduced pathological damage and matrix degradation of articular cartilage in KOA rats.Compared with the KOA group,H-BSQJ rats exhibited downregulated m RNA and protein expression of Wnt-4,β-catenin,Frizzled-2,and caspase-3,as well as upregulated m RNA and protein expression of GSK-3β.In addition,H-BSQJ significantly increased m RNA expression of Col2 a1 and decreased m RNA expression of MMP-1 and MMP-13.CONCLUSION:BSQJ exerted a beneficial effect on KOA by a mechanism involving downregulation of the Wnt/β-catenin pathway,which inhibited both cartilage extracellular matrix degradation and chondrocyte apoptosis to ameliorate KOA in rats.

Coronavirus membrane-associated papain-like proteases induce autophagy through interacting with Beclinl to negatively regulate antiviral innate immunity

Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 （PLP2-TM） of coronaviruses acts as a novel autophagy- inducing protein. Intriguingly, PLP2-TM induces incom- plete autophagy process by increasing the accumula- tion of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2- TM interacts with the key autophagy regulators, LC3 and Beclinl, and promotes Beclinl interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclinl partially reverses PLP2-TM＇s inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results sug- gested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclinl, which in turn modulates coronavirus replication and antiviral innate immunity.