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%。

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.

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.

Immobilization of Papain in Biosilica Matrix and Its Catalytic Property

一个新奇方法通过 papain 导致的一个 biomimetic silicification 过程为 papain 固定被开发。由在一个硅石先锋答案孵化 papain， papain 硅石合成很快形成了， papain 被包含。封装效率和恢复活动分别地是 82.60% 和 83.09% 。与面对另外的猛抛硅石的种类在 biosilica 矩阵使不能调动的酶和 biomolecules 相比，这个 papain 封装过程，一条 biomimetic 途径，由它在温和条件(在中立附近的 pH 和周围的温度) 下面的 autosilification 活动的认识到的高封装效率。而且，包含的 papain 展览提高了热， pH，再循环并且存储稳定性。运动分析证明 biomimetic 硅石矩阵显著地没妨碍底层的集体运输或产品的版本。

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.

Ag-induced Efficient Immobilization of Papain on Silica Spheres

在银和氨基的组之间的 Complexation 和相互作用被使用在硅石范围上导致 papain 的有效固定。在 papain 被联合到硅石范围以前，银 nanoparticles 在硅石范围上被扔。有银 nanoparticles 的硅石范围被高分辨率传播电子显微镜学(HR-TEM ) 描绘， Fournier 变换红外线的光谱学(英尺红外) ，和紫外力的扫描分光计。英尺红外光谱也被用来描绘使不能调动并且免费 papain。在使不能调动的 papain 的活动的一些因素的效果被调查。联合收益和 Ag/SiO2 上的 papain 的相对活动分别地是赤裸的 SiO2 上的那些的 1.17 和 1.86 次，这被观察。在银的最佳集中，使不能调动的 papain 的观察活动在赤裸的硅石上是 2.1 次那。另外，在 Ag/SiO2 上使不能调动的 papain 的最大的特定的活动是 819.9 U 路 m g ? 1，它比免费 papain 的稍微低， 906.2 U 路 m g ? 1。使不能调动的 papain 的稳定性也被检验。结果显示银 nanoparticles 成功地导致 papain 的好固定。

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。

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。

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.

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.

SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3- TBK1 complex

SARS coronavirus （SARS-CoV） develops an antagonis- tic mechanism by which to evade the antiviral activities of interferon （IFN）. Previous studies suggested that SARS-CoV papain-like protease （PLpro） inhibits activa- tion of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism（s） used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro effi- ciently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain （PLpro-TM） from SARS-CoV inhibits STING/ TBKl/IKKE-mediated activation of type I IFNs and dis- rupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBKI. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKK~, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activa- tion of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is dis- rupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3- TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction withSTING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.

多级双水相萃取木瓜蛋白酶的研究

使用双水相萃取大规模生产木瓜蛋白酶时,为获得较高的回收率,通常需要较高的萃取剂浓度和体积比,但这会导致目标物的纯度和浓度降低。该研究采用聚乙二醇和柠檬酸钠组成的双水相体系在多个离心管之间的转移来模拟工业上常用的多级错流萃取和多级逆流萃取过程,以分配系数、酶活性回收率为指标,采用McCabe Thiele图解法得到多级逆流双水相萃取的理论级数,结果表明,当相比为0.5,原料液下相的木瓜蛋白酶的初始质量浓度为0.20 mg/mL时,使用3级逆流双水相萃取木瓜蛋白酶的酶活性回收率达到了96.75%,同时设计出多级逆流双水相萃取木瓜蛋白酶的优化方案。

Application of Enzymatic Promiscuity in Pharmaceutical Synthesis:Papain-catalyzed One-pot Synthesis of 1,4-Dihydropyridine Calcium Channel Antagonists and Derivatives

A new method for the synthesis of 1,4-dihydropyridine(1,4-DHP)calcium channel antagonists felodipme, nitrendipine and their derivatives via papain-catalyzed three-component reactions of aldehyde,methyl acetoacetate and ethyl 3-aminocrotonate was developed.Operational simplicity,mild reaction conditions and eco-friendliness are the key features of this protocol.