碱性蛋白酶提取变性脱脂豆粕中蛋白质

以高温变性脱脂大豆粕为原料,用正交实验法对变性豆粕在蛋白酶作用下的水解特性进行了深入研究。选用国产胰蛋白酶为水解酶对变性豆粕进行水解,研究了变性豆粕中蛋白质溶出率随温度,pH值,时间,低物浓度及用酶量的变化规律,找到了水解变性豆粕的最佳实验条件,结果表明,胰蛋白酶水解高温变性豆粕的最佳条件为,温度50℃,时间60h,底物浓度11%,用酶量10000u/g,pH值8.0,在此条件下,变性豆粕中蛋白质可有69.34%水解溶出。     

半胱氨酸组织蛋白酶与免疫调节

半胱氨酸组织蛋白酶(CC)属于蛋白酶clanCA中的木瓜蛋白酶家族(C1),由组织蛋白酶B、L、H、S、K、F、V、X、W、O和C等组成。这些蛋白酶参与了各种生理和病理过程。     

毕赤酵母水解工艺的研究

在发酵过程中产生的总体积为30%～40%的毕赤酵母菌体，大部分在发酵后，会直接把酵母菌体排入环境中，不仅浪费资源，还污染环境，因此，实现资源再利用是文章的研究目的。最终结果表明，对毕赤酵母自溶破壁的最佳自溶水解条件为50℃、pH 6.0，作用时间为30 h、4%Na Cl，酵母悬浮液终体积分数为10%。此外，通过对木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和酸性蛋白酶进行研究发现，添加木瓜蛋白酶效果最佳，添加后的酵母水解物氨基氮得率为4.5%，固形物得率为59%，粗蛋白含量为45.38%。     

组织蛋白酶B的新型天然抑制剂

组织蛋白酶B是木瓜蛋白酶类半胱氨酸蛋白酶家族的重要成员,它与人类多种疾病相关,尤其是在恶性肿瘤的侵袭转移过程中扮演了重要角色.通过随机筛选,发现了五个对组织蛋白酶B具有较好抑制活性的天然化合物prodelphinidin B-23'-O-gallate(1),prodelphinidin B-2(2),ImJcyarddin B-2(3),puexin A(4)和(-)epigallocatechin-3-O-gallate(5),其IC50值分别为0.58,0.44,0.76,2.07和0.96umol/L.这五个抑制剂为黄烷醇类化合物,均为组织蛋白酶B的新型天然抑制剂.     

双酶法制备螺旋藻多肽的工艺研究

选用碱性蛋白酶和木瓜蛋白酶结合的双酶法对螺旋藻蛋白进行水解。其中,对木瓜蛋白酶水解螺旋藻蛋白的工艺进行优化。以水解度为指标,研究了酶解时间、酶与底物比、pH和酶解温度4种因素对酶解反应的影响。在此基础上设计了3因素(加酶量、酶解温度和pH)3水平的响应面试验。结果表明碱性蛋白酶水解螺旋藻蛋白的最佳酶解条件为:加酶量4300 U/g,pH 7.0,酶解温度55℃,酶解时间160 min;木瓜蛋白酶的最佳酶解条件为:酶底比为4.5%,酶解温度60℃,pH 6.5,酶解时间210 min。利用碱性蛋白酶和木瓜蛋白酶结合的双酶法制得的多肽水解度可达32.90%,与单酶法相比,水解度明显提高。     

复合酶法水解香菇工艺的研究

【目的】高效提取香菇中的功能性成分,进一步开发利用香菇的食用价值。【方法】以香菇为原料,在单因素试验和正交试验的基础上,研究了复合酶法(纤维素酶、木瓜蛋白酶和5-磷酸二酯酶)水解香菇的工艺条件。【结果】第一步为纤维素酶和木瓜蛋白酶共同水解,加酶量分别为0.2%和0.4%,水解温度55°C,水解时间3 h,初始pH 5.5;第二步为5-磷酸二酯酶水解,加酶量为0.2%,水解温度70°C,水解时间2 h,初始pH 7.0。用此法得到的香菇水解液水解度为39.48%,游离氨基酸得率10.25%,5-核苷酸得率0.768%,多糖得率8.67%。【结论】此香菇水解液富含呈鲜味物质和其它营养物质,可进一步深加工为香菇调味品。     

菓菠萝蛋白酶的分子构象与活力变化的研究

发现CBZ-Lys·pNP能有效地被菓菠萝蛋白酶(Fruit Bromelain E.C.3.4.22.5)作用,测得Km为4.167×10~(-4)mol/L,k_(cat)为742min~(-1)。以荧光和紫外差示光谱为监测手段,对酶分子构象变化进行研究。酶的荧光强度随胍浓度增大而逐渐下降,4mol/L胍变性时,发射峰自332nm红移到353nm,并在310nm处出现新的发射峰。酶的荧光强度都因SDS存在而下降,SDS浓度大于3.47mmol/L有所回升,并出现红移,同时在315nm处出现新的发射肩;紫外差示光谱显示在236nm有一个较显著的员峰,此峰与β-螺旋结构变化有关,278、286和295nm出现三个负峰,260nm有较小正峰,说明酶分子中Tyr、Trp和Phe的微环境发生了明显的变化。测定酶在不同浓度胍和SDS中的变性和失活速度常数,对酶构象变化及催化活力的关系作了比较研究,酶的失活速度均大于变性速度。     

水稻巯基蛋白酶抑制剂的纯化及其性质研究

水稻的糠皮和胚经生理盐水浸取、离心后的上清液加热至８０℃处理１０ｍｉｎ,离心获得的上清液调ｐＨ至８．０,得到沉淀。沉淀溶解于０．０１ｍｏｌ／ＬＨＣｌ,经透析冷冻干燥得水稻巯基蛋白酶抑制剂（ＣＰＩ）粗品;粗品再经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱线性离子梯度洗脱和ＳｅｐｈａｄｅｘＧ－１００柱分子筛层析,即可获得在ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和ＨＰＬＣ上均为单一蛋白带的ＣＰＩ样品。经上述步骤,ＣＰＩ可被纯化５８倍。经ＳｅｐｈａｄｅｘＧ－１００和ＳＤＳ－ＰＡＧＥ测定其分子量均为１２０００,Ｎ末端氨基酸为Ｐｒｏ,等电点５．６．水稻ＣＰＩ经１００℃处理１０ｍｉｎ后,其抑制活性无任何变化,在ｐＨ２．０～９．０之间,活性也不发生改变,但ｐＨ在９．０以上,其活性逐渐下降,水稻ＣＰＩ对木瓜蛋白酶是一种高亲和性的抑制剂,它对木瓜蛋白酶和无花果蛋白酶有强抑制作用,对菠萝蛋白酶仅有弱抑制作用,但对胰蛋白酶则全无抑制作用;其抑制类型属竞争性抑制剂类型,Ｋ＿ｉ值约３．５×１０￣（－８）ｍｏｌ／Ｌ对木瓜蛋白酶的抑制摩尔比约为１：１。     

百合巯基蛋白酶抑制剂的纯化及部分性质研究

百合的鳞茎中含有一种对木瓜蛋白酶有强抑制作用的巯基蛋白酶抑制剂．百合的鳞茎经浸取加热处理,木瓜蛋白酶偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析和ＳｅｐｈａｄｅｘＧ－１００分子筛层析,可获得在ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ均为单一蛋白带的百合巯基蛋白酶抑制剂（ＣＰＩ）．此ＣＰＩ为单链蛋白,含有０．３０７％的中性糖;Ｎ端氨基酸为Ｉｌｅ;ＳＤＳ－ＰＡＧＥ测得亚基分子量为１２０００;ＳｅｐｈａｄｅｘＧ－１００测得分子量为１２５００．百合ＣＰＩ在１００℃内和ｐＨ２～１２范围内非常稳定;对木瓜蛋白酶的抑制属竞争性抑制类型,其Ｋｉ值为１．１５×１０~（－９）ｍｏｌ／Ｌ,对木瓜蛋白酶的抑制摩尔比为８．５：１．     

半胱氨酸蛋白酶拟肽抑制剂设计新进展

半胱氨酸蛋白酶包括多种酶,这些酶在广泛的生命过程中发挥作用。人类正常的半胱氨酸蛋白酶表达失调,寄生虫、病毒的半胱氨酸蛋白酶表达与多种病理情况相关。对于这类疾病,抑制半胱氨酸蛋白酶是一个可行的药物治疗策略。当前这类药物设计的目标是3种结构不同的半胱氨酸蛋白酶,即木瓜蛋白酶家族、半胱氨酸-天冬氨基特异性蛋白酶家族(caspases)和小核糖核酸病毒科半胱氨酸蛋白酶抑制剂家族。本文综述了近年来有关半胱氨酸蛋白酶抑制剂的设计思路。     

Monoclonal antibodies to the two most basic papaya proteinases

The proteinases fromCarica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A.     

Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains.

The protein crystals found in potato (Solanum tuberosum L.) tuber cells consist of a single 85-kD polypeptide. This polypeptide is an inhibitor of papain and other cysteine proteinases and is capable of binding several proteinase molecules simultaneously (P. Rodis, J.E. Hoff [1984] Plant Physiol 74: 907-911). We have characterized this unusual inhibitor in more detail. Titrations of papain activity with the potato papain inhibitor showed that there are eight papain binding sites per inhibitor molecule. The inhibition constant (Ki) value for papain inhibition was 0.1 nM. Treatment of the inhibitor with trypsin resulted in fragmentation of the 85-kD polypeptide into a 32-kD polypeptide and five 10-kD polypeptides. The 32-kD and 10-kD fragments all retained the ability to potently inhibit papain (Ki values against papain were 0.5 and 0.7 nM, respectively) and the molar stoichiometries of papain binding were 2 to 3:1 and 1:1, respectively. Other nonspecific proteinases such as chymotrypsin, subtilisin Carlsberg, thermolysin, and proteinase K also cleaved the 85-kD inhibitor polypeptide into functional 22-kD and several 10-kD fragments. The fragments obtained by digestion of the potato papain inhibitor with trypsin were purified by reverse-phase high-performance liquid chromatography, and the N-terminal amino acid sequence was obtained for each fragment. Comparison of these sequences showed that the fragments shared a high degree of homology but were not identical. The sequences were homologous to the N termini of members of the cystatin superfamily of cysteine proteinase inhibitors. Therefore, the inhibitor appears to comprise eight tandem cystatin domains linked by preteolytically sensitive junctions. We have called the inhibitor potato multicystatin (PMC). By immunoblot analysis and measurement of papain inhibitory activity, PMC was found at high levels in potato leaves (up to 0.6 microgram/g fresh weight tissue), where it accumulated under conditions that induce the accumulation of other proteinase inhibitors linked to plant defense. PMC may have a similar defensive role, for example in protecting the plant from phytophagous insects that utilize cysteine proteinases for dietary protein digestion.     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.     

Proteinase binding and inhibition by the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3

The inhibitory capacity of the alpha-macroglobulins resides in their ability to entrap proteinase molecules and thereby hinder the access of high molecular weight substrates to the proteinase active site. This ability is thought to require at least two alpha-macroglobulin subunits, yet the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3 (alpha 1I3) also inhibits proteinases. We have compared the inhibitory activity of alpha 1I3 with the tetrameric human homolog alpha 2-macroglobulin (alpha 2M), the best known alpha-macroglobulin, in order to determine whether these inhibitors share a common mechanism. alpha 1I3, like human alpha 2M, prevented a wide variety of proteinases from hydrolyzing a high molecular weight substrate but allowed hydrolysis of small substrates. In contrast to human alpha 2M, however, the binding and inhibition of proteinases was dependent on the ability of alpha 1I3 to form covalent cross-links to proteinase lysine residues. Low concentrations of proteinase caused a small amount of dimerization of alpha 1I3, but no difference in inhibition or receptor binding was detected between purified dimers or monomers. Kininogen domains of 22 and 64 kDa were allowed to react with alpha 1I3- or alpha 2M-bound papain to probe the accessibility of the active site of this proteinase. alpha 2M-bound papain was completely protected from reaction with these domains, whereas alpha 1I3-bound papain reacted with them but with affinities several times weaker than uncomplexed papain. Cathepsin G and papain antisera reacted very poorly with the enzymes when they were bound by alpha 1I3, but the protection provided by human alpha 2M was slightly better than the protection offered by the monomeric rat alpha 1I3. Our data indicate that the inhibitory unit of alpha 1I3 is a monomer and that this protein, like the multimeric alpha-macroglobulins, inhibits proteinases by steric hindrance. However, binding of proteinases by alpha 1I3 is dependent on covalent crosslinks, and bound proteinases are more accessible, and therefore less well inhibited, than when bound by the tetrameric homolog alpha 2M. Oligomerization of alpha-macroglobulin subunits during the evolution of this protein family has seemingly resulted in a more efficient inhibitor, and we speculate that alpha 1I3 is analogous to an evolutionary precursor of the tetrameric members of the family exemplified by human alpha 2M.     

Proteolytic enzymes; further studies on protein,polypeptide, and other inhibitors of serum proteinase,leucoproteinase, trypsin,and papain

1. Serum proteinase precursor was found in plasma protein fractions I and III of Cohn. Inhibitors of serum proteinase, leucoproteinase, trypsin, and papain were found in fractions IV-1 and IV-4, and to a lesser extent in fractions V and I. 2. Pancreatic, soy bean, lima bean, and egg white inhibitors inhibited trypsin stoichiometrically. Pancreatic inhibitor had comparable inhibitory activity against serum proteinase; soy bean inhibitor had somewhat less, lima bean inhibitor even less, and egg white inhibitor very little. None of these inhibitors appreciably inhibited leucoproteinase or papain. 3. Serum and fractions IV - 1 and IV - 4 had marked inhibitory activity against trypsin and leucoproteinase, and somewhat less against serum proteinase and papain. The inhibitory activity of the plasma proteins against trypsin and leucoproteinase was due almost entirely to fractions IV - 1 and IV - 4; against serum proteinase and papain fraction V was slightly more important. The "reconstituted plasma proteins" accounted for 8 to 25 per cent of the proteinase-inhibitory activity of whole serum or plasma. 4. The proteinase-inhibitory activity of serum, plasma protein fractions, and soy bean inhibitor was heat labile, while that of pancreatic, lima bean, and egg white inhibitors was relatively heat stable. 5. Reducing and oxidizing agents, in very high concentration, inhibited serum proteinase, as well as trypsin and leucoproteinase. These proteinases were not influenced by mercurial sulfhydryl inhibitors, indicating that free sulfhydryl groups do not play an important part in their activity.     

Circular dichroism of cysteine proteinases from papaya latex. Evidence of differences in the folding of their polypeptide chains.

Two forms of proteinase omega were isolated from a commercial preparation of chymopapain (EC 3.4.22.6) by means of cation-exchange liquid chromatography. Their circular dichroism (CD) spectra in the 182-320 nm region indicated that the two forms possess closely related structures. For comparison, we also recorded the CD spectra of chromatographically purified samples of papain (EC 3.422.2) and the most abundant form of chymopapain. According to the qualitative criteria proposed by Manavalan and Johnson (1983) Nature 305, 831-832), the spectral characteristics of papain correctly indicate that this protein belongs to the alpha + beta class. Proteinase omega is also placed in the alpha + beta category, while chymopapain seems to be an alpha/beta protein. Quantitative estimation of secondary structures yielded contents of helices and parallel beta-sheet that were higher in the case of chymopapain. Thus, the results of this work suggest that there are some differences in the folding pattern of chymopapain with respect to the other two proteinases. This proposal seems unexpected when the high amino acid sequence identity among these enzymes is considered.     

Inhibitory mechanism of a cross-class serpin,the squamous cell carcinoma antigen 1

The squamous cell carcinoma antigen (SCCA) 1 and its homologous molecule, SCCA2, belong to the ovalbumin-serpin family. Although SCCA2 inhibits serine proteinases such as cathepsin G and mast cell chymase, SCCA1 targets cysteine proteinases such as cathepsin S, K, L, and papain. SCCA1 is therefore called a cross-class serpin. The inhibitory mechanism of the standard serpins is well characterized; those use a suicide substrate-like inhibitory mechanism during which an acyl-enzyme intermediate by a covalent bond is formed, and this complex is stable against hydrolysis. However, the inhibitory mechanism of cross-class serpins remains unresolved. In this article, we analyzed the inhibitory mechanism of SCCA1 on a cysteine proteinase, papain. SCCA1 interacted with papain at its reactive site loop, which was then cleaved, as the standard serpins. However, gel-filtration analyses showed that SCCA1 did not form a covalent complex with papain, in contrast to other serpins. Interaction with SCCA1 severely impaired the proteinase activity of papain, probably by inducing conformational change. The decreased, but still existing, proteinase activity of papain was completely inhibited by SCCA1 according to the suicide substrate-like inhibitory mechanism; however, papain recovered its proteinase activity with the compromised level, when all of intact SCCA1 was cleaved. These results suggest that the inhibitory mechanism of SCCA1 is unique among the serpin superfamily in that SCCA1 performs its inhibitory activity in two ways, contributing the suicide substrate-like mechanism without formation of a covalent complex and causing irreversible impairment of the catalytic activity of a proteinase.     

Induction of cystatin S in rat submandibular glands by papain.

1. Papain (a cysteine proteinase) were administered into the oral cavity of rats twice daily for 5 days. This treatment caused a dramatic increase in the level of cystatin S (a cysteine proteinase inhibitor belonging to family 2 of cystatin superfamily) in enlarged submandibular glands. 2. Immunochemical analysis using antibody against rat cystatin S and electrophoretic analysis confirmed that the protein induced by papain was identical to that induced by isoproterenol. 3. Induction of the cystatin S in the submandibular glands by oral administration of papain suggested a biological response which plays a role in preventing injury exogenous proteinase.     

Purification and characterisation of a novel papain inhibitor from common bean (Phaseolus vulgaris) seed

A proteinaceous inhibitor of papain was purified to apparent homogeneity from mature seeds of common bean ( Phaseolus vulgaris L.). After four chromatographic steps, the papain inhibitor was purified 219‐fold with 12% recovery. On the basis of papain inhibitory activity, cystatins have been estimated to account for about 0.1% of the total protein content of mature common bean seeds. The purified protein, as other plant cystatins, is an acidic protein, heat stable and insensitive to reducing agents. Its molecular mass is about 37 kDa as judged by size exclusion chromatography and SDS‐PAGE. Moreover it is immunologically related to oryzacystatins, since it is recognised by a specific oryzacystatin I antiserum. Based on its biochemical properties the papain inhibitor described here belongs to the phytocystatin family. Papain inhibitory assays carried out during seed development showed that bean cystatin is active since early maturation stages. Our results suggest that, in common bean seed, cysteine proteinase inhibitors are important during seed development with a putative role in the control and regulation of endogenous thiol protease activity.     

Papaya proteinase IV amino acid sequence

The amino acid sequence of papaya proteinase IV (PPIV), a major proteinase from the latex of Carica papaya [(1989) Biochem. J. 261, 469-476] is described. The enzyme has a high degree of sequence identity with papaya proteinase III, chymopapain and papain (81, 70 and 67%, respectively), and is clearly a member of the papain superfamily of cysteine proteinases. Nevertheless, the sequence shows substitution of certain residues conserved in all other known members of the superfamily. It is suggested that some of these substitutions may account for the unusual specificity of PPIV.