首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 31 毫秒
1.
A method was developed for production of sesame cystatin, a thermostable cysteine protease inhibitor. Sesame cystatin was first expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a short hydrophilic linker peptide. Stable artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble oleosin-cystatin fusion protein. After centrifugation, the oleosin-cystatin fusion protein was exclusively found in the artificial oil bodies. Proteolytic cleavage with papain, a cysteine protease effectively inhibited by cystatin, separated soluble cystatin from oleosin that was firmly embedded in the artificial oil bodies. After recentrifugation, papain that coexisted with cystatin in the collected supernatant was denatured by incubating at 55 degrees C for 30 min. The insoluble denatured papain was removed by one more centrifugation, and the expressed cystatin of high yield and purity was harvested simply by concentrating the ultimate supernatant. Comparable inhibitory activity toward papain was observed between the expressed cystatin and the native one purified from sesame seeds. This method is presumably applicable to production of other protease inhibitors whose target proteases are economically available.  相似文献   

2.
Cystatins are protein inhibitors of cysteine proteinases of the papain family, and those of animal origin have long been studied from medical and physiological aspects. In the meantime, oryzacystatin cloned from rice seeds in 1987 was recognized as the first well-defined cystatin of plant origin. Cloning studies followed to disclose various plant cytstatins including those of corn and soybean origin, their similarities to and differences from animal cystatins being analyzed in detail. Plant seed cystatins are now understood as factors controlling germination by inhibition of endogenous cysteine proteinases. They can also recognize insect midgut proteinases as exogenous target enzymes to control. This paper discusses chemical and phytophysiological relationships between cystatins and their targets.  相似文献   

3.
Vital gluten was used as an ideal substrate to investigate the role of some proteases in storage protein degradation. Aspartic proteinase and carboxypeptidase were identified as endogenous enzymes adsorbed on gluten and their optimum pH values determined. SDS-PAGE of soluble products released by gluten digestion revealed that the activity of these proteases plays a minor role in protein mobilization, whereas cysteine proteinase, purified from wheat seeds at the fourth day of germination, is extremely effective, producing a remarkable protein degradation in short times. Synergistic effects of aspartic and cysteine proteinase were not observed. Spin labeling of the sulfhydryl groups of gluten proteins enabled a comparative EPR investigation of the consequences of proteolytic degradation on gluten elasticity. It was found that storage protein mobilization brings a loss of elasticity to the polymeric network of gluten, which is particularly marked when the hydrolysis is performed by cysteine proteinase.  相似文献   

4.
The recombinant and glycosylation chicken cystatins were expressed and secreted in the broth of Pichia pastoris X-33 transformant with apparent molecular masses (M) of 14 and 55 kDa, respectively. The glycosylation cystatin (glycocystatin) contained a polysaccharide chain that was composed of 50 DP of mannose residues. Because of the polymannosyl chain, the inhibitory ability in glycocystatin was 90.8% of recombinant cystatin. In addition to freeze-thawing stability, the thermal and pH stabilities as well as the susceptibility of glycocystatin were also enhanced. Both cystatins could improve the mackerel surimi gel by inhibiting the gel softening, which was derived from the hydrolysis of catheptic cysteine proteinases. Despite the additional amount of glycocystatin (8 units), twice that of recombinant cystatin, the 40 and 15% increases in breaking force and deformation of gels were also observed. Accordingly, the surimi gel was further improved by enhancing the stability of chicken cystatin.  相似文献   

5.
Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.  相似文献   

6.
During oat seed germination, the insoluble storage proteins must be solubilized and transported to the embryo for use by the developing plantlet. We showed earlier that pH 6.2 active serine and metalloproteinases were the predominant gelatin‐hydrolyzing enzymes of oats, while the oat globulins were degraded by pH 3.8 active cysteine proteases. The pH of the endosperms of germinating oats is 6.2. We have continued our characterization of the germinated oat proteinases by determining which hydrolyze avenins, the oat storage prolamins. Avenins of resting seeds were purified and hydrolyzed with proteinases that were extracted from oat seeds that were germinated for various periods. The peptides released were analyzed using SDS‐PAGE. The α‐avenins were hydrolyzed at pH 3.8 by cysteine proteinases from four‐day germinated seeds and the β‐avenins were hydrolyzed by similar enzymes from eight‐day germinated seeds. At pH 6.2 or pH 5.0, the avenins were not degraded by any of the germinated oats endoproteinases. It is probable that some kind of pH compartmentalization occurs within germinating oat seed. After four days of germination, either new proteinases form or some preexisting proteinases are activated. The cysteine proteinases are apparently responsible for the majority of the storage protein hydrolysis that occurs during oat germination.  相似文献   

7.
A novel trypsin inhibitor (PFTI) was isolated from Plathymenia foliolosa (Benth.) seeds by gel filtration chromatography on a Sephadex G-100, DEAE-Sepharose, and trypsin-Sepharose columns. By SDSPAGE, PFTI yielded a single band with a M(r) of 19 kDa. PFTI inhibited bovine trypsin and bovine chymotrypsin with equilibrium dissociation constants (K(i)) of 4 x 10(-8) and 1.4 x 10(-6) M, respectively. PFTI retained more than 50% of activity at up to 50 degrees C for 30 min, but there were 80 and 100% losses of activity at 60 and 70 degrees C, respectively. DTT affected the activity or stability of PFTI. The N-terminal amino acid sequence of PFTI showed a high degree of homology with various members of the Kunitz family of inhibitors. Anagasta kuehniella is found worldwide; this insect attacks stored grains and products of rice, oat, rye, corn, and wheat. The velvet bean caterpillar (Anticarsia gemmatalis) is considered the main defoliator pest of soybean in Brazil. Diatraea saccharalis, the sugar cane borer, is the major pest of sugar cane crops, and its caterpillar-feeding behavior, inside the stems, hampers control. PFTI showed significant inhibitory activity against trypsin-like proteases present in the larval midguts on A. kuehniella and D. saccharalis and could suppress the growth of larvae.  相似文献   

8.
A recombinant Bacillus subtilis producing soy cystatin was developed by subcloning with a soy cystatin gene cloned in Escherichia coli. An active form of cystatin against the cysteine protease from Pacific whiting fillets contaminated with Myxosporidia parasite was constitutively expressed and secreted extracelluarly into the medium. Two gene fragments of signal peptides from kerA and sacB were introduced and compared for secretion efficiency of cystatin. The secretion level of active cystatin improved with the signal peptide of kerA when compared to that of sacB. Inhibitor activity was reduced rapidly after peak expression of the target protein at 36 h of fermentation. The addition of 1% glucose, a suppressor of protease, into the medium sustained the increase of the cystatin activity during fermentation. This study introduced a potential new method for fermentation production of cystatin.  相似文献   

9.
关于牧草辐射育种几个问题的探讨   总被引：25，自引：4，他引：21  
本文以牧草为材料 ,阐述了辐射敏感性的类型划分方法。将异花授粉牧草沙打旺辐照当代的种子直接播种在≥ 1 0℃ (年积温 2 3 0 8℃ )的低温下 ,提高了突变体的适应性 ,增加了选择的准确性 ,缩短了育种周期 ;M1代产生了显性早熟突变 ,M2 代有 2 7 2 %的植株保持早熟 ,比对照早开花 1 6～ 5 1d ,但仍有分离 ;5年育成彭阳早熟沙打旺 ,能在≥ 1 0℃ (年积温 1 847℃ )的地区开花结籽 ;播种当年始花序着生叶位与出苗至开花天数、株高和一级分枝呈极显著正相关。选育产草量高且早熟的沙打旺新品种 ,始花序叶位以在 9～ 1 0个为宜  相似文献   

10.
优化高压电场处理对谷子种子萌发期内源激素的影响   总被引：2，自引：2，他引：0  
为了探讨高压电场处理谷子种子内源激素含量动态变化对其萌发活力的调控影响,以农大八号谷子种子为试材,运用二次通用旋转组合设计与主成分分析相结合的方法,建模寻优。在优化电场处理条件下,研究种子萌发过程中内源激素含量动态变化。结果表明:高压电场处理谷子种子显著影响其萌发活力(P0.05);模型决定系数R2为0.979 2,表明模型拟合较好;模型耦合效应表明,高压电场处理谷子种子存在阈值效应,且电场强度影响效应大于处理时间;模型解析得到高压电场优化条件为电场强度340 k V/m,处理时间14 min。谷子种子萌发期内源激素含量动态变化结果表明,优化电场处理条件诱导吲哚乙酸(indole-3-acetic acid,IAA)、玉米素核苷(zeatin riboside,ZR)和赤霉素(gibberellin,GA)含量增加,抑制脱落酸(abscisic acid,ABA)含量增加;种子萌动前后,内源IAA、GA迅速上升、ABA快速下降,ZR在萌动时也达到极值,有利于胚根突破种皮,促进种子的萌动;GA/ABA、IAA/ABA、ZR/ABA比值处理均高于对照(未经电场处理),表明高压电场处理谷子种子后内源激素比值的变化是种子萌发活力提高的诱因,胚根达种子的2倍长后,GA/ABA、IAA/ABA比值都呈现增大趋势,表明幼苗生长与其调控密不可分。研究结果为高压电场技术处理种子在农业生产中的应用提供理论依据。  相似文献   

11.
An improved purification procedure leading to black sesame ( Sesamum Indicum L.) pigment was developed involving fat removal by treatment of ground black sesame seeds with dichloromethane followed by an optimized hydrolytic protocol with 6 M HCl, at 100 °C, overnight. The black pigment thus obtained displayed good antioxidant efficiency by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical assay (82% reduction at 0.5 mg/mL), good ferric ion-reducing capacity (61 μM Trolox equivalent concentration at 0.5 mg/mL), and potent antinitrosating properties (74% inhibition of 2,3-diaminonaphthalene (DAN) nitrosation at gastric pH at 2.5 mg/mL). A synthetic pigment obtained by oxidative polymerization of coniferyl alcohol (polyconiferyl alcohol, PCA), the putative biosynthetic precursor to the sesame pigment, was characterized as a reference standard. FT IR spectra of the purified sesame pigment and PCA supported the structural similarity. HPLC analysis of degradation products by alkaline hydrogen peroxide of purified black sesame pigment showed the formation of vanillic acid (VA) as the main isolable fragment. Similar yields of VA were obtained by degradation of PCA. A positive correlation between VA yields and DPPH activity was determined in samples of different purities. It is suggested that VA is a structural marker of black sesame pigment, confirming the biosynthetic origin from coniferyl alcohol and pointing to the o-methoxyphenol motif as the key factor accounting for the potent antioxidant properties of the pigment.  相似文献   

12.
Two assays were conducted to study the evolution of rye and barley phosphatases (phytase and acid phosphatase) and the degradation of its substrates (inositol phosphate esters) during seed germination. In this manner we could obtain a low-phytate, endogenous phosphatase rich ingredient to be used in animal nutrition. In the first assay, the seeds were soaked for 1 and 14 h and germinated for 3 and 5 days with and without the addition of gibberellic acid (GA3). In the second assay, the seeds were soaked for 1 h and germinated for 1, 3, and 5 days with GA3. Phytase (up to 5739 and 3151 U x kg(-1)) and acid phosphatase (up to 18288 and 3151 U x g(-1)) activities, and IP6 (6.09 and 6.01 mg x g(-1)), IP5 (0.48 and 0.48 mg x g(-1)), and IP4 (0.13 and 0.06 mg x g(-1)) were detected in ungerminated rye and barley, respectively. The germination process caused a significant increase of Phy and AcPh activities in rye (up to 112 and 213%) and barley (up to 212 and 634%) and a reduction in the phytate phosphorus content (up to 84 and 58%, respectively). Phytate phosphorus content was affected only by soaking time in the case of rye. Finally, during the course of germination, IP6 and IP5 were rapidly degraded in rye (88 and 79%) and barley (67 and 52%), and IP4 was only a short-living intermediate, which was increased during hydrolysis and degraded to IP3. In conclusion, a marked increase of Phy and AcPh activities in rye and barley with a concomitant decrease in phytate phosphorus content and an increase in the content of lower inositol phosphates were observed during the rye and barley germination.  相似文献   

13.
Sesame (Sesamum indicum L.) seed has been recognized as a nutritional protein source owing to its richness in methionine. Storage proteins have been implicated in allergenic responses to sesame consumption. Two abundant storage proteins, 11S globulin and 2S albumin, constitute 60-70 and 15-25% of total sesame proteins, respectively. Two gene families separately encoding four 11S globulin and three 2S albumin isoforms were identified in a database search of 3328 expressed sequence tag (EST) sequences from maturing sesame seeds. Full-length cDNA sequences derived from these two gene families were completed by PCR using a maturing sesame cDNA library as the template. The amino acid compositions of these deduced storage proteins revealed that the richness in methionine is attributed mainly to two 2S albumin isoforms and partly to one 11S globulin isoform. The presence of four 11S globulin and three 2S albumin isoforms resolved in SDS-PAGE was confirmed by MALDI-MS analyses. The abundance of these isoforms was in accord with the occurrence frequency of their EST sequences in the database. A comprehensive understanding of these storage proteins at the molecular level may also facilitate the identification of allergens in crude sesame products that have caused severe allergic reactions increasingly reported in the past decade.  相似文献   

14.
A trypsin inhibitor, PdKI, was purified from Pithecellobium dumosum seeds by TCA precipitation, trypsin-sepharose chromatography, and reversed-phase-HPLC. PdKI was purified 217.6-fold and recovered 4.7%. SDS-PAGE showed that PdKI is a single polypeptide chain of 18.9 kDa and 19.7 kDa by MALDI-TOF. The inhibition on trypsin was stable in the pH range 2-10 and at a temperature of 50 degrees C. The Ki values were 3.56 x 10(-8)and 7.61 x 10(-7) M with competitive and noncompetitive inhibition mechanisms for trypsin and papain, respectively. The N-terminal sequence identified with members of Kunitz-type inhibitors from the Mimosoideae and Caesalpinoideae subfamilies. PdKI was effective against digestive proteinase from Zabrotes subfasciatus, Ceratitis capitata, Plodia interpunctella, Alabama argillaceae, and Callosobruchus maculatus, with 69, 66, 44, 38, and 29% inhibition, respectively. Results support that PdKI is a member of the Kunitz inhibitor family and its insecticidal properties indicate a potent insect antifeedant.  相似文献   

15.
The cDNAs encoding chicken cystatin and its N-glycosylation-modified mutant (Asn(106)-Ile(108)-->Asn(106)-Thr(108)) were cloned into the pGAPZ alpha C expression vector, using the GAP as promoter and Zeocin as resistant agent, and transformed into Pichia pastoris X-33 expression host. The effect of N-glycosylation on the stability of recombinant chicken cystatin was investigated. A large quantity of recombinant chicken cystatin and the Asn(106)-glycosylated cystatins were expressed and secreted into broth using alpha-factor preprosequence. The K(i) of the recombinant chicken cystatin (0.08 nM) was similar to that of wild-type chicken cystatin (0.05 nM). They acted as a competitive inhibition reaction against papain. According to the K(i), the inhibition ability of Asn(106)-glycosylated mutant cystatin (K(i) = 9.5 nM) was weaker than that of the wild-type one. However, N-glycosylation at Asn(106) substantially enhanced the freezing stability of recombinant chicken cystatin overexpressed in P. pastoris.  相似文献   

16.
Abstract

Salinity and moisture stress are main limiting factors of agricultural development on arid and semi‐arid lands. The objective of this study was to evaluate the tolerance of sesame (Sesamun indicum L.) genotypes to salinity. Salinity treatments in rooting media were 0.5, 2.0, 3.5, 5.0, and 7.0 dS m?1 using calcium chloride (CaCl2). Germination of 50 genotypes was evaluated. Twenty seeds of each cultivar were set in paper towels and moistened with the saline solutions for 15 days. Five germination‐selected genotypes were seeded in 10‐kg pots, and soil salinity was adjusted to the same treatments. Highly significant effects were found for dry matter at 45 (DM 45) days after planting (DAP), at 90 DAP (DM 90), and leaf area (LA). Growth differences among genotypes were only observed for DM 90. These data suggest that sesame tolerance to CaCl2 salinity improved through the growing season and may be genetically controlled.  相似文献   

17.
本研究以新疆特有濒危保护植物矮沙冬青（Ammmopiptanthus nanus（M．Pop．）Chengf）的种子为材料,用不同渗透势浓度的聚乙二醇（PEG6000）模拟干旱胁迫,探讨干旱胁迫对矮沙冬青种子发芽率、平均发芽速度、胚轴和胚根长度及发芽指数、活力指数的影响。结果表明,不同浓度PEG胁迫处理均降低了种子的发芽率,延缓了矮沙冬青种了萌发进程;种子的发芽率、发芽指数和活力指数均随胁迫强度的增加呈明显下降趋势。当-1．20MPa的PEG胁迫处理的种子在试验结束时仍未能萌发,表明-1．20MPa是矮沙冬青种子萌发的临界水势。PEG模拟十旱胁迫中,当PEG处理为-0．2MPa时,虽然最终发芽率与对照一样,但其胚根、胚轴的长度都比对照短,说明矮沙冬青胚根、胚轴的生长比发芽率对干旱胁迫更敏感。干旱胁迫可能是导致矮沙冬青种群天然更新能力弱的原因之一。本研究将为矮沙冬青种质资源的保护和种群的恢复提供科学依据。  相似文献   

18.
The present study investigated the possible mediator role of salicylic acid (SA) in alleviating cadmium (Cd) toxicity during the germination stage of barley. The exposure of barley seedling to increasing Cd concentrations (25, 50 and 100 µM) during early stages of their establishment, caused a gradual decrease in vigour index, root length, α-amylase, acid phosphatase, alkaline phosphatase activities in endosperms and mitotic index of the root tip. Seed pretreatment with 600 µM SA partially alleviated the negative effects of Cd on germination parameters and increased the hydrolytic enzyme activities and mitotic index. Cadmium treatment increased Cd accumulation in roots. Furthermore, results showed that compared to the cadmium treatment applied alone, SA pretreatment of the seeds did not influenced the Cd concentration in the roots. These results suggest that SA plays a positive role in barley-seed germination and early seedling growth by protecting it against Cd toxicity by moderating its toxic effect on the mobilization of organic reserves.  相似文献   

19.
Six genes involved in anthocyanin biosynthesis in tartary buckwheat have been cloned, namely, FtC4H, Ft4CL, FtCHI, FtF3H, FtF3'H, and FtANS, which encode cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), chalcone isomerase (CHI), flavones 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), and anthocyanidin synthase (ANS), respectively. Then, these cDNAs were used, along with previously isolated clones for phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), to compare gene expression in different organs, flowering stages, and maturing seeds of tartary buckwheat cultivars 'Hokkai T8' and 'Hokkai T10'. Quantitative real-time polymerase chain reaction analysis showed that these anthocyanin biosynthetic genes were most highly expressed in the stems and roots of Hokkai T10. The FtANS gene was more highly expressed than other genes during flowering and maturing seeds. In addition, the anthocyanin concentration was higher in 'Hokkai T10' than in 'Hokkai T8'; however, naringenin chalcone, a flavonoid, was absent from 'Hokkai T10' seedlings based on fluorescence microscopy.  相似文献   

20.
A DNA encoding thioredoxin-mature carp ovarian cystatin (trx-cystatin) fusion protein was ligated into a pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3) expression host. After induction by isopropyl beta-D-thiogalactopyranoside, a high level of the soluble form of recombinant trx-cystatin was expressed in the cytoplasm of E. coli. The recombinant trx-cystatin could be purified by Ni(2+)-NTA agarose affinity chromatography. The molecular mass (M) of the recombinant trx-cystatin was approximately 28 kDa composed of recombinant thioredoxin (16 kDa) and recombinant mature carp ovarian cystatin (12 kDa). Both recombinant trx-fused and mature carp ovarian cystatins were stable at pH 6-11. No obvious decrease in activity was observed even after 5 min of incubation at 60 degrees C. They exhibited papain-like protease inhibition activity comparable to that of the mature carp ovarian cystatin, which could inhibit papain and mackerel cathepsins L and L-like, but not cathepsin B.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号