奇异果甜蛋白及其基因工程

奇异果甜蛋白(thaumatin)是迄今为止最甜的物质之一,对其研究具有很重要的意义。奇异果甜蛋白的生化性质基本清楚,基因序列和氨基酸序列都已测定。它的甜味可能是由奇异果甜蛋白上特定基团和受体结合引起的。对奇异果甜蛋白的生理功能知之甚少。近二十年来,在奇异果甜蛋白的基因工程上取得了一定进展,但仍然存在许多困难。 Abstract:Thaumatin is one of the sweetest substances known to date,it is important to study the thaumatin.The biochemical properties of thaumatin have been clarified clearly.Thaumatin had been isolated and sequenced.The mechanism of the sweetness of thaumatin may be due to the combination of some special groups and the receptors.The exact function of thaumatin is still not clear.Although gene engineering of thaumatin has been carried out for 20 years,there are still some difficulties to be solved for using in the market.     

甜味蛋白及其基因工程

甜味蛋白（ｔｈａｕｍａｔｉｎ）是从一种热带植物ＴｈａｕｍａｔｏｃｏｃｕｓｄｅｎｉｅｌｉＢｅｎｔｈ的胶质假种皮中分离出来的,该植物为竹芋科（Ｍａｒａｂｔａｃｅａｅ）的一种多年生灌木,广泛生长在西非的雨林中。甜味蛋白被认为是世界上已知的最甜的物质,大约２...     

植物甜蛋白Thaumatin研究进展

甜蛋白自 2 0世纪 70年代发现以来 ,一直倍受人们关注 ,而源于自然的Thaumatin是植物甜蛋白中的一种 ,它具有低热量、高甜度、安全无毒 ,并可降解为人体所需的氨基酸等多种优点 ,是一种新型甜味剂。在物质文化生活日益丰富的今天 ,人们越来越重视饮食的科学性 ,吃饱的同时更加关注所摄入食品的品质 ,无疑具多功能的非糖类物质 Thaumatin就是人们所需求的理想食品。因此 ,Thaumatin成为热门研究领域之一也就不足为怪了。1 　植物甜蛋白研究概况迄今为止 ,人们从多种植物中发现并分离出 7种甜味蛋白 [1 ]。更确切地说 ,其中 5种( Thaumatin,…     

植物甜蛋白的研究进展

本文简要介绍了近年来在植物中发现的几种甜味蛋白质的分子结构及其化学性质。讨论了它们在结构上的相关性及可能的甜味机制,并对甜蛋白在食品工业及植物改良方面的应用进行了展望。     

超甜蛋白的基因工程及开发研究进展

甜蛋白是一类高甜度、低热量、多功能的新型超级甜味剂,应用前景广阔。由于它们存在于几种稀有的植物中,产地偏僻,产量低,提取困难,因而不能发挥它们的使用价值。运用基因工程等高新生物技术,将甜蛋白基因克到微生物细胞中,构建产生甜蛋白基因工程菌株,将为工厂化发酵生产甜蛋白开辟一条快速有效的新途径。     

甜味蛋白研究进展

甜味蛋白是一类具有高甜度、低热卡、多功能的天然甜味剂。但要从几种古老的植物中提取甜味蛋白较为困难 ,且难以开发和利用。随着生物技术的发展 ,尤其是利用基因工程技术将甜味蛋白基因克隆到微生物细胞中 ,构建产甜味蛋白的基因工程菌 ,为商品化生产甜味蛋白开辟了一条快速而有效的新途径。     

甜味蛋白研究进展

甜味蛋白是一类具有高甜度,低热卡,多功能的天然甜味剂,但要从几种古老的植物中提取甜味蛋白较为困难,且难以开发利用,随着生物技术的发展,尤其是利用基因工程技术将甜味蛋白基因克隆到微生物细胞中,构建产甜味蛋白的基因工程菌,为商品化生长甜味蛋白开辟了一条快速而有效的新途径。     

马槟榔甜味蛋白的研究Ⅱ.甜蛋白Ⅱ和甜蛋白Ⅰ的比较

从马槟榔(Capparis masaikai Levl.)种子分离鉴定了二种甜蛋白:甜蛋白Ⅰ(mabinlinⅠ或MaⅠ)分子量MW11600,等电点pI 11.8;甜蛋白Ⅱ(mabinlinⅡ或MaⅡ)MW10400,pI 11.3。另一组分mabinlinⅢ MW10200,pI 11.8,可能是提取过程产物。MaⅡ有84个氨基酸残基,比MaⅠ少15个。它们有80个氨基酸残基是共同的,都是缺丝氨酸和蛋氨酸的单一多肽链。MaⅠ还缺赖氨酸和酪氨酸。测不出有自由SH基。在8M尿素中用巯基乙醇还原处理,两种甜蛋白分子都可转变为二聚体而失去甜味。 自去壳脱脂的种子干粉中提取MaⅠ和MaⅡ的得率依所用溶剂和提取条件而异。用50％丙酮水溶液提取,继用CM-纤维素分离,甜蛋白的得率达13％。用水或0.1N pH6.2磷酸缓冲液提取,得率低于1％。     

马槟榔甜味蛋白的研究Ⅱ．甜蛋白Ⅱ和甜蛋白Ⅰ的比较

从马槟榔（Capparis masaikai Levl．）种子分离鉴定了二种甜蛋白：甜蛋白I（mabinlinⅠ或MaⅠ）分子量MWll600,等电点PⅠll.8;甜蛋白Ⅱ（mabinlinⅡ或MaⅡ）MW 10400,PⅠll.3。另一组分mabinlinⅡ MW10200,pl 11.8,可能是提取过程产物。MaⅡ有84个氨基酸残基,比MaⅠ少15个。它们有80个氨基酸残基是共同的,都是缺丝氨酸和蛋氨酸的单一多肽链。MaⅠ还缺赖氨酸和酪氨酸。测不出有自由SH基。在8 M尿素中用巯基乙醇还原处理,两种甜蛋白分子都可转变为二聚体而失去甜味。     

甜蛋与抗虫蛋白

在植物中发现有极甜的蛋白质。植物中也有抗虫的蛋白质。其中一类抗虫蛋白与甜蛋白在结构上极为相似。介绍了它们的研究现状和广泛的应用前景。     

Possible Active Site of the Sweet-tasting Protein Thaumatin

Epitopes on thaumatin and monellin were studied using the PEPSCAN-technology.The antibodies used were raised against thaumatin. Only antibodiesthat, in an ELISA, both recognized thaumatin and monellin wereused in the PEPSCAN-analyses. On thaumatin two major overlappingepitopes were identified. On monellin no epitopes could be identified.The identified epitope region on thaumatin shares structuralfeatures with various peptide and protein sweeteners. It containsan aspartame-like site which is formed by Asp21 and Phe80, tipsof the two extruding loops KGDAALDAGGR_19–29 and CKRFGRPP_77–84,which are spatially positioned next to each other. Furthermore,sub-sequences of the KGDAALDAGGR^19–29 loop are similarto peptide-sweeteners such as L-Asp-D-Ala- L-Ala-methyl esterand L-Asp-D-Ala-Gly-methyl ester. Since the aspartame-like Asp21-Phe80site and the peptide-sweetener-like sequences are also not presentin non-sweet thaumatin-like proteins it is postulated that theKGDAALDAGGR_19–29 and CKRFGRPP_77–84 loop containimportant sweet-taste determinants. This region has previouslynot been implicated as a sweet-taste determinant of thaumatin.Chem. Senses 20: 535–543, 1995.     

The Taste-active Regions of Monellin, a Potently Sweet Protein

Monellin, a protein found in the berries of the West Africanplant Dioscoreophyllum cumminsii, is one of the most potentlysweet compounds known. The native three-dimensional structureof monellin is required for sweetness, and this protein hasbeen the subject of intense research in an attempt at understandingthe structural basis for its taste activity. We have used structure-basedsite-directed mutagenesis to delineate the taste-active site(s)of monellin, and we present these results, along with similarwork from M. Kohmura, Y. Ariyoshi and coworkers, in the lightof the three-dimensional structure of this protein. The mutagenesis work suggests that at least four residues, locatedN-terminal to the     

Complete Amino Acid Sequence of the Sweet Protein Monellin

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of these chains. The complete amino acid sequence of monellin was determined by a combination of FAB- and ESI-mass spectrometry, and by automatic Edman degradation.     

Highly Probable Active Site of the Sweet Protein Monellin

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Synthetic monellin is 4000 times as sweet as sucrose on a weight basis, and the native conformation is essential for the sweet taste. Knowledge of the active site of monellin will provide important information on the mode of interaction between sweeteners and their receptors. If the replacement of a certain amino acid residue in monellin removes the sweet taste, while the native conformation is retained, it may be concluded that the position replaced is the active site. Our previous replacement studies on Asp residues in the A chain did not remove the sweet taste. The B chain contains two Asp residues at positions 7 and 21, which were replaced by Asn. [Asn^B21] Monellin and [Asn^B7]monellin were 7000 and 20 times sweeter than sucrose, respectively. The low potency of the [Asn^B7]monellin indicates that ASp^B7 participates in binding with the receptor. ASp^B7 was then replaced by Abu. [Abu^B7]Monellin was devoid of sweetness, and retained the native conformation. ASp^B7 is located at the surface of the molecule (Ogata et al.). These results suggest that Asp⁷ in the B chain is the highly probable active site of monellin.     

甜蛋白Monellin基因在大肠杆菌中的高效表达

据已报道的单链monellin甜蛋白的氨基酸序列,采用细菌偏爱密码子,人工合成了全长 294bp的 monellin基因。插入到大肠杆菌表达载体Pet_22b中,构建重组分泌型表达载体Petmo。经IPTG诱导Petmo所含有的甜蛋白基因可在大肠杆菌BL21（DE3）中高效表达,表达量占菌体可溶性蛋白的44.8％。且经纯化后测定其甜度是蔗糖的3000倍。得到的甜蛋白热稳性及耐酸性均比天然产物有所提高。     

Bt毒蛋白基因与植物抗虫基因工程

Ｂｔ毒蛋白基因与植物抗虫基因工程林良斌官春云（作物基因工程湖南省重点实验室,湖南农业大学长沙４１０１２８）在世界范围内,害虫造成的损失约占农作物总收获量的１３％,每年大约损失数千亿美元。目前,防治害虫仍然是主要地使用化学农药,但随着时间推移,由此而引起的一些问题日益突出,越来越引起人们的关注。例如,化学农药对人、畜有严重危害,其在自然界中的残留及在食物链中的富积已造成了严重的环境污染;化学农...     

单链莫内甜蛋白基因的合成及其植物表达载体的构建

目的:合成单链莫内甜蛋白基因,构建其植物表达载体。方法与结果:根据已报道的单链莫内甜蛋白的氨基酸序列及甜味机理,重新设计合成了全长294bp的莫内甜蛋白基因,其中单链莫内甜蛋白氨基酸序列中的Asp69(原AspA16)突变为Asn。利用DNA重组技术,将莫内甜蛋白基因克隆到植物表达载体pBl221中,构建了莫内甜蛋白基因的植物表达载体pBI221-monellin。结论:构建了莫内甜蛋白基因的植物表达载体,为转化园艺植物以改善其口感奠定了基础。     

Targeting Sod by Gene and Protein Engineering and Inhibition of Free Radical Injury

Although oxygen toxicity of tissues can be decreased by a variety of antioxidants and some enzymes, such as SOD and catalase, their protective effect on tissue injury in various diseases are fairly small predominantly because of their unfavorable in vivo behavior. To minimize oxidative stress in various diseases. such as ischemic myocardial injury, circulatory disturbance and corneal inflammation, we synthesized three types of SOD derivatives by gene and protein engineering technique. One type of SOD (SM-SOD covalently linked with hydrophobic anions) circulates bound to albumin with a half life of 6 h and accumulates in tissues whose local pH is decreased. The other type of SOD (AC-SOD covalently linked with long chain fatty acids via the ?-amino group of lysyl residues) anchors onto membranc/lipid bilaycrs of various cells. The last type of SOD (HB-SOD synthesized by constructing a fusion gene coding human CuZn-type SOD and a C-terminal heparin-binding domain) binds to heparin-like proteoglycans on vascular cndothelial cell surface. Intravenous administration of either SM-SOD or HB-SOD markedly inhibited postischcmic reflow arrhythmias in the rat. When the left anterior descending artery was occluded permanently. about 65 % of animals died within 30 min predominantly due to irreversible ventricular fibrillation; the motality of animals decreased to 15 % by administering SM-SOD either before or after occlusion. Topically administered AC-SOD bound to the corneal epithelial cell surface and polyrnorp%onuclear leukocytes and efficiently dismutated superoxide radicals at their cell surface. Thus,' endotoxin-induced kcratitis was inhibited markedly by topical instillation of AC-SOD. Unmodified SOD itself failed to inhibit the pathologic events occurring in these disease models. Thus, these SOD derivatives permit in vivo studies on the mechanism and the site for oxygen toxicity in various diseases and provide a new strategy for targeting enzymes and bioactive peptides for medical use to appropriate site(s) of their action.     

叶绿体基因工程简介

叶绿体是植物细胞中一种特殊的细胞器。自1988年开始,人们认识到叶绿体在植物基因工程中的特殊地位。叶绿体基因工程的特点,特别是其高效表达和安全性,使其受到越来越多的重视,本文对叶绿体转化作了较为全面的介绍,包括其优势、方法、用途及不足等内容。 Abstract:Chloroplast is a kind of special cell organin plant cells.Since 1988,Scientists have realized its advantages in plant gene enginearing.It′s high efficient expression and safety made it been attached more and more importance to.This paper introduces the chloroplast transfer mation,including its advantages,methods,uses and defects.