葡糖醛酸酯酶的研究进展

葡糖醛酸酯酶是一种新型碳水化合物酯酶,可以水解4-O-甲基-D-葡萄糖醛酸(Me Glc A)与木质素醇羟基之间的酯键,从而破坏Me Glc A在细胞壁中形成的致密的交联结构。本文主要介绍了葡糖醛酸酯酶的来源、酶活检测底物、基因的克隆与表达、晶体结构方面的研究进展,并对今后的研究方向提出了建议。     

酵母3-脱氧葡糖醛酮代谢酶的分离纯化及部分性质

3-脱氧葡糖醛酮 ( 3- deoxyglucosone)是美拉德反应的主要中间产物 ,对生物体具有毒性作用 .用硫酸铵分部沉淀、DEAE- cellulose52、Hydroxyapatite、DEAE- Sepharose CL- 6B柱层析从酿酒酵母 YBr-M( S.cerevisiae YBr-M)抽提液中分离纯化了 3-脱氧葡糖醛酮代谢酶 (以 NADPH为辅酶 ) .该酶是单一的分子 ,分子量为 44k D,反应最适 p H为 7.0 ,p H6.0～ 8.0之间酶活性相对稳定 ,以 3-脱氧葡糖醛酮为底物的米氏常数 Km 为 2 .2 5mmol/ L.在 35℃以下保温 30 min酶活不变 ,50℃保温 30 min后酶活损失 50 % .该酶对二羰基化合物的活性较高 ,对单羰基化合物则较低 ,其催化作用受碘乙酸、N-乙基顺丁烯二酰亚胺的抑制 ,而被β-巯基乙醇、二硫苏糖醇激活 ,催化作用必须以 NADPH为专一辅酶 ,当用 NADH代替 NADPH时 ,活力只有 5.3% .     

植物中的葡糖磷酸变位酶

葡糖磷酸变位酶是催化葡糖1磷酸与葡糖6磷酸之间可逆性转化的酶类,在有机体内的糖原合成及利用中起中枢作用。本文综述了葡糖磷酸变位酶在植物中的分布、重要性、功能及分子特性等,重点介绍了关于植物葡糖磷酸变位酶在遗传及分子生物学方面的研究进展和热点,并讨论了研究葡糖磷酸变位酶的理论和实际意义。     

植物中的葡糖磷酸变位酶

葡糖磷酸变位酶是催化葡糖－１－磷酸与葡糖－６－磷酸之间可逆性转化的酶类,在有机体内的糖原合成及利用中起中枢作用。本文综述了葡糖磷酸变位酶在植物中的分布、重要性、功能及分子特性等,重点介绍了关于植物葡糖磷酸变位酶在遗传及分子生物学方面的研究进展和热点,并讨论了研究葡糖磷酸变位酶的理论和实际意义。     

细菌3-脱氧葡糖松还原酶产生面及产酶条件

从12株细菌菌株中筛选到一株产3-脱氧葡糖松还原酶的高产菌Bacillussp．2,并研究了该菌株的产酶条件。在所试验的细菌中,3-脱氧葡糖松氧化酶活性较低甚至检测不出,而还原酶活性普遍较高。该菌株最佳产酶条件为：培养基组成（％）：牛肉膏0．3,牛肉蛋白陈1．0,氯化钠0．5,0．5mmol／L3-脱氧葡糖松。培养基起始pH6．6,30℃振荡培养48h,产酶量最高,酶活力可达36．4u／g。3-脱氧葡糖松对产酶有诱导作用,而甲基乙二醛对产酶有抑制作用。     

海洋细菌Bacillus sp.2-羰基还原酶的纯化和性质

从一株海洋细菌Bacillussp .中通过硫酸铵分级盐析、QSepharoseFF阴离子交换层析、Hydroxyapatite柱层析和SephadexG 1 0 0凝胶过滤 ,分离纯化出一种 3 脱氧葡糖醛酮代谢酶 ,定性为 2 羰基醛还原酶。以粗酶液作起始 ,所获样品纯度提高 1 41 4倍 ,活力回收率 1 1 4%。 2 羰基醛化合物对该酶是特异性很好的底物 ,该酶对 3 脱氧葡糖醛酮的Km为 2 5mmol L ,分子量约为 33kD ,反应最适pH为 6 2 ,在pH5 0～ 8 0 ,温度 30℃以下酶活保持稳定。适量的ED TA、巯基乙醇或二硫苏糖醇能提高酶的活性 ,碘乙酸、N 乙基顺丁烯二酰亚胺均造成酶部分失活。     

葡糖酸醋杆菌内源复制片段的克隆

采用改进的碱裂解法提取Gluconacetobacter hansenii ATCC23769自发不产膜突变体的内源隐蔽质粒。用不同的限制性内切酶对混合质粒直接进行酶切,酶切后的片段混合物与pUC18载体连接构建重组载体。重组载体回转入G.hansenii ATCC23769获得隐蔽质粒上具有复制能力的片段,序列结果分析表明:该片段上没有某些其他质粒所具有的Rep蛋白。利用该片段,构建了能同时在大肠杆菌和葡糖酸醋杆菌中复制的质粒载体,体内的抗生素抗性实验证明该载体具有良好的稳定性。     

葡糖杆菌属分类及其主要应用的研究进展

葡糖杆菌是醋酸菌科的一个重要属,与人类关系密切,该属中部分菌株在维生素C、米格列醇的工业生产及合成中起着重要作用;此外,该属菌株还能氧化葡萄糖生成葡糖酸盐和酮基葡糖酸等工业重要中间体。综述了葡糖杆菌的研究进展,主要介绍了葡糖杆菌的生理生化特征、分类进展及其主要应用,可以为广大醋酸菌研究者提供参考。     

海洋细菌Bacillus sp.2—羧基还原酶的纯化和性质

从一株海洋细菌Bacillus sp,中通过硫酸铵分级盐析,Q Sepharose FF阴离子交换层析,Hydroxyapatite柱层析和Sephadex G-100凝胶过滤,分离纯化出一种3－脱氧]葡糖醛酮代谢酶,定性为2－羧基醛还原酶,以粗酶液作起始,所获样品纯度提高141．4倍,活力回收率11．4％,2－羧基醛化合物对酶是特异性很好的底物,该酶对3－脱氧葡糖醛酮的Km和2.5mmol/L,分子量约为33kD,反应最适pH为6．2,在pH5．0－8．0,温度30℃以下酶活保持稳定,适量的ED－TA,巯基乙醇或二硫苏糖醇能提高酶的活性,碘乙酸,N-乙基顺丁烯二酰亚胺均造成酶部分失活。     

葡糖淀粉酶基因的分子生物学研究

从酶活、底物、产物、应用及空间结构等方面论述了葡糖淀粉酶的一般生物学特性及应用,分析了其不同来源的基因同源度。着重从分子生物学角度阐述了葡糖淀粉酶基因在多种表达系统中的分泌表达情况,并对其基因在曲霉及酵母中的高效表达及分泌引导功能进行了初步阐述和探讨。     

甘薯和花生胰蛋白酶抑制剂的初步研究

许多植物蛋白制成品均含有抑制动物消化的蛋白酶抑制剂。目前已从某些豆类及蔬菜种子中分离出多种对胰蛋白酶具有抑制作用的活性物质。该实验以花生、甘薯等为原料,通过DEAE-Sepharose4BFF阴离子交换柱层析分离胰蛋白酶抑制剂,以N-苯甲酰-L-精氨酸乙酯(BAEE)为底物测定其对胰蛋白酶的抑制活性;将具有抑制活性的组分通过SDS-PAGE测定蛋白质相对分子质量(Mr);以聚丙烯酰胺凝胶等电聚焦电泳测定蛋白质等电点(pI)。结果显示,甘薯中至少有4种胰蛋白酶抑制剂组分,相对分子质量为20～25kD、等电点在pH5.0～6.6之间;花生中至少有三种胰蛋白酶抑制剂组分,相对分子质量为30～70kD、等电点在pH5.0～5.8之间。     

豌豆球蛋白及其亚基的分离提纯

本文详细地叙述了提取、分离、提纯豌豆球蛋白及其亚基的方法:用硼酸钠缓冲液提取豌豆球蛋白,用85%饱和度硫酸铵沉淀除去其它蛋白质杂质,再用凝胶柱进一步提纯;提纯的豌豆球蛋白通过DE_(52)-纤维素阴离子树脂柱(8M尿素作为变性分离试剂)和S_(200)凝胶柱(70%甲酸作为变性分离试剂)则可分离出豌豆球蛋白的各个亚基;试验结果还表明:在pH8.5时,33,000的亚基携带少量负电荷;而12,000的亚基携带大量负电荷。     

Isolation and characterization of lectins from Vicia villosa. Two distinct carbohydrate binding activities are present in seed extracts

An uncharacterized lectin from Vicia villosa seeds has been reported to bind specifically to mouse cytotoxic T lymphocytes (Kimura, A., Wigzell, H., Holmquist, G., Ersson, B., and Carlsson, P., (1979) J. Exp. Med. 149, 473-484). We have found that V. villosa seeds contain at least three lectins which we have purified by affinity chromatography on a column of immobilized porcine blood group substances eluted with varying concentrations of N-acetylgalactosamine and by anion exchange chromatography. The three lectins are composed of two different subunits with Mr = 35,900 (subunit B) and 33,600 (subunit A), estimated from their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sedimentation equilibrium analysis suggests that the purified lectins are tetramers. They have been designated B4, A4, and A2B2 to indicate their apparent subunit compositions. The purified B4 and A4 lectins contain 6.7-9.8% carbohydrate by weight; in addition, both are rich in the acidic and hydroxylic amino acids and lack cysteine and methionine. The A4 lectin agglutinates A erythrocytes specifically and binds to A1 erythrocytes (273,000 sites/cell) with an association constant of 1.8 X 10(7) M-1. Although a blood group A agglutinating activity was recognized in the original preparation of V. villosa lectins, lectins with this activity were obtained in relatively small amounts from seed extracts. The predominant lectin in V. villosa seeds, B4, does not agglutinate A, B, or O erythrocytes.     

Chemical composition and nutritional value of para-rubber seed and its products for chickens

Para-rubber (Hevea brasiliensis) seed and its products were subjected to different methods of processing such as decortication, oil extraction, autoclaving and fermentation and assayed for their chemical composition and nutritional value. Peanut oil meal and yellow maize were also assayed similarly for comparison.

Decortication reduced the crude fibre content, with proportionate increases in other nutrients and energy value. Autoclaving and fermentation failed to improve the nutritional value of undecorticated rubber seed oil meal.

Crude protein content of rubber seed and its products ranged from 11.5% in rubber seeds to 27.4% in commercial decorticated rubber seed oil meal. The oil content of the rubber seeds and kernels was 24.0 and 40.1%, respectively. The available carbohydrate content of rubber seed and its products ranged from 6.3% in rubber seeds to 15.9% in commercial decorticated rubber seed oil meal; these values may be compared with the value of 59.0% for yellow maize.

Both undecorticated and decorticated rubber seed oil meals appeared to be deficient in sulphur-containing amino acids and lysine. The gross protein value of undecorticated and decorticated rubber seed oil meals and peanut oil meal was estimated to be 43.6, 47.0 and 49.7, respectively.

Both undecorticated and decorticated rubber seed oils were rich in oleic and stearic acids, but relatively poor in poly-unsaturated fatty acids, compared with peanut oil.

Determined apparent ME (AME) values were (kcal/g dry matter): rubber seeds, 2.91; kernels, 4.70; undecorticated rubber seed oil meal, 2.00; and decorticated rubber seed oil meal, 2.80. The true ME (TME) values were 3.24, 5.16, 2.22 and 3.00 kcal/g dry matter, respectively. In general, TME values were about 10% higher than the AME values.     

Isolation and properties of a lectin from sainfoin (Onobrychis viciifolia, Scop.)

A glycoprotein capable of binding simple carbohydrates and causing hemagglutination has been isolated from seeds of the legume plant sainfoin (Onobrychis viciifolia, Scop. var Eski). The phytolectin was prepared by affinity chromatography of pH 7.0 sodium phosphate extracts on columns of Sepharose-4B containing covalently attached D-mannose. Molecular weight determinations showed the lectin to be a dimer consisting of 26 000 dalton, non-covalently associated monomers. Amino acid analyses indicated high amounts of aspartate, glutamate, threonine and serine which accounted for 41% of all amino acids. One residue of cysteine was present and methionine was totally absent. The lectin contained 2.6% (w/w) neutral carbohydrate and two residues of N-acetylglucosamine/monomer. Carbohydrate-binding specificity was directed toward D-mannose and D-glucose and their alpha-glycosidic derivatives. The purified protein agglutinated cat erythrocytes at 5 micrograms/ml. Antiserum to seed lectin showed a single common immunoprecipitation line in Ouchterlony double diffusion against both the seed and root antigen. Lectin isolated from sainfoin seedling roots showed molecular weight, amino acid and carbohydrate values similar to that of the seed lectin.     

Cathepsins B and H from porcine spleen. Purification, polypeptide chain arrangements, and carbohydrate content

Procedures for the purification of cathepsins B and H from porcine spleens have been described. The purified porcine cathepsin B (Mr = 27,000) is predominantly a two-chain enzyme with a heavy chain (Mr = 22,000) and a light chain (Mr = 5,000). It also contains two minor forms of cathepsin B with different chain structures. Porcine cathepsin H is a single-chain enzyme with a molecular weight of 25,000. The carbohydrate analyses showed that these enzymes were glycoproteins. A glycopeptide containing 3 amino acids, 2 glucosamines, and 6 mannoses was isolated from cathepsin H. Proton NMR studies revealed that it contained a mixture of 4 high mannose-type of oligosaccharides characteristic of those found on lysosomal enzymes. The carbohydrate of cathepsin B consisted of a single residue of glucosamine and trace mannose. This sugar content is in agreement with the finding that about 80% of the porcine spleen cathepsin B contained a single N-acetylglucosamine while 20% of the enzyme contained a 5-sugar oligosaccharide (Takahashi, T., Schmidt, P. G. and Tang, J. (1984) J. Biol. Chem. 259, 6059-6062). Thus, the studies on carbohydrate contents also indicated the good purity of the enzymes.     

An amphiphilic structure of the ninth component of human complement. Evidence from analysis of fragments produced by alpha-thrombin

Purified human C9 was treated separately with three proteolytic enzymes: trypsin, plasmin, and alpha-thrombin, and the digestion products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Trypsin initially cleaved the Mr = 71,000 C9 to produce a Mr = 47,000 fragment plus numerous smaller fragments and prolonged digestion reduced the molecule to small polypeptides. Plasmin produced a Mr = 37,000 fragment which was stable to further digestion, plus fragments smaller than Mr = 10,000. Human alpha-thrombin cleaved C9 (7.8% carbohydrate) at a single internal site to produce a Mr = 37,000 fragment (11.3% carbohydrate) and a Mr = 34,000 fragment (3.9% carbohydrate). Statistical analysis of the amino acid compositions of the fragments and alkaline polyacrylamide gel electrophoresis showed that C9 is highly amphiphilic; the Mr = 34,000 fragment contains a majority of the acidic amino acids and migrates rapidly on alkaline gels; the Mr = 37,000 fragment is hydrophobic with a slow electrophoretic mobility. The two fragments remain noncovalently associated, but were separated by sodium dodecyl sulfate-hydroxylapatite chromatography. The NH2-terminal sequence analysis of native C9, of alpha-thrombin-cleaved C9, and for the isolated fragments showed that the acidic Mr = 34,000 fragment is the NH2-terminal C9a domain and the more hydrophobic Mr = 37,000 fragment is the carboxyl-terminal C9b domain. Hemolytic activity of C9 was unaffected by alpha-thrombin cleavage.     

荔枝胚败育过程中内源激素与蛋白质含量的变化

连续3年(1999-2001年)对典型的荔枝焦核品种桂味、糯米糍和大核品种黑叶、怀枝花后10-40d的幼胚和胚乳内源激素、多酚含量及蛋白质动态变化进行研究。结果表明,焦核品种幼胚及胚乳中的IAA、GAs和ABA含量低于大核品种;多酚类物质含量在胚中低于大核品种,胚乳中则高于大核品种;胚和胚乳中的蛋白质含量均低于大核品种。蛋白质电泳结果显示,22．5、28．5和45kD这3类蛋白质在怀枝和黑叶的胚蛋白质代谢过程中表现出较高的稳定性,桂味和糯米糍胚蛋白质中的28．5kD蛋白质也有相似的特性。     

Polyembryony in Citrus. Accumulation of seed storage proteins in seeds and in embryos cultured in vitro.

Citrus exhibits polyembryonic seed development, an apomictic process in which many maternally derived embryos arise from the nucellus surrounding the developing zygotic embryo. Citrus seed storage proteins were used as markers to compare embryogenesis in developing seeds and somatic embryogenesis in vitro. The salt-soluble, globulin protein fraction (designated citrin) was purified from Citrus sinensis cv Valencia seeds. Citrins separated into two subunits averaging 22 and 33 kD under denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cDNA clone was isolated representing a citrin gene expressed in seeds when the majority of embryos were at the early globular stage of embryo development. The predicted protein sequence was most related to the globulin seed storage proteins of pumpkin and cotton. Accumulation of 33-kD polypeptides was first detected in polyembryonic Valencia seeds when the majority of embryos were at the globular stage of development. Somatic Citrus embryos cultured in vivo were observed to initiate 33-kD polypeptide accumulation later in embryo development but accumulated these peptides at only 10 to 20% of the level observed in polyembryonic seeds. Therefore, factors within the seed environment must influence the higher quantitative levels of citrin accumulation in nucellar embryos developing in vivo, even though nucellar embryos, like somatic embryos, are not derived from fertilization events.     

Some Properties and Characterization of Rice Seed Hemagglutinin

The phytohemagglutinin of rice seed has been purified by a sequence of steps involving fractionation with ammonium sulfate and successive chromatography on DEAE-and eMcellulose and finally gel filtration on Bio-Gel P-100. The purified rice seed hemagglutinin was shown to be homogeneous by electrophoresis on polyacrylamide gel and its molecular weight was 10,000, calculated from both the Ve/Vo value of gel filtration on Bio-Gel P-100 and the sum of the individual constituents (amino acids, sugars and metals). In addition to amino acid, the rice seed hemagglutinin contained 26.8% covalently bound carbohydrate which was identified and quantitated by gas chromatography of the acetylated alditols. Glucose was the predominant sugar with lesser amounts of glucosamine, xylose, and mannose also being present. And the rice seed hemagglutinin contained 1 g atom of calcium per molecule. The molecular weight of the rice seed hemagglutinin is smallest compared with some of phytohemagglutinins isolated from leguminous seeds and other plant sources. The rice seed hemagglutinin has the blastogenetic activity for human peripheral lymphocytes as well as Phaseolus vulgaris phytohemagglutinins or concanavalin A, jack bean hemagglutinin.