Isolation and Characterization of Acidic Peptides in Soy Sauce

A soy sauce sample was fractionated by gel filtration on a Sephadex G–15 column, then the fractions were subfractionated on the basis of acidity by ion exchange chromatography on a QAE-Sephadex A–25 column. The acidic subfractions with various acidities were further fractionated, using a preparative amino acid analyzer and by paper chromatography to separate the acidic peptide components.

Four dipeptides and sugar derivatives of ten dipeptides and two tripeptides were isolated and characterized as the major acidic peptides in soy sauce. However, it was difficult to anticipate any direct contribution of these peptides to the flavor construction in soy sauce on the basis of their contents and taste intensities.     

Mapping of Peptides in Soy Sauce by an Ion Exchange Chromatography

Pattern analysis of peptides in soy sauce was tried by gel filtration and ion exchange chromatography. In gel filtration through Sephadex G-15, the separation of smaller peptides and amino acids in soy sauce was found to be not so critical. Subsequently the mapping of peptides in soy sauce was done by ion exchange chromatography with Dowex 50-X2. Consequently it was estimated that there existed more than ten kinds of peptides in soy sauce.

Moreover, in the mapping of peptides, the automated measurement by the use of a discrete type analyzer, EEL Auto Chemist, was tried. The method developed here was not fully automated but available and effective for the mapping of peptides eluted from an ion exchange resin column.     

Isolation of Alkaline and Neutral Proteases from Aspergillus flavus var. columnaris, a Soy Sauce Koji Mold

Two different extracellular proteases, protease I (P-I), an alkaline protease, and protease II (P-II) a neutral protease, from Aspergillus flavus var. columnaris were partially purified by using (NH(4))(2)SO(4) precipitation, diethylaminoethyl-Sephadex A-50 chromatography, carboxymethylcellulose CM-52 chromatography, and Sephadex G-100 gel filtration. The degree of purity was followed using polyacrylamide gel electrophoresis. The activity of P-I was completely inhibited by 0.1 mM phenylmethylsulfonyl fluoride, and that of P-II was completely inhibited by 1 mM ethylenediaminetetraacetate. By using these inhibitors with extracts of wheat bran koji, the proportions of total activity that could be assigned to P-I and P-II were 80 and 20%, respectively. This compared favorably with activities estimated by using polyacrylamide gel electrophoresis slices (82 and 18%, respectively). Extracts from factory-run soybean koji gave comparable results. Both enzymes demonstrated maximum activity at 50 to 55 degrees C and only small changes in activity between pH 6 and 11. For P-I, activity was somewhat higher from pH 8.0 to 11.0, whereas for P-II it was somewhat higher from pH 6 to 9. In the presence of 18% NaCl, the activities of both P-I and P-II dropped by approximately 90 and 85%, respectively. P-I was inferred to possess aminopeptidase activity since it could hydrolyze l-leucyl-p-nitroanilide hydrochloride. P-II was devoid of such activity. The ramifications of the results for factory-produced soy sauce koji are discussed.     

Isolation and Analysis of Molds from Soy Sauce Koji in Thailand

Five different isolates of Aspergillus and one of Mucor were compared with a Japanese commercial strain of Aspergillus oryzae for proteolytic activity on wheat bran substrate. One isolate of Aspergillus with superior protease production, identified as Aspergillus flavus var. columnaris, showed no detectable aflatoxin production on glutinous rice or soybean substrate. Preliminary tests using this fungus as a koji mold in a traditionally operated factory resulted in a soy sauce superior in quality to that usually produced.     

从老抽酱醪中分离耐盐性酵母的研究

以老抽酱醪为实验材料进行耐盐性酵母菌种分离,并做菌种鉴定。分析了在不同盐度条件下耐盐性酵母菌的生长情况和生长过程中培养基总糖的消耗,可以发现实验得到的酵母在22％（质量与体积}E）盐度下依然能够良好生长。结果表明,实验分离出的No．2菌在同级盐度的条件下的生长量要明显高于No．1菌,但在乙醇产率方面,两株菌在相同的含盐量为16％（质量与体积比）的麦芽汁培养基中发酵8d,No．1菌的乙醇产率为3．1％（体积比）,No．2菌的乙醇产率2．9％（体积比）。     

胀罐酱油中耐渗透压酵母菌分离及鉴定

从广东某调味食品厂的3批次的胀罐酱油中分离到3株引起胀罐的耐渗透压的酵母菌,这些酵母菌在含有50％～60％葡萄糖和15％NaCl的培养基上生长较快,在普通酵母培养基上生长缓慢。通过对其进行形态特征观察和生理生化的测定,初步鉴定这3株酵母菌为埃切毕赤酵母（Pichia etchellsii）。     

Effects of Chemical Modification of Carboxyl Groups in the Hemolytic Lectin CEL-III on Its Hemolytic and Carbohydrate-Binding Activities

Effects of chemical modification of carboxyl groups in the hemolytic lectin CEL-III on its activities were investigated. When carboxyl groups were modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine methyl ester, hemolytic activity of CEL-III decreased as the EDC concentration increased, accompanied by reduction of oligomerization ability and hemagglutinating activity. However, binding ability of CEL-III for immobilized lactose was retained fairly well after modification, suggesting that one of two carbohydrate-binding sites might be responsible for such inactivation of CEL-III.     

Isolation and Characterization of Opioid Peptides from Rabbit Cerebellum

The rabbit cerebellum has been shown to contain significant quantities of opioid receptors consisting of both mu- and kappa-subtypes. To determine the nature of the endogenous opioid ligands in this tissue, extracts from rabbit cerebellum were separated by various chromatography techniques and fractions were assayed initially for opioid peptides with a radioimmunoassay capable of detecting all peptides with an amino-terminal Tyr-Gly-Gly-Phe sequence. This sequence is common to all mammalian opioid peptides and is critical for recognition by all known opioid receptors. Each of the three immunoreactive opioid peptide peaks detected was purified to homogeneity and subjected to amino acid composition and sequence analysis. One peak was analyzed further by mass spectrometry. This identified the major opioid peptides in the cerebellum as [Met5]enkephalin, [Leu5]enkephalin, and heptapeptide [Met5]enkephalyl-Arg6-Phe7. The comprehensiveness of this initial detection scheme in identifying biologically active opioid peptides was substantiated through subsequent analysis. Using specific radioimmunoassays for representative opioid peptides of the three opioid systems currently known, no other peptides of either the proenkephalin, proopiomelanocortin, or prodynorphin series were detected in any appreciable amounts. Collectively, these results are consistent with the position that rabbit cerebellar opioids are derived from proenkephalin. However, given that no appreciable quantities of either [Met5]enkephalyl-Arg6-Arg7-Val8-NH2 (metorphamide) or [Met5]enkephalyl-Arg6-Gly7-Leu8 were detected suggests that rabbit proenkephalin may have a slightly altered sequence and/or is differentially processed relative to other mammalian species studied.     

Evaluation of Soy Sauce Flavor by Stepwise Multiple Regression Analysis of Gas Chromatographic Profiles

The linear correlation was found by the stepwise multiple regression analysis between the sensory test of soy sauce flavor and the gas Chromatographic (GLC) data transformed with arc-sine and logarithm. GLC data will possibly be used for objective evaluation of soy sauce flavor. A multiple correlation coefficient or of a determination coefficient of more than 0.9 was respectively obtained at the 5 th or 10th of 47 steps. The fact that the minimum standard error of an estimate was found at the 24th step suggests the importance of selecting proper peaks from the whole gas chromatogram. High estimated accuracy was acquired by application of GLC data to the calculated multiple regression model.     

植物凝集素的功能

植物凝集素是来源于植物的一类能凝集细胞和沉淀单糖或多糖复合物的非免疫来源的非酶蛋白质。由于其对于单糖或糖复合物特异性结合的能力,使得其在如信号转导、免疫反应、植物防御等诸多信号过程中均具有重要作用。同时植物凝集素具有细胞凝集、抗病毒、抗真菌及诱导细胞凋亡或自噬等多种能力,因此在生命科学、医学及农业方面均有较好的研究价值和应用前景。综述了植物凝集素的研究历史和凝集素的主要功能,并对现阶段凝集素的重点应用做简要介绍。     

短肽蝎毒素的结构分类与功能特征

大量的资料已证实蝎毒中主要致死成分是一类由60～70个残基组成, 选择性地作用于电压门控Na⁺通道的长肽毒素.另一类由30～40个残基组成的短肽蝎毒素,由于其具有结构致密,易于合成改造的优点,特别是具有选择性地阻遏K⁺或Cl^－通道的特异药理功效,近年来倍受学术界的关注,并在结构与功能方面取得了很大的研究进展.     

中药桑寄生凝集素的分离及体外抗肿瘤活性的研究

采用硫酸铵分级沉淀和酸化的Sepharose-4B亲和层析分离得到桑寄生凝集素;通过A-PAGE和SDS-PAGE电泳分析,确定所得凝集素组分和亚基情况;通过MTT法对所得到的凝集素进行体外抗肿瘤活性研究。研究结果表明,桑寄生凝集素包含两种组分,共含有四个亚基,亚基分子量分别为36.8 kDa、33.4 kDa,31.3 kDa和29.3 kDa;凝集素具有抗肿瘤效果,对肝癌BEL-7402细胞和胃癌MGC-823细胞的IC50分别为24.2μg/mL和20.9μg/mL。     

Structure and ligand binding of carbohydrate-binding module CsCBM6-3 reveals similarities with fucose-specific lectins and "galactose-binding" domains

Carbohydrate-binding polypeptides, including carbohydrate-binding modules (CBMs) from polysaccharidases, and lectins, are widespread in nature. Whilst CBMs are classically considered distinct from lectins, in that they are found appended to polysaccharide-degrading enzymes, this distinction is blurring. The crystal structure of CsCBM6-3, a "sequence-family 6" CBM in a xylanase from Clostridium stercorarium, at 2.3 A reveals a similar, all beta-sheet fold to that from MvX56, a module found in a family 33 glycoside hydrolase sialidase from Micromonospora viridifaciens, and the lectin AAA from Anguilla anguilla. Sequence analysis leads to the classification of MvX56 and AAA into a family distinct from that containing CsCBM6-3. Whilst these polypeptides are similar in structure they have quite different carbohydrate-binding specificities. AAA is known to bind fucose; CsCBM6-3 binds cellulose, xylan and other beta-glucans. Here we demonstrate that MvX56 binds galactose, lactose and sialic acid. Crystal structures of CsCBM6-3 in complex with xylotriose, cellobiose, and laminaribiose, 2.0 A, 1.35 A, and 1.0 A resolution, respectively, reveal that the binding site of CsCBM6-3 resides on the same polypeptide face as for MvX56 and AAA. Subtle differences in the ligand-binding surface give rise to the different specificities and biological activities, further blurring the distinction between classical lectins and CBMs.     

X-ray crystal structure of a non-crystalline cellulose-specific carbohydrate-binding module: CBM28

Natural cellulose exists as a composite of different forms, which have historically been broadly characterized as "crystalline" or "amorphous". The recognition of both of these forms of cellulose by the carbohydrate-binding modules (CBM) of microbial glycoside hydrolases is central to natural and efficient biotechnological conversion of plant cell wall biomass. There is increasing evidence that, at least some, individual binding modules target distinct and different regions of non-crystalline "amorphous" cellulose. Competition experiments show that CBM28 modules do not compete with CBM17 modules when binding to non-crystalline cellulose. The structure of the BspCBM28 (http://afmb.cnrs-mrs.fr/CAZY/) module from the Bacillus sp. 1139 family GH5 endoglucanase, comprising a 191 amino acid protein, has therefore been determined at 1.4A resolution using single isomorphous replacement with anomalous scattering methods. The structure reveals a "beta-jelly roll" topology, with high degree of similarity to the structure of CBM17 domains. Sequence and structural conservation strongly suggests that these two families of domains have evolved through gene duplication and subsequent divergence. The ligand-binding site "topographies" of CBMs from families 28, 17 and 4 begins to shed light on the differential recognition of non-crystalline cellulose by multi-modular plant cell wall-degrading enzymes.     

The Thermostable Direct Hemolysin Gene (tdh) of Vibrio hollisae Is Dissimilar in Prevalence to and Phylogenetically Distant from the tdh Genes of Other Vibrios: Implications in the Horizontal Transfer of the tdh Gene

Vibrio hollisae strains isolated recently from patients in various locations were examined for the presence of the thermostable direct hemolysin gene (tdh) using nucleic acid hybridization and polymerase chain reaction assays. The results were consistent with the previous finding that all strains of V. hollisae carry the tdh gene. In contrast, the tdh gene has been detected in a minority of strains for other Vibrio species (V. parahaemolyticus, V. cholerae non-O1, and V. mimicus). Detailed phylogenetic analysis showed that the tdh genes of the non-V. hollisae species were very closely related to each other and that the tdh gene of V. hollisae was distantly related to the tdh genes of the non-V. hollisae species. These results and the proposed insertion sequence-mediated tdh transfer mechanism suggest that the tdh gene may have been maintained stably in V. hollisae and that the tdh genes of the non-V. hollisae species may have been involved in recent horizontal transfer.     

从抗TNF抗体的CDR肽库中获得拮抗TNF的短肽

为设计来自抗体的短肽 ,以抗肿瘤坏死因子 (TNF)嵌合抗体 (cA2 )CDRs为模板 ,在其两侧各加 3个随机氨基酸残基 ( X3 CDR X3 ) ,构建了 6个以CDR为基础的肽库 .经过 3轮亲和选择 ,挑取单克隆 ,进一步经ELISA检测TNF阳性噬菌体克隆 ,分离得到 7个ELISA阳性较好的噬菌体肽克隆 ,分别命名为CDR2L1、CDR2L2、CDR2L3、CDR1L1、CDR2H1、CDR3H1、CDR3H 2 .应用MTT方法 ,检测 7个克隆对TNF生物学活性的拮抗作用 .结果显示 :来自CDR2L ,CDR3H肽库中的CDR2L2、CDR2L3,CDR3H2噬菌体肽具有明显的拮抗TNF诱导L92 9细胞的细胞毒作用 ,其中以CDR2L2噬菌体肽的拮抗活性最强 .而来源于CDR1L ,CDR2H肽库的CDR1L1和CDR2H1噬菌体肽和来自CDR2L ,CDR3H肽库中的CDR2L1和CDR3H1噬菌体肽没有明显的拮抗TNF作用 .研究结果初步表明 :从cA2抗体CDR肽库中筛选得到的噬菌体CDR模拟肽具有亲本抗体相似的结合活性和生物学效应 ,从而为开发已知抗体 (特别是治疗用抗体 )CDR为基础的肽药物创建一个技术平台奠定基础     

长裙竹荪凝集素的分离纯化与部分生化性质

凝集素是一类与糖专一结合的蛋白质或糖蛋白 ,具有众多的生物学性质[1～ 5] ,在细胞凝集、鉴别人类血型物质和分离纯化某些高分子化合物等都有着非常重要的作用 ,已成为生物化学、细胞学、免疫学及医学等领域中有用的科研材料 ,并被应用于临床诊断、治疗和某些工业生产[1] .自 1888年Ｈ .Ｓｔｉｌｌｍａｒｋ首次从蓖麻籽中发现凝集素以来 ,已分离纯化 10 0多种凝集素 ,约有 60种已成为商品 ,其研究开发日益受到人们的重视 .竹荪是一种著名的食、药兼用菌 ,具有许多药用功效 .由于竹荪含有多种生理活性物质 ,从竹荪子实体或菌丝体中分离到的…     

Studies on the Activities of Bacteria in Soy Sauce Brewing

The activity of proteinases in soy masy juice was measured by a modification of the Anson’s method. Compared to proteinases of purely cultured soy koji digest which consisted of only Aspergillus proteinases, no difference was observed with the proteinases of usual soy mash through the studies of pH-proteinese activity curves, the inhibition test, and inactivation by acid and alkali treatment. It is supposed, therefore, that the proteinases in soy mash are mostly of Aspergillus origin. Viable counts on salted media, heat shocking at 80°C for thirty minutes, and the microscopic counts proved that there are very few Bacilli vegetative cells in soy mash. From these facts the author assumed that Bacilli mainly grow in the koji and survive in the soy mash mostly in the form of spores. This work also suggested that the so-called “Aspergillus alkaline proteinase” consists of two distinct proteinases, and further the existence of an inhibitor which only acts at pH8.     

Physicochemical ProPerties and binding-site amino acid residues of galactoside-binding Protein of human Placenta

The galactose-binding lectin of human Placenta has been Purified to homogeneity by affinity chromatograPhy on asialo-fetuin column. The Protein, extractable from the tissue only with lactose is aPParently membrane-bound. Molecular weight determination of native Protein and subunit indicated a dimer of l3.4 kDa subunits. Inhibition of haemagglutination with various saccharides indicate that thiodigalactoside is the best inhibitor followed by lactose. However,P-nitroPhenyl-and 1-O-methyl derivatives of galactose showed that α-anomers inhibited slightly better than β-anomer. Modification of amino acid residues indicated involvement of arginine, lysine and histidine residues at the saccharidebinding site. Cysteine residue modificatioin also abolished haemagglutinating activity. Amino acid comPosition of the lectin is also Presented.