Isolation and enzymatic fragmentation of the coeliac-active gliadin peptide CT-1.

Investigations on the structure/toxicity relationships of gliadin peptides were continued with the coeliac-active gliadin peptide CT-1, which is derived from the N-terminal portion (residues 3-24 of the amino acid sequence) of alpha-gliadins [this journal (1986) 182:115-117]. CT-1 was produced by chymotryptic digestion and reversed-phase (RP) HPLC from the peptide fraction G3 [this journal (1992) 194:1-6] and digested with the proteases endoproteinase Glu-C, pancreatin, papain and thermolysin. The fragment peptides were separated by preparative RP-HPLC and characterized by amino acid analysis. On the basis of the specificity of the enzymes for CT-1 and the toxic effect of enzymatic hydrolysates of gliadin described in the literature, the significance of partial sequences, in particular of the sequence -Pro-Ser-Gln-Gln-Gln-Pro- for the coeliac-toxicity effect, is discussed.     

Coeliac activity of the gliadin peptides CT-1 and CT-2

Summary The coeliac active peptide B 3142, which has been isolated from a peptic-tryptic digest of gliadin [1] and which consists of 53 amino-acid sequences [2], was partially hydrolyzed with -chymotrypsin. The two fragment peptides CT-1 (positions 1–22 of B 3142) and CT-2 (positions 23–53) were separated by high-performance liquid chromatography on octadecyl silica gel and purified by gel filtration on Biogel P2. The examination in the organ-culture test including 18 coeliac patients on normal diet and 7 control persons have shown that the toxicity is preserved after the chymotryptic treatment and that the peptides B 3142, CT-1 and CT-2 do not significantly differ from one another according to their coeliac-specific effect.

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Coeliakieaktivitat der Gliadinpeptide CT-1 und CT-2

Zusammenfassung Das coeliakieaktive Peptid B 3142, das aus einem peptisch-tryptischen Partialhydrolysat von Gliadin gewonnen wurde [1] und aus einer Sequenz von 53 Aminosäureresten besteht [2], wurde mit -Chymotrypsin partiell hydrolysiert. Die beiden Fragment-peptide CT-1 (Positionen 1–22 von B 3142) und CT-2 (Positionen 23–53) wurden durch Hochdruckflüssig-keitschromatographie an Octadecyl-Kieselgel aufgetrennt und an Biogel P2 gereinigt. Die Prüfung im Organkultur-Test unter Einbeziehung von 18 Coeliakie-patienten unter Normalkost und von 7 Kontrollpersonen zeigte, daß die Toxizität nach chymotryptischer Spaltung erhalten bleibt, und daß sich die Peptide B 3142, CT-1 und CT-2 in ihrer coeliakiespezifischen Wirkung nicht wesentlich unterscheiden.

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Supported by a grant from Deutsche Forschungsgemeinschaft. We gratefully acknowledge the excellent assistance given by Mrs. U. Schützler and Ms. B. Mosler     

Coeliac activity of the gliadin peptides CT-1 and CT-2

The coeliac active peptide B 3142, which has been isolated from a peptic-tryptic digest of gliadin and which consists of 53 amino-acid sequences, was partially hydrolyzed with alpha-chymotrypsin. The two fragment peptides CT-1 (positions 1-22 of B 3142) and CT-2 (positions 23-53) were separated by high-performance liquid chromatography on octadecyl silica gel and purified by gel filtration on Biogel P2. The examination in the organ-culture test including 18 coeliac patients on normal diet and 7 control persons have shown that the toxicity is preserved after the chymotryptic treatment and that the peptides B 3142, CT-1 and CT-2 do not significantly differ from one another according to their coeliac-specific effect.     

Isolation of coeliac active peptide fractions from gliadin

For the isolation of coeliac active peptide fractions the peptic tryptic digest of whole gliadin was successively separated by ultrafiltration, gel filtration, cation-exchange chromatography, anion exchange chromatography and high-performance liquid chromatography. After each separation step the peptide fractions obtained were characterized by amino acid analysis and examined for coeliac activity in an immunological test (LIF test) and in an organ-culture test. The most active fractions have molecular weights ranging from 7,000 to 14,000 daltons, high contents of Glx (greater than 40 mol-%), Pro (greater than 20 mol-%) and Phe (greater than 5 mol-%) and low contents of S-containing and basic amino acids (0 and less than 2.0 mol-%, respectively). The peptide fraction B3142 obtained after five separation steps seems to be a pure peptide, which shows activity in the immunological test for all coeliac patients examined in very low concentrations. This peptide consists of 53 amino acid residues and has the composition Glx24, Pro15, Val4, Phe3, Ser2, Leu2, Asx1, Gly1, Tyr1.     

Isolation of a Cappelle-Desprez gliadin.

A Cappelle-Desprez gliadin, previously unreported, has been isolated. It appears to be a single-chain protein with a molecular weight of only 18 000, considerably lower than that of any other gliadin so far isolated. Its amino acid composition, though broadly typical of the class, has surprising characteristics, such as 7 Met, 7 CySSCy, but less Gln and Pro and no Lys or His. Although at acid pH the mobility is less than that of the γ-gliadin group, at pH 8.9 the molecule is still positively charged. The high isoelectric point implies that almost all the carboxyl side-chains are amidated. The gliadan contains no SH groups and does not appear to be a glycoprotein. The amino acid analysis suggests that microheterogeneity is present.     

Oxidative modification of a proline-rich gliadin peptide

Prolamins are proline-rich proteins occurring in cereal grains. Prolamins of wheat, barley and rye, or gluten protein, can cause coeliac disease in individuals not tolerating gluten. Degrading harmful prolamins can reduce their toxicity. A model peptide sequenced in α-gliadin, 33-mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), was chosen for our study. The metal-catalysed oxidation of 33-mer was studied, instead of enzymatic hydrolysis. Peptide 33-mer was treated in several oxidative systems. Iron-catalysed hydrogen peroxide-induced oxidation showed the greatest modification of 33-mer. Carbonyl groups and dityrosine cross-links were readily formed. At best, the immunological activity of 33-mer was reduced to 18% of its initial level after 24 h of oxidation. Oxidative treatment can be further applied for the modification of cereal prolamin proteins, since it appears to be a potential alternative for reduction of coeliac immunological activities in gluten proteins.     

Amino-acid sequence of the coeliac active gliadin peptide B 3142

Summary Peptide B 3142, which has been isolated from a peptic tryptic digest of whole gliadin by several separation steps [1], was examined for coeliac activity in an immunological test and in an organ-culture test, comparing enlarged groups of coeliac patients and control persons. In both test systems the peptide shows a coeliac specific effect.The N-terminal sequence analysis (EDMAN degradation), the C-terminal sequence analysis (incubation with carboxypeptidase Y) and the sequence determination of peptides, obtained from B 3142 by digestion with papain and chymotrypsin, result in the following total amino-acid sequence: H-Val-Pro-Val-Pro-Gln-Leu-Gln-Pro-Gln-Asn-Pro-Ser-Gln-Gln residues correspond to a molecular mass of 6,129 g/mol.

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Aminosäuresequenz des coeliakieaktiven Gliadinpeptids B 3142

Zusammenfassung Das in vorangegangenen Arbeiten [1] aus einem peptisch-tryptischen Partialhydrolysat von Gliadin über einen mehrstufigen Trennprozeß gewonnene Peptid B 3142 wurde an einem erweiterten Patientenkreis im immunologischen Test und im Organkultur-Test auf Coeliakie-Aktivität geprüft. In beiden Tests zeigt das Peptid eine Coeliakie-spezifische Wirkung. Die N-terminale Sequenzanalyse (EDMAN-Abbau), die C-terminale Sequenzanalyse (Umsetzung mit Carboxypeptidase Y) sowie die Sequenz-analyse von Spaltpeptiden aus den mit Papain und Chymotrypsin erhaltenen Partialhydrolysaten ergeben folgende, aus 53 Aminosäureresten bestehende Gesamtsequenz: H-Val-Pro-Val-Pro-Gln-Leu-GlnPro-Gln-Asn-Pro-Ser-Gln-Gln-Gln-Pro-Gln-Glu Gln-Val-Pro-Leu-Val-Gln-Gln-Gln-Gln-Phe-Pro-Gly-Gln-Gln-Gln-Pro-Phe-Pro-Pro-Gln-Gln-Pro-Tyr-Pro-Gln-Pro-Gln-Pro-Phe-Pro-Ser-Gln-Gln-Pro-Tyr-OH. Daraus errechnet sich eine Molmasse von 6129 g/mol.

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Supported by grants from Stiftung Volkswagenwerk and from Deutsche Forschungsgemeinschaft

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We gratefully acknowledge the excellent assistance given by Mrs. Schiltzler     

Foaming properties of tryptic gliadin hydrolysate peptide fractions

A tryptic gliadin hydrolysate was separated into central domain (CD) or terminal domain (TD) related peptide fractions. Whereas the initial foam volume (FV) of CD peptide fractions remained constant as a function of pH, FV of TD peptide fractions increased from acidic to alkaline pH. Foam stability (FS) of CD peptide fractions was maximal near neutral pH. For TD peptide fractions, one fraction showed maximal FS at strongly alkaline pH, while the other showed no clear maximal FS. CD related peptide foams contained higher levels of hydrophobic peptides than the respective solutions, while small differences were observed for TD peptide fractions. Peptide compositions of foams did not vary with pH, indicating that the foaming properties of gliadin peptides are mainly dictated by charges. As the pH dependent foaming properties of TD related peptides resemble best those of gliadin, it was concluded that the pH dependent foaming properties of gliadins are mainly determined by their TDs.     

Relationship between gliadin peptide structure and their effect on the fetal chick duodenum

Summary The tendency to form a-turn in-gliadin was estimated using the B-cell determinant prediction program based on the Chou and Fasman probability of-turn formation. Six sequences possessing a high probability of-turn formation were found. A statistically high agreement was found between these six sequences and three areas in-gliadin with the occurrence of Pro-Ser-Gln-Gln sequence which has recently been considered responsible for toxicity in coeliac disease. By means of solid-phase synthesis seven peptides were obtained covering the above-mentioned regions. Their toxicity was tested using the fetal chick duodenum. The results support the suggestion that peptides containing the sequences Pro-Ser-Gln-Gln and Gln-Gln-Gln-Pro may be involved in the pathogenesis of coeliac disease.

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Beziehung zwischen der Gliadin-Peptid-Struktur und ihr Einfluß auf den fetalen Kückendarm

Zusammenfassung Die Tendenz zur Bildung einer-Umwandlung im-Gliadin wurde bei Anwendung eines mathematischen Programms zur Vorhersage von B-Zelldeterminanten bestimmt, welches auf der Wahrscheinlichkeit der-Umwandlung nach Chou und Fasman basiert. Es wurden 6 Sequenzen gefunden, die eine hohe Wahrscheinlichkeit für die Bildung von-Umwandlungen aufwiesen. Zwischen diesen 6 Sequenzen und 3 Regionen im-Gliadin mit der Sequenz Pro-Ser-Gln-Gln, die kürzlich als verantwortlich für die Toxizität bei Cöliakie angesehen wurden, konnte eine statistisch gesicherte Beziehung gefunden werden. Mittels Festphasensynthese wurden 7 Peptide erhalten, die die oben genannten Regionen überdeckten. Ihre Toxizität wurde im fetalen Kückendarm getestet. Die Ergebnisse weisen darauf hin, daß Peptide, welche die Sequenz Pro-Ser-Gln-Gln und Gln-Gln-Gln-Pro enthalten, an der Pathogenese der Cöliakie beteiligt sein könnten.

     

Relationship between gliadin peptide structure and their effect on the fetal chick duodenum

The tendency to form a beta-turn in alpha-gliadin was estimated using the B-cell determinant prediction program based on the Chou and Fasman probability of beta-turn formation. Six sequences possessing a high probability of beta-turn formation were found. A statistically high agreement was found between these six sequences and three areas in alpha-gliadin with the occurrence of Pro-Ser-Gln-Gln sequence which has recently been considered responsible for toxicity in coeliac disease. By means of solid-phase synthesis seven peptides were obtained covering the above-mentioned regions. Their toxicity was tested using the fetal chick duodenum. The results support the suggestion that peptides containing the sequences Pro-Ser-Gln-Gln and Gln-Gln-Gln-Pro may be involved in the pathogenesis of coeliac disease.     

Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides from wheat gliadin hydrolysate

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from wheat gliadin hydrolysate prepared with acid protease. Consecutive purification methods were used for peptide isolation including ion-exchange chromatography, size-exclusion chromatography, and reverse-phase high-performance liquid chromatography. The amino acid sequence of this peptide was identified as Ile-Ala-Pro, and the ACE inhibitory activity (IC50 value) was 2.7 microM. The hypotensive activity of Ile-Ala-Pro on spontaneously hypertensive rats was investigated. This peptide inhibited the hypertensive activity of angiotensin I with intravenous injection, and decreased the blood pressure significantly with intraperitoneal administration.     

生淀粉酶产生菌的筛选和研究

利用透明圈法再经单菌分离纯化从土壤中筛到一株产酶为158U/mL的生淀粉酶产生菌TCCC451031,经16S rDNA鉴定为Paenibacillus属。最适发酵培养基:碳源为2%的玉米粉,最佳氮源为酵母粉与玉米浆混用比例为1∶2,用量为1%。最适发酵条件:35℃、pH值为7.0、转速160r/min,发酵5d酶活达312U/mL。该酶最适作用温度40℃,最适作用pH值为4.2,Ca2+、Mg2+、Zn2+对酶有一定的激活作用,Cu2+、Mn2+、K+、Fe2+、Ba2+对酶活有抑制作用。     

酶法制备香菇多肽的研究

目的:为了使香菇蛋白更易被人体所吸收,提高香菇蛋白在人体内的利用率。方法:以香菇为原料,用酶法制备香菇多肽。实验以香菇多肽得率作为指标,比较了胃蛋白酶、3.350酸性蛋白酶、菠萝蛋白酶、木瓜蛋白酶等多种酶的酶解效果。结果:香菇多肽工艺的最佳制备条件为:加酶量3000 U/g,p H9.0,温度45℃,酶解时间4 h,在此条件下酶解得率为92.7%。G25凝胶层析色谱分析表明,香菇多肽分子量主要为1000~5000 u和1000 u两部分,其中分子量1000 u的寡肽的比例占总多肽的68.38%。香菇多肽的氨基酸分析可知,香菇多肽保留了蛋白的大部分营养物质。结论:胰蛋白酶的酶解效果最佳。在最佳酶解条件下,营养物质得到了充分的保留,所得香菇多肽多为寡肽,利于人体吸收。     

产普鲁兰酶蜡样芽孢杆菌的分离鉴定及酶学性质研究

利用选择性培养基从广西南宁淀粉加工厂附近土壤中分离出一株具有产普鲁兰酶能力的野生菌株GXBC-2。该菌株革兰染色为阳性,形成芽孢,菌体细胞杆状,结合生理生化特征和16S rDNA序列分析,初步鉴定该菌株为Bacillus cereus GXBC-2。对该菌所产普鲁兰酶的性质研究表明:酶的最适反应温度为50℃,在30～50℃范围内稳定,最适pH为7.0,稳定pH范围为6～8.5。产物经HPLC分析证明,该酶能水解普鲁兰糖产生麦芽三糖,对可溶性淀粉不起作用,所以该酶属于I型普鲁兰酶。     

Fingerprinting of glutenin and gliadin

Probability considerations applied to fingerprints of enzymic digests of Conley glutenin and gliadin have shown a significant relationship between them. Acetic acid-soluble proteins of wheat and rye also gave closely similar fingerprints. It is tentatively concluded that there may be a basic similarity in the aminoacid sequences of considerable portions of the polypeptide chains present in glutenin, gliadin and in rye proteins. This possibility is not incompatible with modern theories of the effect of mutations on protein structure. A protein with low mobility and alcohol solubility has been detected in Conley flour.     

酶法水解芝麻粕制备芝麻多肽

以芝麻制油后副产物芝麻粕为原料,采用蛋白酶水解法制取芝麻多肽。在水解酶筛选、单因素试验基础上,以多肽产率为指标,设计正交试验对碱性蛋白酶酶解芝麻粕制取芝麻多肽工艺进行了优化。结果表明,最佳工艺参数为:pH 9.0,料液比1∶20,加酶量11 000 U/g,酶解时间2.5 h,酶解温度65℃。在此条件下,多肽产率达到3.51%。     

酶法水解血红蛋白制备亚铁血红素肽

以新鲜猪血为原料制备血红蛋白采用中性蛋白酶和风味酶两步水解工艺，并通过除氧和添加抗氧化剂保护亚铁离子，水解产物经过10kDa和3kDa的微孔滤膜超滤，得到分子量在3～10kDa的亚铁血红素肽，其中亚铁血红素与肽的比值达到9．92％。通过微胶囊包埋，得到稳定的红色产品。     

高温酸性脂肪酶产生菌Acinetobacter sp. Lip-55的筛选、鉴定及其酶学性质研究

从云南大理市弥渡县石夹泉热泉的55℃底泥中筛选到1株高温脂肪酶的高产菌株,进行显微形态及生理生化特征、16S rRNA基因序列分析,将其初步鉴定为不动杆菌属(Acinetobacter sp.)的一株菌,命名为Acinetobacter sp. Lip-55。对其生长条件及酶学性质进行了研究,结果表明,该菌株耐高温性较好,菌株在55℃仍能生长,菌株最适生长温度为37℃。所产脂肪酶最适酶活温度为55℃,最适反应pH值为5.0。该酶在25℃以下,能保持良好的稳定性,Zn~(2+)、Ca~(2+)、Mg~(2+)对该酶有一定的抑制作用,Mn~(2+)、K~+、Fe~(2+)、Cu~(2+)均对该酶活力起到促进作用,其中Fe~(2+)的促进效果最为显著。     

酶法制备核桃多肽研究进展

近年科学研究发现,人体摄取蛋白质经酶消化后,并非主要以氨基酸形式吸收,而以肽的形式吸收;一些肽不仅能提供人体生长、发育所需营养物质,同时还具有多种生理功能。核桃蛋白属于高蛋白食品,利用蛋白酶水解核桃蛋白生产核桃多肽,对促进核桃蛋白精深加工发展具有积极作用。该文介绍目前酶法制备核桃多肽研究进展,讨论影响酶解效果因素,阐述核桃多肽营养价值及发展前景。     

南瓜籽仁蛋白多肽的酶法制备和抗氧化活性研究

以酸溶性氮得率为评价指标,用木瓜蛋白酶制备南瓜籽仁蛋白多肽。通过正交实验优化酶解条件,确定其最佳酶解工艺为:底物浓度1%、加酶量4%、酶解温度60℃、酶解时间4h,经验证实验所得南瓜籽多肽酶解液的酸溶性氮得率为43.52%±0.39%,其多肽的抗氧化活性可达到VC的20%以上。