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.     

酶解法制备核桃谷蛋白-1 ACE抑制肽的工艺优化

为获得优质的核桃蛋白血管紧张素转化酶(ACE)抑制肽的制备原料及优化其制备工艺,采用连续提取法从脱脂核桃粕中依次分离出清蛋白、球蛋白、醇溶蛋白、谷蛋白-1和谷蛋白-2 5种组分蛋白,测定5种组分蛋白的占比及ACE抑制率,以ACE抑制率最大的组分蛋白作为原料采用酶解法制备ACE抑制肽,在筛选出最适酶解用酶基础上,采用单因素试验与响应面试验优化酶解制备核桃蛋白ACE抑制肽的工艺。结果表明：5种组分蛋白中谷蛋白-1占比仅次于谷蛋白-2,且其ACE抑制率最高,以核桃谷蛋白-1为原料,在筛选出胃蛋白酶作为酶解用酶基础上,经工艺优化得到最优的酶解法制备核桃谷蛋白-1 ACE抑制肽的工艺条件为酶解温度46℃、酶解时间6 h、酶用量4.2%(以底物质量计)、酶解pH 1.6,在该条件下所得核桃谷蛋白-1酶解液的ACE抑制率为(50.08±2.34)%。因此,核桃谷蛋白-1经胃蛋白酶酶解可生产ACE抑制活性较高的核桃多肽。     

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。     

高效产锰氧化酶鞘细菌的分离鉴定及酶学特性

从活性污泥中分离筛选锰氧化酶鞘细菌及研究酶学特性,利用尿素富集培养基、CGY平板及摇瓶复筛从水样中分离产锰氧化酶菌株,通过菌落形态观察、生理生化实验和16S rRNA基因序列对菌株进行鉴定,并对其发酵条件及所产锰氧化酶的酶学性质进行研究。结果表明,分离筛选到一株高产锰氧化酶的菌株FM-3,被鉴定为鞘细菌属纤发菌（Leptothrix）。该菌株的最适产酶条件为甘油添加量0.8%,蛋白胨添加量0.15 g/L,吐温-80添加量0.2%,30 ℃ 150 r/min振荡培养72 h,此时发酵液的锰氧化酶活性可达2 977.44 IU/mL。该菌株产锰氧化酶的最适温度和pH分别为40 ℃和7.0,添加一定量的Mg2+对锰氧化酶活性有增强作用,Ca2+、Cu2+对锰氧化酶影响不大,Pb2+、Zn2+、Ag+对锰氧化酶具有一定的抑制作用。     

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。      

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。     

一株高产果胶酶的真菌分离鉴定及酶学特性

以果胶为唯一碳源,对采集自湖南武冈市脐橙果园土样（pH 5.5～6.5）进行富集筛选,分离得到一株产果胶酶的菌株,编号为BM201。根据形态学观察、ITS rDNA及28S rDNA D1/D2序列同源性比对及系统进化分析对菌株进行多相鉴定,初步将该菌株鉴定为尖孢镰刀菌（Fusarium oxysporum）BM201。该菌株发酵产酶条件为30 ℃、160 r/min摇床发酵培养3 d,在该条件下获得的果胶酶酶活为421 U/L。该酶最适反应温度为50 ℃,在30～50 ℃条件下处理60 min,该酶的相对酶活在80%以上,表明该酶的热稳定性较好;该酶的最适作用pH值为5.0,在pH 4.0～7.0下处理60 min,该酶的相对酶活在50%以上,表明该酶在酸性条件下的稳定性较好。     

高温碱性果胶酶产生菌的筛选及部分酶学特性研究

从高温堆肥中分离到一株高产碱性果胶酶的菌株DG-3,经形态、生理生化及16SrDNA序列分析将其鉴定为Bacillus licheniformis DG-3,对DG-3菌株所产碱性果胶酶的初步研究表明,该酶适宜温度范围60～70℃,适宜pH为9.8～10.5。温度稳定性实验表明,碱性果胶酶具有较高的耐热性;6mmol/L的Ca2+对碱性果胶酶具有显著的激活作用。在最适作用条件下,发酵液中碱性果胶酶的酶活达42U/mL,在国内尚未见报道。      

Isolation and characterisation of a novel antibacterial peptide from bovine αS1-casein

Bovine casein was hydrolysed with a range of proteolytic enzymes including pepsin, trypsin, α-chymotrypsin and β-chymotrypsin, and assessed for antibacterial activity. The pepsin digest of bovine casein, which showed antibacterial activity, was fractionated using reverse phase high performance liquid chromatography and the antibacterial peptides isolated were characterised using electrospray ionisation mass spectrometry. Two antibacterial peptides were identified, a novel peptide (Cp1) which corresponded to residues 99–109 of bovine α_S1-casein and a previously reported peptide (Cp2) which corresponded to residues 183–207 of bovine α_S2-casein. The minimum inhibitory concentration (MIC) of Cp1 and Cp2 were determined against a range of bacterial cultures. Cp1 exhibited an MIC of 125 μg mL⁻¹ against all Gram-positive bacteria tested, and MIC ranging between 125 and >1000 μg mL⁻¹ against the Gram-negative bacteria tested. Cp2 was generally far more potent against the Gram-positive bacteria, exhibiting an MIC of 21 μg mL⁻¹, compared to MICs ranging from 332 to >664 μg mL⁻¹ against most of the Gram-negative bacteria tested.     

蚕蛹蛋白肽酶水解条件优化

为了降低蚕蛹过敏风险,提高蚕蛹优质蛋白利用价值并制备免疫活性肽,对蚕蛹蛋白进行了酶解优化。本文选用菠萝蛋白酶、碱性蛋白酶、中性蛋白酶、风味蛋白酶和木瓜蛋白酶这5种酶对脱脂蚕蛹蛋白进行酶解。通过对小鼠脾细胞增殖和水解度的检测,选取最佳蛋白酶。在此基础上对最佳蛋白酶的四个单因素（酶和底物量,初始p H,水解温度和底物浓度）进行优化,从而确定最佳的酶解条件。结果表明,5种蛋白酶中碱性蛋白酶对脱脂蚕蛹蛋白的酶解效果最好。对碱性蛋白酶进一步优化,通过Box-Behnken实验和回归分析获得碱性蛋白酶酶解脱脂蚕蛹蛋白的最佳条件为加酶量=6.0%,温度=55℃,酶解时间=2.00 h,p H=8.0,水∶底物=20∶1,测得的水解度为19.96%±1.02%,免疫活性OD490 nm为0.2512±0.0125。