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1.
A glutenin hydrolysing enzyme (bug proteinase), present in New Zealand wheat damaged by Nysius huttoni, was purified 50000-fold by anion exchange, hydrophobic interaction, immobilized metal ion affinity and gel filtration chromatography. The enzyme had an apparent Mr of 14·1k as determined by gel filtration chromatography. SDS-PAGE showed a major protein band of Mr 30k and six minor bands of Mr 13·2-28·5k, none of which was a glycoprotein. Isoelectric focusing revealed two major enzyme active bands (pI 9·6 and 9·2) and three minor activity bands (pI 9·9, 8·8 and 8·2). IEF showed no protein contaminants in the most purified sample. The enzymes had optimum activity at pH 8·9 and 45°C. The activity was stable in the pH range 4·5-11 and at 50°C for 20 min at pH 8·9. The bug proteinase was shown to be a serine proteinase by inhibition with phenylmethylsulphonyl fluoride and potato proteinase inhibitors (POT-IC and POT-ID). Thirty other proteinaceous serine proteinase inhibitors did not inhibit the enzyme. Bread baking with partially purified enzyme produced loaves with the poor quality characteristics of loaves made with bug-damage wheat.  相似文献   

2.
Five amylose preparations of different origins (cassava, potato, smooth seeded pea, wheat and maize), obtained from native starch granules by thymol complexation, were ultracentrifuged as the final decontamination step to remove a high molecular weight population contaminating the amylose solutions. The efficiency of this ultracentrifugation procedure was assessed by dynamic light scattering (DLS): decreases in apparent hydrodynamic radii,R?H, from 46·1–72·6 nm before ultracentrifugation to 16·1–29·3 nm after were observed. Amylose solutions were then characterised by size exclusion chromatography coupled on-line to multi-angle laser light scattering (SEC–MALLS). Under these conditionsM?wof cassava, potato, smooth seeded pea, wheat and maize amyloses were, respectively, 1·05×106, 7×105, 6·2×105, 5·1×105and 3·4×105g/mol. Using a specific optimisation algorithm, experimental molecular weight distributions (MWD) were fitted by two mathematical models of ‘Most Probable’ distribution and ‘log-normal’ distribution. The best fit was obtained for the second model, but fittedM?wwere higher than experimentalM?w. When a ‘Most Probable’ model was used, the fittedM?wwere consistent with experimentalM?wbut with a lower quality of fit. Exponentscin the power lawRG=KMcwere between 0·6 and 0·7, indicating an extended linear random coil in the range of MW analysed (3×105–9×106g/mol). This procedure was also applied to the characterisation of different commercial amylose products.  相似文献   

3.
《Plant Production Science》2013,16(3):314-321
Abstract

Kunitz soybean trypsin inhibitor (KSTI) is hydrolyzed during seed germination to yield amino acids needed to support initial seedling growth. The type of KSTI from Glycine max (L.) Merrill cv. Toyokomachi is KSTI-Ti b. The KSTI-Ti b from 4-day-old post-germination cotyledons (KSTI-Ti b’) has 3 or 4 amino acid residues cleaved off at the C-terminus. This KSTI modification is important to understand the mechanism of degradation in seed reserve proteins by proteases. Protease K1 also cleaves amino acid residues at the C-terminus of KSTI but it removes 5 amino acid residues. Therefore, we presumed the KSTI-Ti b’ was produced by a protease other than protease K1. In this study, the protease T1 responsible for cleavage of KSTI-Ti b at the C-terminus was purified. The enzyme was estimated to have a molecular mass of 33 kDa from its mobility on SDS-PAGE gels. The N-terminal amino acid sequence of the purified protease T1 corresponded to amino acids Phe-73 to Phe-92 of both thiol protease isoforms A and B from the soybean leaf, and shared 83% identity with the partial amino acid sequence of the membrane-associated cysteine protease from mung bean seedlings, a protease known to perform post-translational cleavage of C-terminal peptides of target proteins. Finally, this enzyme was shown to convert KSTI-Ti b to KSTI-Ti b’.  相似文献   

4.
Dough strength, as determined by Alveograph W, was studied in Italian bread wheat cultivars grown at several locations over 21 years of testing. Broad sense heritability of Alveograph W was found to range between 0·40 and 0·82, variation in this parameter being affected by genotype×year interactions. Standardised Alveograph W values (Wst) across 40 environments (location–year combinations) were computed for 54 cultivars with reference to the long-term control (cv. Mec), and a quality score based on the Wst value was given to each gliadin allele occurring in at least four of the 54 cultivars analysed. Significant differences in Wst values were observed at theGli-B1,Gli-B2andGli-A2loci. AllelesGli-B1b,Gli-B2candGli-A2bwere significantly related to high gluten strength. An overall quality score based on allelic composition at these loci explained 41% of the observed Wst variation in the 54 cultivars used for the score definition, and 23% to 48% of W value variation in three sets of independent data. The combination of this score with a quality score based onGlu-1alleles coding for highMrglutenin subunits could explain, on average, about 50% of gluten strength variation.  相似文献   

5.
An enzyme-linked immunosorbent assay (ELISA) was used to determine the bifunctional alpha-amylase/subtilisin inhibitor (BASI) content of barley grain from 11 cultivars grown in six diverse locations in Australia. The inhibitor ranged from 119 to 254 μg/g in 57 barley samples. Genotype had a significant (P<0·05) effect on BASI content but there was no effect due to environment. Total protein varied independently of BASI and was influenced by environment and genotype. BASI content was higher (P<0·05) in malting barley than in feed barley and was correlated positively (r=0·29;P<0·05) with alpha-amylase activity in corresponding malts. The ELISA used monoclonal and polyclonal antibodies raised against purified BASI. In immunoblot analysis the monoclonal antibody showed high specificity for the inhibitor in barley and also detected the inhibitor in wheat. Low levels of inhibitor (mean 3·2 μg/g) were found in 12 Australian wheat cultivars using the ELISA developed for barley. The assay had a linear working range of 5–50 ng/mL with a detection limit of 2 ng/mL. Reproducibility between assays was good (CV=4·9%) but mean recoveries were high, ranging from 116–129% when purified inhibitor was added to barley extracts. The ELISA may have useful applications in brewing research and barley breeding programmes.  相似文献   

6.
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7.
Thermostability assays in conjunction with IEF and molecular mapping were used to identify three beta-amylase alleles (Bmyl-Sd1, -Sd2L, -Sd2H) in cultivated barley and an additional allele (Bmy1-Sd3) in an accession of wild barley Hordeum vulgare ssp. spontaneum. The four forms of beta-amylase exhibit different rates of thermal inactivation in barley extracts. This variation was shown to persist after the proteolytic processing of the enzyme that occurs during germination. Three forms of beta-amylase representing the range of thermostabilities were purified and shown to have T50 temperatures of 56·8°C for the Sd2L enzyme, 58·5°C for the Sd1 enzyme, and 60·8°C for the Sd3 beta-amylase from wild barley. Analysis of the relationship between beta-amylase thermostability and fermentability, i.e. the yield of fermentable sugars obtained from starch hydrolysis during brewing in 42 commercial malt samples suggests that increased thermostability results in more efficient starch degradation. Screening for specific beta-amylase alleles is proposed as a method for increasing fermentability in malting barley.  相似文献   

8.
Seed nitrogen content, protein composition and free amino acid content were determined in rice mutant lines with altered storage protein composition and their parental cultivars. The Lgc1 gene, which causes low glutelin content and high content of 13 kDa prolamin and 26 kDa globulin, did not affect grain weight, nitrogen content, or free amino acid content. The glb1 gene, which lacks the 26 kDa globulin, did not affect grain weight or nitrogen content, but in mutant lines with glb1 gene the content of the major free amino acids was significantly (1.3–1.5 times) higher than those of their parental cultivars. These results suggest that absence of 26 kDa globulin is accompanied by an accumulation of a high level of free amino acids in rice grains.  相似文献   

9.
Three hundred and eighty four immobilised overlapping nonapeptides, corresponding to the full amino acid sequences of three high Mr subunits of glutenin from bread wheat (Triticum aestivum) grain, were used to determine the linear epitopes recognised by four monoclonal antibodies. These antibodies were selected on the basis of significant and positive correlations between their binding to wheat flour extracts in a two-site ('sandwich') enzyme immunoassay and rheological measures of dough strength, an important aspect of bread wheat quality. The antibodies did not bind to a single, specific sequence but bound a series of related peptides in each high Mr glutenin subunit examined. The sequences recognised were not identical for the four antibodies, but in each case were in the central repeating domain of the high Mr glutenin subunits, and usually comprised regions that overlapped the degenerate repeat nonamer and hexamer sequences. High Mr glutenin subunits that have been associated with greater dough strength, such as the D-genome allelic products 1Dx5 and 1Dy10, displayed an increased number of the epitope sequences. The location of the epitopes in sequences of overlapping β-turns in the repetitive region supports the hypothesis that dough elasticity arises partly from β-turn-forming secondary structure in the repeat regions of the Mr glutenin subunits. Additional β-turn within high Mr subunits may extend their structure to allow increased interaction between the glutenin subunits and with the other proteins of the gluten complex, thus improving dough strength.  相似文献   

10.
The x- and y-type high molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe investigations on the HMW glutenin subunits from several Pseudoroegneria accessions. The electrophoretic mobilities of the HMW glutenin subunits from Pd. stipifolia, Pd. tauri and Pd. strigosa were much faster than those of orthologous wheat subunits, indicating that their protein size may be smaller than that of wheat subunits. The coding sequence of the Glu-1St1 subunit (encoded by the Pseudoroegneria stipifolia accession PI325181) was isolated, and found to represent the native open reading frame (ORF) by in vitro expression. The deduced amino acid sequence of Glu-1St1 matched with that determined from the native subunit by mass spectrometric analysis. The domain organization in Glu-1St1 showed high similarity with that of typical HMW glutenin subunits. However, Glu-1St1 exhibited several distinct characteristics. First, the length of its repetitive domain was substantially smaller than that of conventional subunits, which explains its much faster electrophoretic mobility in SDS-PAGE. Second, although the N-terminal domain of Glu-1St1 resembled that of y-type subunit, its C-terminal domain was more similar to that of x-type subunit. Third, the N- and C-terminal domains of Glu-1St1 shared conserved features with those of barley D-hordein, but the repeat motifs and the organization of its repetitive domain were more similar to those of HMW glutenin subunits than to D-hordein. We conclude that Glu-1St1 is a novel variant of HMW glutenin subunits. The analysis of Glu-1St1 may provide new insight into the evolution of HMW glutenin subunits in Triticeae species.  相似文献   

11.
Silkworm has great potential as production system of recombinant mammalian proteins. When the protein products are used for medical purpose, it is required to reduce the risk of an allergy, the content of core alpha 1,3-fucosyl residue attached to the N-glycan of proteins, for example. We isolated the gene of an enzyme responsible for the transfer of core alpha 1,3-fucosyl residue, core alpha 1,3-fucosyltransferase (Fuc-T C3), from silkworm. A candidate cDNA for silkworm Fuc-T C3 was isolated as a homolog of the fruit fly enzyme gene fucTA. The gene was located on chromosome 7 of the silkworm genome and was composed of seven exons, which spanned approximately 10 kb on the genome. The coding region of the gene was 1,350 bp and encoded a 450-amino acid protein with a molecular mass of 52.2 kDa. Deduced amino acid sequence of the coding region showed one transmembrane domain in its N-terminal and typical motifs common to fucosyltransferases including Fuc-T C3s of other organisms in its C-terminal. The extract of CHO cells transfected with the cDNA showed Fuc-T C3 activity using GDP-fucose and DABS-GnGn peptide as substrates. These results showed this cDNA clone actually encodes silkworm Fuc-T C3.  相似文献   

12.
硒是人类不可缺少的重要微量元素之一,人类必须补充足够的硒才能保证身体健康。同型半胱氨酸甲基转移酶(homocysteine-S-methyltransferase,HMT)基因与植物富硒关键酶硒代半胱氨酸甲基转移酶(selenocysteine methyltransferase,SMT)基因同源。为了发掘并利用麦类作物中的富硒关键酶基因,基于NCBI已公布的节节麦(Aegilops tauschii Coss.)同型半胱氨酸甲基转移酶1基因(AetHMT1)的序列设计HMT1基因的特异性引物H1-F/H1-R,克隆四倍体小麦Langdon HMT1的开放阅读框(open reading frame,ORF)后进行序列分析,并通过原核表达验证该基因。结果表明,利用H1-F/H1-R从四倍体小麦Langdon(LDN)克隆出两条长度均为975bp的序列,分别命名为LDNHMT1-1和LDNHMT1-2。LDNHMT1-1、LDNHMT1-2与AetHMT1基因一样,均编码342个氨基酸,分子量大小分别为35.19kDa和35.18kDa。通过氨基酸序列比对和进化树分析发现,LDNHMT1-1、LDNHMT1-2与其他HMT、SMT有较高的相似性。构建原核表达载体,并通过SDS-PAGE鉴定,成功获得LDNHMT1-1、LDNHMT1-2的原核表达菌株,且原核表达的蛋白质与预测分子量大小一致。  相似文献   

13.
Using a PCR-based strategy, four new 75K γ-secalin genes were isolated from Secale cereale, Secale vavilovii, Secale sylvestre, and Secale strictum in genus Secale (rye). Based on amino acid sequences, the primary structure of the 75K γ-secalin subunits was demonstrated, which was composed of four main structural regions: (a) a conservative 19 amino acids signal peptide, (b) a steady short N-terminal region of 12 amino acids containing a cysteine residue, (c) a repetitive domain, which began with the conservative tetrapeptides PQ3 and was rich in glutamine and proline. PFPQ1−2(PQQ)1−2 was the core repeat motif in the repetitive region. Besides amino acid substitutions, this region showed variations in length due to the insertion and deletion events. In the repetitive region of EF432549 (Secale strictum), there were two octapeptides (PFPQQPQQ and PVPQQSQQ) insertions. On the contrary, deletion events of two residues (QT) took place in EF432546 (Secale sylvestre). Accounting for the amino acid replacement, an extra cysteine residue appeared in the repetitive region of EF432546, which did not exist in other genes, and (d) a conserved 143 amino acids C-terminal domain including eight cysteine residues. The implications of the results for quality improvement are discussed.  相似文献   

14.
The analysis of bulk seed samples of some selected Swedish cultivars showed thatBrassica campestris cultivars contain higher amounts of alanine, valine and aspartic acid than those ofB. napus, whereasB. sinapis cultivars contain higher amounts of glycine and aspartic acid and slightly more threonine and phenylalanine. The amino acid analysis was carried out by gas chromatography.
Zusammenfassung Die Analyse von Samenproben einiger selektierter Sorten zeigte, dasz Sorten vonBrassica campestris höhere Gehalte an Alanin, Valin, Asparaginsäure besitzen als Sorten vonB. napus. Dagegen verfügen Sorten vonB. sinapis über höhere Gehalte an Glycin und Asparaginsäure und etwas mehr an Threonin und Phenylalanin. Die Aminosäurenbestimmungen erfolgten gaschromatographisch.
Résumé Lés analyses d'échantillons massifs de graines de certains cultivars selectionnes en Suéde ont montré que les cultivars deBrassica campestris contiennent des taux plus élevés d'alanine, valine, acide aspartique, que ceux deB. napus, tandisque les cultivars deB. sinapis contiennent des taux plus élevés de glycocolle et d'acide aspartique, et un peu plus de thréonine et de phénylalanine. Les analyses d'aminoacides ont été faites par chromatographie en phase gazeuse.
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15.
Thirty-seven varieties of a Mediterranean durum wheat collection grown in Tunisia and Spain were analysed for their allelic composition in prolamins, as well as their protein concentration, sodium dodecyl sulphate sedimentation (SDSS) test and mixograph parameters. Genotype was a greater source of variation in all measurements than locality. Uncommon high and low molecular glutenin subunits (HMW-GS and LMW-GS) were found (V and 2•• subunits at Glu-A1, 13 + 16 at Glu-B1, 5* subunit and ax allele at Glu-A3). The rare combinations 2 + 4+14 + 18 and 8 + 9+13 + 16+18 subunits at the Glu-B3 locus were found. Glu-A3ax had a positive influence on SDSS and mixograph parameters. Of all the prolamins, those that have the B-LMW-GS composition aaa (for Glu-A3, Glu-B3 and Glu-B2 loci, respectively), when associated with the Glu-A1c and Glu-B1d gave the best semolina quality. By contrast, semolina quality is poor when this same composition is associated with the Glu-A1c and Glu-B1e and even poorer when associated with the Glu-A1c and Glu-B1f. In addition, the cultivars with B-LMW-GS allelic composition aab (for Glu-A3, Glu-B3 and Glu-B2 loci, respectively), when associated with the Glu-A1c and Glu-B1d, gave high quality, whereas when associated with the Glu-A1c and Glu-B1e or with Glu-A1o and Glu-B1f, the quality was very poor.  相似文献   

16.
The acid extract viscosities and β-glucan contents of ten two- and six-rowed barley cultivars grown at seven locations in three consecutive years in Spain were studied in the present work. The viscosities varied from 2·4 to 24·8 centistokes (cSt) and the mean value was 6·4 cSt. The average β-glucan content of barleys determined by HPLC was 3·5% with a range of 1·9–5·5%. Significant differences were found in both β-glucan content and acid extract viscosity between different cultivars, locations and years. The β-glucan contents and viscosities of winter cultivars were higher than those of spring. Cvs. Barbarrosa and Hatif de Grignon were the genotypes with the highest values for both parameters, while cv. Beka had the lowest viscosity and β-glucan content. Environmental factors influenced both parameters. The acid extract viscosities of barleys were correlated negatively with the amount of precipitation (r=−0·754;P<0·05). Barleys grown in wet and rainy areas (Girona and La Coruña) had lower viscosity values.  相似文献   

17.
A sensory profiling analysis of heat-processed oats (Avena sativaL.) was performed. The oat grains were of three varieties (Kapp, Mustang and Svea), stored at different relative humidities (30, 55 or 80%) for 3·5 or 15·5 months and heat-treated either with or without prior dehulling. The main differences in sensory attributes were related to the various thermal processes. Processed dehulled samples, particularly of theMustangvariety, had the highest intensities of oat odour, oat flavour, fresh odour and fresh flavour. Samples processed with hulls, in particular the varieties Kapp and Svea had the highest intensities of odour and flavour, rancid odour and flavour and bitterness. The sensory profile of processed oats were described partly by variations in phenolic compounds of low molecular weight, fat acidity and moisture content after processing. Twenty-nine per cent of the variation in odour and flavour attributes was explained by the 11 phenolic compounds analysed. Water content and the phenolic compoundsp-coumaric acid, vanillin,p-hydroxybenzaldehyde and coniferyl alcohol were significantly correlated (P<0·05) to high levels of rancidity, flavour intensity and bitterness and low levels of freshness, oat odour and flavour. The avenanthramides were related mainly to low levels of flavour intensity and rancid odour and flavour. Caffeic acid and fat acidity were related to low intensities of sweetness and aftertaste.  相似文献   

18.
The nutrient composition, ofColocasia esculenta flowers (CF) and leaves (CL), and the green fruits ofSolanum melongena (SM) were carried out as a means to determine their nutritional potential. Results showed that these food materials had high moisture and fiber levels which ranged between 888 and 906 g·kg–1; and 204 and 303 g·kg–1 dry weight (dw) for moisture and fiber respectively. The calorific values were between 3889 and 4001 kcals·kg–1 dw, while the total lipids ranged from 53 in CF to 71 g·kg–1 dw in SM. The leaves ofColocasia esculenta had the highest crude protein value of 307 g·kg–1 dw. The flowers had 149 g·kg–1 dw while SM had 138 g·kg–1 dw. The amino acid profile in the flowers and leaves ofColocasia esculenta in contrast to SM were balanced comparable to the reference FAO pattern. Ash values were high (ranging from 76 in SM to 98 g·kg–1 in CL) with potassium being the principal element. Iron and Zinc levels were also high especially in CF (with 303 and 82 mg·kg–1 dw respectively). These foods also contained moderate quantities of calcium, phosphorus and magnesium but were poor in manganese and copper.  相似文献   

19.
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20.
为了建立准确有效小麦HMW-GS的检测方法,提高优质小麦品种鉴定和筛选效率,以已知HMW-GS组成的16份小麦品种为对照,优化完善SDS-PAGE结合分子标记检测小麦HMW-GS的方法,并对103份宁夏小麦品种进行了验证和分析。结果表明,8.5%的分离胶可以有效区分除了 Glu-B1位点的7*、7OE与8*亚基之外的其他亚基,分辨效果优良;SDS-PAGE结合 Bx7OE、 Bx7*/Bx7和 By8基因的分子标记可以准确鉴定小麦HMW-GS。在103份宁夏小麦品种中,发现15种HMW-GS和29种组合类型;首次在该地区小麦品种的 Glu-B1位点检测出了携带7OE、7*和8*亚基的品种;1/17+18/5+10为当地小麦HMW-GS的优势亚基组合类型,占20%。自1970年至2010年,HMW-GS的优质亚基（1、17+18和5+10）的出现频率呈明显增长趋势;宁夏小麦的HMW-GS种类和优质亚基出现频率随品种更换呈增加趋势。综上所述,分离胶浓度为8.5% 的SDS-PAGE和 Bx7OE、 Bx7*/Bx7和 By8分子标记的方法可准确有效地检测小麦HMW-GS组成,此方法可用于优质小麦品种的鉴定和筛选。  相似文献   

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