Debranning of wheat prior to milling reduces xylanase but not xylanase inhibitor activities in wholemeal and flour

Debranning of wheat to remove the outer 7% of the kernel, prior to grinding or milling reduced xylanase activity in wheat wholemeal and wheat endosperm flour by up to 80 and 60%, respectively, whereas there was no significant reduction of xylanase inhibiting activity. Flours obtained after debranning and milling showed no major differences in moisture content, whereas ash content decreased and protein and arabinoxylan content decreased slightly with increasing debranning degree. Part of the xylanase activity in the flour was lost on addition of Triticum aestivum xylanase inhibitor (TAXI). Since TAXI specifically inhibits glycosyl hydrolase family 11 xylanases and since endogenous cereal xylanases belong exclusively to family 10, part of the xylanase activity in the flour is most likely of microbial origin. Debranning also significantly reduced alpha-amylase activities in wheat wholemeal and wheat flour. Debranning prior to milling can, therefore, impact on flour functionality.     

A Wheat Xylanase Inhibitor Protein (XIP-I) Accumulates in the Grain and has Homologues in Other Cereals

We have measured xylanase inhibitor activity against an Aspergillus niger xylanase in different parts of the wheat plant at different stages of development and used immunodetection to determine the spatial and temporal distribution of xylanase inhibitor protein I (XIP-I) in Triticum aestivum var. Soisson. Although xylanase inhibitor activity was detected in all parts of the wheat plant throughout development, XIP-I was located predominantly in the grain tissue, where it appears at a late stage in grain development and persists after germination, indicating that the different xylanase inhibitor proteins are under different regulatory controls. (1,4)-β-Xylanase activity was detected in wheat grains during development and post-germination. Pure XIP-I and a crude sample containing TAXI inhibitors but not XIP-I did not have the ability to inhibit this endogenous (1,4)-β-xylanase activity. Protein extracts from the seeds of various monocots were also tested for the presence of XIP-I, where it was found to be present in the grains of several wheat varieties, rye and barley, but was not detected in rice, sorghum or maize.     

Variation in the levels of the different xylanase inhibitors in grain and flour of 20 French wheat cultivars

A xylanase from Aspergillus aculeatus (glycoside hydrolase family 10), uninhibited by TAXI, and a xylanase from Bacillus subtilis (family 11), uninhibited by XIP-I, were selected to quantify the respective apparent levels of XIP-I and TAXI inhibitors, in flours and grains of 20 wheat varieties. The apparent TAXI amount ranged from 0.05 to 0.19 mg/g in flour (mean: 0.11 mg/g) and from 0.07 to 0.2 mg/g in grain (mean: 0.14 mg/g). The range observed for XIP-I was 0.12–0.6 mg/g in flour (mean 0.32 mg/g), and 0.21–0.56 mg/g in grain (mean: 0.41 mg/g). The inhibition profile of the xylanase from A. aculeatus by a crude inhibitor preparation suggested the presence of an additional component in wheat flour, responsible for an increase in inhibition.     

XIP-type endoxylanase inhibitors in different cereals

Highly pure XIP-type (for Xylanase Inhibiting Protein) endoxylanase inhibitor fractions were selectively obtained with a high yield from rye, durum wheat, barley, and maize extracts by affinity chromatography with immobilised Aspergillus niger endoxylanase Xyn1 following removal of the TAXI-type (for Triticum aestivum xylanase inhibitor) endoxylanase inhibitors by affinity chromatography with immobilised Bacillus subtilis endoxylanase XynA. No inhibitors belonging to the XIP family occur in rice, oats, and buckwheat. N-terminal amino acid sequences of the non-wheat XIP-type inhibitors were very similar or identical to those of wheat XIP-1, a chitinase homologue. The isolated inhibitors are basic, monomeric proteins of ca. 30 kDa with pI values of at least 8.5 (rye, durum wheat, and barley XIP) and ca. 7.0 (maize XIP). They are, in general, active against fungal endoxylanases and do not hydrolyse chitin. SDS–PAGE analysis and high-resolution cation exchange chromatography suggest the presence of multiple XIP-type isoinhibitors in the different cereals.     

Antibodies against wheat xylanase inhibitors as tools for the selective identification of their homologues in other cereals

Polyclonal antibodies (PAbs) were raised against wheat (Triticum aestivum L.) TAXI- and XIP-type xylanase inhibitors by rabbit immunization. A small contaminant in both antigens, not detected by SDS-PAGE and later identified through Western blot as a recently discovered third type of xylanase inhibitor from wheat, i.e. thaumatin-like xylanase inhibitor (TL-XI), led to the coproduction of PAbs against this protein in the rabbits. To obtain inhibitor-specific PAbs, the PAbs against TAXI, XIP and TL-XI were separated by affinity chromatography using immobilised recombinant and native xylanase inhibitors. The purified PAbs allowed the immunoquantification of each type of wheat xylanase inhibitor using Western blot and densitometric analysis against purified inhibitor standards. The method allowed the detection of the purified inhibitors at the 20 ng level. As the PAbs against the wheat xylanase inhibitors cross-reacted with their homologous targets from other cereals, immunoprobing allowed identification of XIP homologues in oats (Avena sativa L.) and TL-XI homologues in durum wheat (Triticum durum Desf.) and rye (Secale cereale L.).     

Isolation and characterisation of a xylanase inhibitor Xip-II gene from durum wheat

Cereals contain xylanase inhibitor proteins (XIPs) which inhibit microbial xylanases from glycoside hydrolase families 10 and 11. Here, we report for the first time the isolation and characterisation of a genomic clone containing a xylanase inhibitor gene. This gene, Xip-II, isolated from a durum wheat genomic library (Triticum durum Desf.) encodes a mature protein of 307 amino acid (aa) residues that shares highest aa sequence identity (64%) with the rice RIXI xylanase inhibitor. XIP-II showed inhibition against family 11 xylanases and no chitinase activity. In silico analysis of the 5′ promoter region of Xip-II revealed sequences with similarity to known cis regulatory elements upstream from the initiation codon. In particular, the identification of a number of cis-acting elements controlling the expression of defence and seed-specific genes supports the role for this class of inhibitors in plant defence against pathogens but also provides new clues on a potential role in plant development.     

Specificity of feruloyl esterases for water-extractable and water-unextractable feruloylated polysaccharides: influence of xylanase

Representatives of three types of feruloyl esterases were examined for their ability to release mono- and di-meric ferulic acid from water-extractable and water-unextractable cereal cell wall material, either alone or in the presence of a family 10 or family 11 xylanase. A type-C feruloyl esterase from Talaromyces stipitatus (TsFaeC) released 100% of the ferulic acid from water-extractable wheat endosperm arabinoxylan when acting in combination with a xylanase from Trichoderma longibrachiatum. The type-A esterase from Aspergillus niger, AnFaeA, was most effective in releasing ferulic acid from wheat bran and brewers' spent grain, with over 50% of the available ferulic acid being released from wheat bran in the presence of a xylanase from Bacillus subtilis. In general, family 11 xylanases were the preferred synergistic partners with feruloyl esterases for the release of ferulic acid, while family 10 xylanases were preferred for the liberation of diferulic acid, with only the 5,5′ form being released by the action of AnFaeA alone. This suggests that ferulic acid may be located in regions of low substitution on arabinoxylans while the 5,5′ diferulate moiety is located in more branched regions of the xylan chain.     

Affinity Chromatography with Immobilised Endoxylanases Separates TAXI- and XIP-type Endoxylanase Inhibitors from Wheat (Triticum aestivum L.)

An affinity-based purification procedure allowed the resolution of two distinct groups of endoxylanase inhibitors with different molecular structures and endoxylanase specificities from wheat wholemeal. The first group comprises the so-called Triticum aestivum L. Endoxylanase inhibitor (TAXI)-type proteins which are of approx. M_r 40 000 and occur in two different molecular forms. These inhibitors were removed from a concentrated cation exchange chromatography fraction from wheat wholemeal on a Bacillus subtilis endoxylanase affinity column. The second group of structurally different endoxylanase inhibitors, the so-called xylanase inhibiting protein (XIP)-type, of approx.M_r 29 000–32 000, with pI values varying between 8·8 and 9·2, was purified from the unbound fraction from the B. subtilis endoxylanase affinity column by chromatography on an Aspergillus niger endoxylanase affinity column followed by gel permeation chromatography. The XIP-type inhibitors are not active against the B. subtilis endoxylanase and were consequently not retained on the B. subtilis endoxylanase column. Further analysis of the XIP-type proteins by high-resolution cation exchange chromatography, SDS-PAGE and iso-electrofocusing, revealed several forms. They had similar endoxylanase specificities and N-terminal amino acid sequences.     

Endo-β-1,4-xylanase inhibitors in leaves and roots of germinated maize

Extracts of both leaves and roots of germinated maize were found to contain endo–β–1,4–xylanase inhibitors, previously reported only from whole maize meal. The inhibitors seem to be of the xylanase inhibitor protein (XIP) type, since they inhibit endoxylanases of families 10 and 11 and also show some other characteristics similar to XIP inhibitors described in other cereals. Inhibitors from leaves and roots appeared to be similar. A novel property of the inhibitors described in this work is their unusual thermostability. The half-life of inhibitors at pH 4.5 and 100 °C is greater than 10 h. However, the inhibitors are less thermostable at higher pH levels. Because they did not inhibit a plant endoxylanase, the inhibitors may play a role in maize defense against phytopathogens.     

木聚糖酶抑制剂基因与小麦白粉病病情指数的关系

为了解木聚糖酶抑制剂基因(TAXI型、XIP型和TLXI型)与小麦白粉病病情指数的关系,选取来源于不同麦区的192份小麦品种,以特异PCR方法检测木聚糖酶抑制剂基因,田间自然诱发鉴定白粉病病情指数。结果表明,含有TAXI-I基因的77份品种的平均病情指数(8.99)比不含有TAXI-I基因的115份品种的病情指数(11.89)低24%,差异显著;含有XIP-I基因的115份品种和不含XIP-I基因的77份品种的平均病情指数分别为10.86和10.53,差异不明显;含有TLXI基因的116份品种和不含TLXI基因的76份品种的平均病情指数分别为10.33和11.33,差异不显著。说明TAXI-I基因可能是一个有效的白粉病抗性基因。     

The combined use of hull-less barley flour and xylanase as a strategy for wheat/hull-less barley flour breads with increased arabinoxylan and (1→3,1→4)-β-D-glucan levels

Bread-making with a composite flour (CF) consisting of 60% wheat flour (WF) and 40% hull-less barley flour, increased the total and soluble (1→3,1→4)-β-D-glucan and total arabinoxylan (AX) contents of dough and bread samples, but decreased the specific bread loaf volume. A xylanase insensitive to inhibition by Triticum aestivum L. xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP), increased loaf volume by 8.8 and 20.1% for WF and CF breads, respectively. Xylanase addition not only markedly improved loaf volume of CF bread, but also increased the soluble AX content of the WF and CF dough and bread samples because of conversion of water-unextractable AX into soluble AX. The xylanase had no impact on the extractability and molecular weight of (1→3,1→4)-β-D-glucan, but (1→3,1→4)-β-D-glucan was degraded during bread-making probably because of endogenous β-glucanase activity. Taken together, the results clearly show that the combined use of hull-less barley flour and a xylanase active during bread making, lead to palatable breads with high total and soluble AX and (1→3,1→4)-β-D-glucan contents. The sum of total AX and (1→3,1→4)-β-D-glucan was 1.70% for WF bread and 3.06% for CF bread, while the sum of soluble AX and (1→3,1→4)-β-D-glucan was 0.49 and 1.41% for control WF and CF xylanase supplemented breads, respectively.     

Gene Cloning,Expression and Characterization of a Novel Xylanase from the Marine Bacterium,Glaciecola mesophila KMM241

Marine xylanases are rather less studied compared to terrestrial xylanases. In this study, a new xylanase gene, xynB, was cloned from the marine bacterium, Glaciecola mesophila KMM241, and expressed in Escherichia coli. xynB encodes a multi-domain xylanase XynB of glycoside hydrolase (GH) family 8. The recombinant XynB comprises an N-terminal domain (NTD) with unknown function and a catalytic domain, which is structurally novel among the characterized xylanases of GH family 8. XynB has the highest identity (38%) to rXyn8 among the characterized xylanases. The recombinant XynB showed maximal activity at pH 6–7 and 35 °C. It is thermolabile and salt-tolerant. XynB is an endo-xylanase that demands at least five sugar moieties for effective cleavage and to hydrolyze xylohexaose and xylopentaose into xylotetraose, xylotriose and xylobiose. NTD was expressed in Escherichia coli to analyze its function. The recombinant NTD exhibited a high binding ability to insoluble xylan and avicel and little binding ability to chitosan and chitin. Since the NTD shows no obvious homology to any known carbohydrate-binding module (CBM) sequence in public databases, XynB may contain a new type of CBM.     

Arabinoxylan and hydroxycinnamate content of wheat bran in relation to endoxylanase susceptibility

Hydroxycinnamate and protein contents and monosaccharide composition were determined for 11 industrial wheat (Triticum aestivum) brans and related to the susceptibility of their arabinoxylans (AX) to enzymatic degradation. There was significant variation in carbohydrate, A/X ratio, protein, hydroxycinnamic acid and diferulic acid (DiFA) content among the wheat brans. In addition, a strong correlation was found between AX and ferulic acid contents. Brans were extracted with water followed by a dry-thermal treatment to remove 90% starch and to inactivate endogenous enzymes and proteinaceous inhibitors. Treated brans were compared with respect to AX degradation by a family 11 xylanase. Digestion with xylanase had a strong impact on the chemical composition of the residual bran. The A/X ratio changed from 0.60 to 1.07. The solubilised AX had an A/X ratio of 0.32. The A/X ratio of enzyme-depleted bran was negatively correlated with the loss of AX. Total DiFA in destarched brans was negatively correlated with the amount of soluble AX. These relationships indicate that structural features of AX and the extent of its cross-linking in the cell walls of the bran tissues influence its susceptibility to xylanase treatment. Thus, the amount of enzyme-solubilised AX was not directly related to the content of AX in the destarched bran. However, brans differed in their susceptibility to xylanase attack.     

The combined use of hull-less barley flour and xylanase as a strategy for wheat/hull-less barley flour breads with increased arabinoxylan and (1→3,1→4)-β-D-glucan levels

Bread-making with a composite flour (CF) consisting of 60% wheat flour (WF) and 40% hull-less barley flour, increased the total and soluble (1→3,1→4)-β-D-glucan and total arabinoxylan (AX) contents of dough and bread samples, but decreased the specific bread loaf volume. A xylanase insensitive to inhibition by Triticum aestivum L. xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP), increased loaf volume by 8.8 and 20.1% for WF and CF breads, respectively. Xylanase addition not only markedly improved loaf volume of CF bread, but also increased the soluble AX content of the WF and CF dough and bread samples because of conversion of water-unextractable AX into soluble AX. The xylanase had no impact on the extractability and molecular weight of (1→3,1→4)-β-D-glucan, but (1→3,1→4)-β-D-glucan was degraded during bread-making probably because of endogenous β-glucanase activity. Taken together, the results clearly show that the combined use of hull-less barley flour and a xylanase active during bread making, lead to palatable breads with high total and soluble AX and (1→3,1→4)-β-D-glucan contents. The sum of total AX and (1→3,1→4)-β-D-glucan was 1.70% for WF bread and 3.06% for CF bread, while the sum of soluble AX and (1→3,1→4)-β-D-glucan was 0.49 and 1.41% for control WF and CF xylanase supplemented breads, respectively.     

Substrate selectivity and inhibitor sensitivity affect xylanase functionality in wheat flour gluten–starch separation

Addition of xylanases (EC 3.2.1.8) that varied in their substrate selectivities and/or wheat xylanase inhibitor sensitivities in dough batter gluten–starch separation of wheat flour showed the importance of these enzyme characteristics for their functionality in this process. A xylanase from Aspergillus aculeatus (XAA) with selectivity for hydrolysis of water extractable arabinoxylan (WE-AX), which is not inhibited by wheat flour xylanase inhibitors decreased batter viscosity and improved gluten agglomeration behaviour. In contrast, a xylanase from Bacillus subtilis (XBS_i) with selectivity for hydrolysis of water unextractable arabinoxylan (WU-AX), which is in vitro inhibited by wheat flour xylanase inhibitors had a negative effect on gluten agglomeration at low enzyme dosages. As expected, solubilisation of WU-AX increased batter viscosities. At higher dosages however, this enzyme also improved gluten agglomeration because of degradation of both WE-AX and enzymically solubilised AX. A mutated B. subtilis xylanase (XBS_ni) with selectivity for hydrolysis of WU-AX comparable to XBS_i but which is not inhibited by wheat flour xylanase inhibitors, increased the level of large gluten aggregates as well as the total gluten protein recovery, even at lower dosages. Because of its inhibitor insensitivity, the solubilisation and degradation of AX proceeded further. An XBS_ni dosage approximately 4 times lower than XBS_i performed as well as its inhibited counterpart. The degradation of both WE-AX and WU-AX by XBS_ni improved the gluten agglomeration behaviour to a larger extent than the XAA treatment which primarily resulted in hydrolysis of WE-AX. The results confirm the detrimental impact not only of WE-AX, but also of WU-AX, on gluten agglomeration in a dough batter gluten–starch separation process. At the same time, they provide firm evidence that xylanases are not only inhibited by xylanase inhibitors in vitro, but are also partly inhibited in the industrial process in which they are used.     

Xylanases from microbial origin induce syrup formation in dough

Syrup formation in refrigerated doughs is a problem since it reduces the doughs’ shelf life. Microbial exogenous xylanases associated with wheat kernels were found to play a role in this syruping phenomenon. Using xylanase-producing microorganisms isolated from wheat kernels, we investigated their potency to induce syruping in dough. Growth of the fungal xylanase producer Fusarium sp. (10² colony forming units (CFU)/g dough) and the bacterial xylanase producer Paenibacillus sp. (10⁴ CFU/g dough) in synthetic media and their respective addition to wheat dough could not bring about a significant amount of syruping. However, when these species were grown on moist wheat kernels and an extract of these kernels containing both the organisms and its xylanases was made and added to dough, intensive syruping was noted. This effect was primarily attributed to the xylanases present in the extract. These findings suggest that the involvement of xylanase-producing microorganisms in the syruping phenomenon is situated prior to harvest. Additional quantitative analyses of microbial biomass present on wheat kernels revealed that the fungi in particular could be correlated to higher microbial exogenous xylanase activities on wheat. Our results indicate that the syruping is linked to fungal xylanase production on the wheat kernels in the field.     

Interaction of water unextractable solids and xylanase with gluten protein: effect of wheat cultivar

A previously proposed explanation for the change in gluten properties on addition of pentosans to doughs was based on data for only one wheat cultivar. Using three wheat cultivars, Scipion, Soissons and Amazon, differing in technological quality from weak to strong we have obtained results that support the previous explanation. In addition to standard techniques for characterizing gluten and glutenin macropolymer (GMP) yield, composition and properties, a new technique (particle size analysis) was applied that provides further detail on GMP particle size distribution. For each of the three wheat cultivars the effect of WUS and xylanase on gluten and GMP yield, composition and properties followed the trend previously observed. However, WUS and xylanase affected gluten yield and properties more strongly for Scipion and Soissons than for Amazon. Amazon flour contains more protein and less pentosans. The analysis of GMP particles demonstrates that the volume surface average particle diameter D_3,2 of GMP particles from Amazon wheat is larger than those from Scipion and Soissons. Amazon has the ability to form larger and stickier particles. These factors may explain why the effects of pentosans and xylanase on gluten yield and properties are smaller for this wheat.     

普通小麦-滨麦及其衍生系的染色体组成分析

滨麦[Leymus mollis(Trin.)Pilger,NsNsXmXm,2n=28]属禾本科(Poaceae)小麦族(Triticeae)大麦亚族(Hordinae)赖草属(Leymus Hochst.)的一个多年生四倍体植物,蕴含着丰富的小麦改良优异基因,是小麦育种的重要三级基因资源。为给普通小麦-滨麦种质类型的创制、筛选和鉴定提供参考依据,本研究通过细胞学观察、顺序荧光原位杂交(fluorescence in situ hybridization,FISH)-基因组原位杂交(genomic in situ hybridization,GISH)及分子标记等技术分别对普通小麦7182、滨麦及其高世代衍生系的染色体组成进行了研究。采用寡核苷酸探针pSc119.2和pTa-535相结合的技术绘制出普通小麦7182所有染色体的FISH标准核型图。初步推断出滨麦的染色体核型公式2n=4x=28=22m(6sat)+6sm。同时通过FISH-GISH技术鉴定出两种类型的普通小麦-滨麦高世代衍生系M47和M39,染色体组成分别表示为2n=56=42T.a+14L.m、2n=56=44T.a+12L.m。     

小麦CO-like基因TaCO9的克隆及分析

CO蛋白(CONSTANS)是光周期途径中重要的调控因子,为了从普通小麦中进一步挖掘COlike基因,利用同源克隆的方法得到与大麦HvCO9基因同源的小麦TaCO9基因。结果表明,在冬性品种西农889中,初步克隆得到TaCO9基因的三个同源序列,分别命名为TaCO9-1、TaCO9-2、TaCO9-3。其cDNA序列全长均为870bp,开放阅读框为1 977bp,编码289个氨基酸,含有CO-like蛋白家族典型的CCT结构域,但不含B-box结构域;而在春性品种中发现TaCO9-1序列的第二外显子区域有6个碱基的插入,利用中国春缺-四体材料将该序列定位于小麦的1A染色体,命名为TaCO9-1A。系统发育分析表明,TaCO9蛋白与水稻Ghd7及大麦HvCO9位于同一分支。空间结构分析表明,其CCT结构域的NF-YA2区域较为保守,而该区域与CCAAT box互作相关。本研究克隆得到的TaCO9基因可能是小麦CO-like基因家族的新成员,与大麦HvCO9基因的结构相似,可作为新的小麦光周期候选基因加以研究利用。     

西农538 LMW-GS基因的克隆、原核表达及功能鉴定

为了探索普通小麦品种西农538的LMW-GS对面粉加工品质的影响,根据NCBI中已公布的LMW-GS基因序列,设计了一对特异性引物,从西农538基因组DNA中克隆出LMW-GS基因后,对其进行原核表达及掺粉试验。序列分析表明,克隆得到的LMW-GS基因序列(GenBank登录号为KX452081)有单一完整的开放阅读框,编码区长915bp,无内含子。同源性比对及进化树分析发现,该基因属于Glu-D3、Type V(Group 10)、m型、C组LMW-GS基因。SDS-PAGE和Western blot分析表明,该基因原核表达成功。微量掺粉试验表明,诱导表达的蛋白对小麦面粉加工品质有负效应。