首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
The effect of high hydrostatic pressure (HHP) on the susceptibility of potato starch (25%, w/v) suspended in water to degradation by exposure to bacterial α-amylase (0.02%, 0.04% and 0.06%, w/v) for 40 min at 25°C was investigated. Significant differences (p < 0.05) in the structure, morphology and physicochemical properties were observed. HHP-treated potato starch (PS) exposed to α-amylase (0.06%, w/v) showed a significantly greater degree of hydrolysis and amount of reducing sugar released compared to α-amylase at a concentration of 0.04% (w/v) or 0.02% (w/v). Native PS (NPS) granules have a spherical and elliptical form with a smooth surface, whereas the hydrolyzed NPS (hNPS) and hydrolyzed HHP-treated PS granules showed irregular and ruptured forms with several cracks and holes on the surface. Hydrolysis of HHP-treated PS by α-amylase could decrease the average granule size significantly (p <0.05) from 29.43 to 20.03 μm. Swelling power decreased and solubility increased with increasing enzyme concentration and increasing pressure from 200–600 MPa, with the exception of the solubility of HHP-treated PS at 600 MPa (HHP600 PS). Fourier transform infrared spectroscopy (FTIR) showed extensive degradation of the starch in both the ordered and the amorphous structure, especially in hydrolyzed HHP600 PS. The B-type of hydrolyzed HHP600 PS with α-amylase at a concentration 0.06% (w/v) changed to a B+V type with an additional peak at 2θ = 19.36°. The HHP600 starch with 0.06% (w/v) α-amylase displayed the lowest value of T o (onset temperature), T c (conclusion temperature) and ΔH gel (enthalpies of gelatinization). These results indicate the pre-HHP treatment of NPS leads to increased susceptibility of the granules to enzymatic degradation and eventually changes of both the amorphous and the crystalline structures.  相似文献   

2.
The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation.  相似文献   

3.
Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.  相似文献   

4.
Sun Z  Henson CA 《Plant physiology》1990,94(1):320-327
The initial hydrolysis of native (unboiled) starch granules in germinating cereal kernels is considered to be due to α-amylases. We report that barley (Hordeum vulgare L.) seed α-glucosidases (EC 3.2.1.20) can hydrolyze native starch granules isolated from barley kernels and can do so at rates comparable to those of the predominant α-amylase isozymes. Two α-glucosidase charge isoforms were used individually and in combination with purified barley α-amylases to study in vitro starch digestion. Dramatic synergism, as much as 10.7-fold, of native starch granule hydrolysis, as determined by reducing sugar production, occurred when high pl α-glucosidase was combined with either high or low pl α-amylase. Synergism was also found when low pl α-glucosidase was combined with α-amylases. Scanning electron micrographs revealed that starch granule degradation by α-amylases alone occurred specifically at the equatorial grooves of lenticular granules. Granules hydrolyzed by combinations of α-glucosidases and α-amylases exhibited larger and more numerous holes on granule surfaces than did those granules attacked by α-amylase alone. As the presence of α-glucosidases resulted in more areas being susceptible to hydrolysis, we propose that this synergism is due, in part, to the ability of the α-glucosidases to hydrolyze glucosidic bonds other than α-1,4- and α-1,6- that are present at the granule surface, thereby eliminating bonds which were barriers to hydrolysis by α-amylases. Since both α-glucosidase and α-amylase are synthesized in aleurone cells during germination and secreted to the endosperm, the synergism documented here may function in vivo as well as in vitro.  相似文献   

5.
Germinating rice seeds were fed with [35S]methionine and the incorporation of 35S into β-amylase demonstrated by quantitative immunoprecipitation using rabbit anti-β-amylase immunoglobulin G fraction. Separation of the antigen-antibody complex by Na-dodecylsulfate gel electrophoresis and subsequent radioautography clearly showed the radioactive labeling of the β-amylase molecule. The specific radioactivity of β-amylase derived from scutellum by immunoprecipitation was significantly greater than that of the endosperm. The results strongly indicate that at the onset of germination of rice seeds β-amylase is synthesized de novo in the scutellum and that in later stages there occurs activation of an inactive, latent form of the enzyme associated with starch granules in the endosperm. In later stages of germination this activated form of the enzyme becomes dominant.  相似文献   

6.
Features of programmed cell death (PCD) and dynamic changes of starch accumulation in developing pericarp cells of wheat (Triticum aestivum L.) were observed and analyzed by periodic acid–Schiff/toluidine blue O double staining, fluorescence staining, terminal deoxynucleotidyl transferase-mediated fluorescein deoxyuridine triphosphate nick-end labeling (TUNEL) and transmission electron microscopy. The results showed that cellular organelles were orderly disintegrated. TUNEL-positive nuclei were detected at 0 day after flowering (DAF), whereas nuclei showed significant features of degradation at 2 DAF, such as chromatin condensation, nuclei condensation, and nuclei deformation. Then, heterochromatin gradually disappeared and the cellular nucleus was completely degraded. The mitochondria degradation and vacuolation also were detected at 15 DAF. These results indicated that the development of pericarp cells was a typical process of PCD. However, the PCD in pericarp cells had their own characteristics: PCD started early and lasted for a considerable time. In the delayed process of PCD, starch granules were synthesized, deposited, and degraded temporarily in amyloplasts or chloroplasts. The delay of PCD in pericarp cells may be due to sufficient photosynthetic assimilates and energy supply. Besides, normal mitochondria were required for pericarp cells to survive. Pericarp cells contained only compound starch granules. Starch was massively synthesized from 0 to 11 DAF, but it was rapidly degraded after 11 DAF. Therefore, apart from protection, pericarp cells played essential roles in starch synthesis, storage, and degradation, as well as nutrient transportation.  相似文献   

7.
Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher α-amylaseactivity than the parent strain under submerged culture at 30°C for 24 h. About 70% of the total α-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable α-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable α-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal α-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.  相似文献   

8.
Graham Dunn 《Phytochemistry》1974,13(8):1341-1346
An in vitro system for the breakdown of starch granules by mixtures of α- and β-amylase is developed and discussed with reference to information concerning the degradation of starch in vivo. β-Amylase has no action on starch granules and has very little effect on the rate of starch granule digestion by α-amylase. It does, however, affect the product distribution in an α-amylase digest and is considered to attack dextrin intermediates produced by the action of α-amylase on the starch granules.  相似文献   

9.
Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear α-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases α-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism.  相似文献   

10.

Key message

The cytological and physiological features of developing wheat pericarp were clearly characterized in this report. Our results may be helpful to articulate the functions of pericarp during the seed development.

Abstract

Although wheat pericarp has been well studied, knowledge of the sequence of events in the process of pericarp development is incomplete. In the present study, the structural development process of wheat (Triticum aestivum L.) pericarp was investigated in detail using resin microtomy and microscopy. Chlorophyll contents, and photosynthetic and respiratory rates, in pericarp were determined using spectrophotometer and an oxygen electrode, respectively. Mineral nutrient contents were also determined using scanning electron microscopy. The main results are as follows: (1) based on the structures and physiological characteristics observed, the developmental process of pericarp was divided into four stages, growth, formation, extinction and maturation stages, pericarp exhibited specific features at each stage. (2) Pericarp development differed in different parts, or varieties, of wheat. The dorsal pericarp had fewer starch grains and slower rates of apoptosis than the abdominal mesocarp. The cross cells in dorsal pericarp had an irregular outline. When compared with soft wheat cv. Yangmai 11, mesocarp cells in hard wheat cv. Xumai 30 had more starch grains, larger cell size and longer development duration. (3) The chlorophyll content, photosynthesis rate and respiratory rate in pericarp increased gradually, reaching a maximum about 16 days after anthesis, and later decreased continually. The photosynthetic rate in pericarp was lower than the respiration rate. (4) The contents of mineral elements in pericarp, such as calcium, zinc, iron and potassium were higher than those in the inner endosperm. The data indicate that wheat pericarp has many functions, e.g. protection, photosynthesis, mineral accumulation, synthesis and degradation of starch.  相似文献   

11.
α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp666), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5–8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys499 and Cys587 is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.  相似文献   

12.
An α-amylase gene (AMY) was cloned from Schwanniomyces occidentalis CCRC 21164 into Saccharomyces cerevisiae AH22 by inserting Sau3AI-generated DNA fragments into the BamHI site of YEp16. The 5-kilobase insert was shown to direct the synthesis of α-amylase. After subclones containing various lengths of restricted fragments were screened, a 3.4-kilobase fragment of the donor strain DNA was found to be sufficient for α-amylase synthesis. The concentration of α-amylase in culture broth produced by the S. cerevisiae transformants was about 1.5 times higher than that of the gene donor strain. The secreted α-amylase was shown to be indistinguishable from that of Schwanniomyces occidentalis on the basis of molecular weight and enzyme properties.  相似文献   

13.
Naoko Fujita  Tomoaki Taira 《Planta》1998,207(1):125-132
A novel 56-kDa granule-bound starch synthase (GBSS; NDPglucose-starch glucosyltransferase, EC 2.4.1.21) responsible for amylose synthesis was found in the pericarps, aleurone layers and embryos of immature diploid wheat (Triticum monococcum L.). The GBSS and other proteins bound to starch granules of various tissues of immature normal and waxy diploid wheat seeds were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and their activities were examined. In the waxy mutant, the waxy protein (59.5 kDa, GBSSI) was absent, but amylose and GBSS activity were evident in all tissues except the endosperm. Of the proteins bound to starch granules, only the 56-kDa protein was associated with the presence of amylose and GBSS activities in the pericarps, aleurone layers and embryos. Mutations at the waxy locus did not affect the 56-kDa protein in these tissues. Changes in the amount of 56-kDa protein during the course of seed development, and the distribution of the 56-kDa protein in each tissue of immature seeds were quite different from those of the waxy protein. On the other hand, the N-terminal amino acid sequence of the 56-kDa protein had a 40–50% similarity to GBSSI of some other plant species and was antigenically related to the waxy protein. These results strongly suggest that the 56-kDa protein in diploid wheat is a GBSSI class enzyme and, hence, an isoform of the waxy protein. The waxy protein and 56-kDa protein, however, are expressed in different seed tissues and at different stages of seed development. Received: 15 May 1998 / Accepted: 18 June 1998  相似文献   

14.
α-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55°C and 5.5, respectively, and the apparent Km for starch was 0.8 mg ml−1. The products of α-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of α-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial α-amylase. Activities of α-amylase up to 4.4 U ml−1 (1 U represents 1 μmol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml−1 in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml−1), α-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed α-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of α-amylase (initial concentration, 2.5 mg ml−1), they were found to be less effective than starch, but maltose was almost as effective. The fungal α-amylase was found to be stable at 60°C in the presence of low concentrations of starch (≤5%), suggesting that it may be suitable for industrial application.  相似文献   

15.
We have examined the occurrence/disappearance, tissue location, and posttranslational modification of β-amylase proteins in rye (Secale cereale L.) kernels at three physiological stages (development, maturity, germination) with a normal inbred line and a mutant line exhibiting a high but incomplete β-amylase deficiency. This deficiency corresponds to a lack of accumulation of β-amylase activity in the endosperm and does not affect the level of activity in the outer pericarp and green tissues as compared to the normal line. Two antigenically related but distinct β-amylases (I and II) were detected in the normal line (II being the major constituent) and only one (I) in the mutant line. I and II display very similar electrophoretic polymorphism. In both lines, I appears to be ubiquitous, although it disappears from the outer pericarp during ripening. Antigen II was present only in the normal line and appears to be specific for the endosperm and perhaps for the maternal green tissues of the seed. Posttranslational modifications occurring during germination, which are mimicked by the action of papain, affect II but not I. The two groups of β-amylases are discussed in relation to recent reports indicating the presence of two types of β-amylase with different functions and gene loci in barley and wheat.  相似文献   

16.
Decline of proteasome activity has been reported in mammals, flies and yeasts during aging. In the yeast Saccharomyces cerevisiae, the reduction of proteolysis in stationary phase is correlated with disassembly of the 26S proteasomes into their 20S and 19S subcomplexes. However a recent report showed that upon entry into the stationary phase, proteasome subunits massively re-localize from the nucleus into mobile cytoplasmic structures called proteasome storage granules (PSGs). Whether proteasome subunits in PSG are assembled into active complexes remains an open question that we addressed in the present study. We showed that a particular mutant of the RPN11 gene (rpn11-m1), encoding a proteasome lid subunit already known to exhibit proteasome assembly/stability defect in vitro, is unable to form PSGs and displays a reduced viability in stationary phase. Full restoration of long-term survival and PSG formation in rpn11-m1 cells can be achieved by the expression in trans of the last 45 amino acids of the C-terminal domain of Rpn11, which was moreover found to co-localize with PSGs. In addition, another rpn11 mutant leading to seven amino acids change in the Rpn11 C-terminal domain, which exhibits assembled-26S proteasomes, is able to form PSGs but with a delay compared to the wild type situation. Altogether, our findings indicate that PSGs are formed of fully assembled 26S proteasomes and suggest a critical role for the Rpn11 protein in this process.  相似文献   

17.
The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.  相似文献   

18.
Kim HR  Im YK  Ko HM  Chin JE  Kim IC  Lee HB  Bai S 《Biotechnology letters》2011,33(8):1643-1648
Industrial strains of a polyploid, distiller’s Saccharomyces cerevisiae that produces glucoamylase and α-amylase was used for the direct fermentation of raw starch to ethanol. Strains contained either Aspergillus awamori glucoamylase gene (GA1), Debaryomyces occidentalis glucoamylase gene (GAM1) or D. occidentalis α-amylase gene (AMY), singly or in combination, integrated into their chromosomes. The strain expressing both GA1 and AMY generated 10.3% (v/v) ethanol (80.9 g l−1) from 20% (w/v) raw corn starch after 6 days of fermentation, and decreased the raw starch content to 21% of the initial concentration.  相似文献   

19.
Mutants of Arabidopsis thaliana (L.) Heynh. with altered regulation of starch degradation were identified by screening for plants that retained high levels of leaf starch after a period of extended darkness. The mutant phenotype was also expressed in seeds, flowers, and roots, indicating that the same pathway of starch degradation is used in these tissues. In many respects, the physiological consequences of the mutations were equivalent to the effects observed in previously characterized mutants of Arabidopsis that are unable to synthesize starch. One mutant line, which was characterized in detail, had normal levels of activity of the starch degradative enzymes α-amylase, β-amylase, phosphorylase, D-enzyme, and debranching enzyme. Thus, it was not possible to establish a biochemical basis for the phenotype, which was due to a recessive mutation at a locus designated sex1 at position 12.2 on chromosome 1. This raises the possibility that hitherto unidentified factors, altered by the mutation, play a key role in regulating or catalyzing starch degradation.  相似文献   

20.
Lim MH  Lee OH  Chin JE  Ko HM  Kim IC  Lee HB  Im SY  Bai S 《Biotechnology letters》2008,30(12):2125-2130
Phytase liberates inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) which is the major phosphate reserve in plant-derived foods and feeds. An industrial strain of Saccharomyces cerevisiae expressing the Debaryomyces castellii phytase gene (phytDc) and D. occidentalis α-amylase gene (AMY) was developed. The phytDc and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae by using the δ-integration system, which contains DNA derived exclusively from yeast. The recombinant industrial strain secreted both phytase and α-amylase for the efficient degradation of phytic acid and starch as main components of plant seeds. This new strain hydrolyzed 90% of 0.5% (w/v) sodium phytate within 5 days of growth and utilized 100% of 2% (w/v) starch within 48 h simultaneously.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号