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1.
The upper leaf sheath of rice (Oryza sativa L.) serves as a temporary starch sink before heading, subsequently becoming a carbon source tissue to the growing panicle
at the post-heading stage. The time of sink–source transition in upper leaf sheaths is highly correlated to the panicle exsertion.
Here, we found that the expression profiles of starch synthesis genes such as ADP-glucose pyrophosphorylase large subunit 2, granule-bound starch synthase II, soluble starch synthase I, starch branching enzyme (SBE) I, SBEIII, and SBEIV were highly correlated with starch content changes during the heading period in the second leaf sheath below the flag leaf.
In addition, the α-amylase2A and β-amylase were considered as major genes that were in charge of starch degradation at the post-heading period. Of the five sucrose
transporter (OsSUT) genes, OsSUT1 and OsSUT4 appeared to play an important role in sucrose loading into the phloem of source leaf sheaths. Moreover, the microarray-based
data implied that the dominant processes associated with functional leaf sheath transition from sink to source were carbohydrate
metabolism and the translocation of the carbon and nitrogen sources and inorganic phosphate. 相似文献
2.
Raw starch-digesting amylases (RSDAs) in many microorganisms convert starch granules into maltodextrins and simple sugars.
We cloned and sequenced from Cytophaga sp. an RSDA with an excellent raw starch digestion activity. This RSDA was highly inducible by raw starch, but not by other
sugars, suggesting that an unknown signal transduction mechanism is involved in the degradation of raw starch. We used a proteomic
approach to investigate the effect of raw starch on protein expression in Cytophaga sp. Using MALDI–TOF MS protein analysis, we have identified three proteins up-regulated by raw starch, i.e., a 60-kDa chaperonin
(cpn60), glutaminase, and pyruvate phosphate dikinase (PPDK). Subsequent time-course studies detected an increased expression
of RSDA as well as the highest expression of PPDK occurring 6 h post-incubation with raw corn starch, implying that the latter
enzyme may work along with RSDA on the digestion of raw starch. Finding these proteins up-regulated by raw starch may provide
an insight into how Cytophaga sp. cells respond to raw starch stimulation. 相似文献
3.
Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes α-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the
chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures
in the range 30 to 40°C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37°C, respectively. The α-amylase activity using raw
corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient
simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence. 相似文献
4.
The cloning of α-amylase gene ofS. occidentalis and the construction of starch digestible strain of yeast,S. cerevisiae AS. 2. 1364 with ethanol-tolerance and without auxotrophic markers used in fermentation industry were studied. The yeast/E.coli shuttle plasmid YCEp1 partial library ofS. occidentalis DNA was constructed and α-amylase gene was screened in S.cerevisiae by amylolytic activity. Several transformants with amylolysis were obtained and one of the fusion plasmids had an about 5.0
kb inserted DNA fragment, containing the upstream and downstream sequences of α-amylase gene fromS. occidentalis. It was further confirmed by PCR and sequence determination that this 5.0 kb DNA fragment contains the whole coding sequence
of α-amylase. The amylolytic test showed that when this transformant was incubated on plate of YPDS medium containing 1 %
glum and 1 % starch at 30°C for 48 h starch degradation zones could be visualized by staining with iodine vapour. α-amylase
activity of the culture filtratate is 740–780 mU/mL and PAGE shows that the yeast harboring fusion plasmids efficiently secreted
α-amylase into the medium, and the amount of the recombinant α-amylase is more than 12% of the total proteins in the culture
filtrate. These results showed that α-amylase gene can be highly expressed and efficiently secreted inS. cerevisiae AS. 2.1364, and the promotor and the terminator of α-amylase gene fromS. occidentalis work well inS. cercvisiac AS. 2.1364. 相似文献
5.
Okano K Kimura S Narita J Fukuda H Kondo A 《Applied microbiology and biotechnology》2007,75(5):1007-1013
To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from
efficient (0.09 g l−1 h−1 lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought
to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture,
and, as a result, a more rapid fermentation was accomplished (1.31 g l−1 h−1 lactate). Maximum volumetric lactate productivity was further increased (1.57 g l−1 h−1 lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high
optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation. 相似文献
6.
The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH4)2SO4 fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS,
indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate
content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C.
The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and
xylose (64%). The addition of Mn++ activated the enzyme by 45%, while Li+, Zn++, Mg++, Cu+, Ca++, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose,
respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with
the hydrolysis of starch granules using 10× the amount of GLA. Apparent Km and Vmax for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent kcat of 2.9×105 min−1. 相似文献
7.
Kortessa Dimassi-Theriou Artemios M. Bosabalidis 《Plant Cell, Tissue and Organ Culture》1997,47(2):127-134
Shootlets of kiwifruit plants (Actinidia deliciosa) were culturedin vitro. Combinations of light intensity, Mg and sucrose in the cultures showed that an increase of light intensity resulted in a
corresponding increase of the relative size of the leaf mesophyll cells and in a decrease of the numbers of chloroplasts and
contained starch grains. The addition of sucrose to the substrate media negatively affected the size of the mesophyll cells
under normal Mg concentration (35 mg l−1), and positively under high Mg concentration (105 mg l−1 ). Sucrose further resulted in an increase in the numbers of chloroplasts and contained starch grains. The photosynthetic
capacity of leaves greatly increased when Mg concentration was enhanced and sucrose was excluded from the nutrient substrate.
Total sugar accumulation in all treatments was favoured by normal light intensity and addition of sucrose. 相似文献
8.
The starch-binding domain of Bacillus sp. strain TS-23 α-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel–chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70°C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K
m value. Starch-binding assays showed that the K
d and B
max values for the fusion enzyme were 2.3 μM and 0.35 μmol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris–HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption–elution cycle with a purification of 11.4-fold. 相似文献
9.
F. Pérez-Pomares S. Díaz V. Bautista C. Pire G. Bravo J. Esclapez B. Zafrilla María-José Bonete 《Extremophiles : life under extreme conditions》2009,13(4):633-641
Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic
archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an α-amylase into the external medium
in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use
of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase
on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes
on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3)
have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed
maximal activity at 3 M NaCl, and 50°C. The purification steps and molecular masses have also been reported. The other activity
(AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium.
This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt
requirement in the range 1.5–3 M NaCl.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
10.
A novel bacterial cell-surface display system was developed in Escherichia coli using omp1, a hypothetical outer membrane protein of Zymomonas mobilis. By using this system, we successfully expressed β-amylase gene of sweet potato in E. coli. The display of enzyme on the membrane surface was also confirmed. The recombinant β-amylase showed to significantly increase
hydrolytic activity toward soluble starch. Our results provide a basis for constructing an engineered Z. mobilis strain directly fermenting raw starch to produce ethanol. 相似文献
11.
Lee MH Yang SJ Kim JW Lee HS Kim JW Park KH 《Extremophiles : life under extreme conditions》2007,11(3):537-541
A gene that encodes the enzyme Pyrococcus furiosus cyclodextrin glucanotransferase (PFCGT) was cloned in Escherichia
coli. PFCGT was highly expressed in recombinant E. coli after compensation for codon usage bias using the pRARE plasmid. Purified PFCGT was extremely thermostable with an optimal
temperature and pH of 95°C and 5.0, respectively, retaining 97% of its activity at 100°C. Incubation at 60°C for 20 min during
the purification process led to a 1.5-fold increase in enzymatic activity. A time course assay of the PFCGT reaction with
starch indicated that cyclic α-1,4-glucans with DPs greater than 20 were produced at the beginning of the incubation followed
by an increase in β-CD. The major final product of PFCGT cyclization was β-CD, and thus the enzyme is a β-CGTase. 相似文献
12.
We demonstrated the new target for lodging resistance in rice (Oryza sativa L.) by the analysis of physiological function of a locus for lodging resistance in a typhoon (lrt5) with the near isogenic line under rice “Koshihikari” genetic background (tentatively named S1). The higher lodging resistance
of S1 was observed during a typhoon in September 2004 (28 days after heading), when most other plants in “Koshihikari” became
lodged. Visual observations showed that bending of the upper stems triggered lodging during the typhoon; the upper stem of
“Koshihikari” buckled completely, whereas that of S1 remained straight. In addition to the strong rain and winds during the
typhoon, the weight of the buckled upper plant parts increased the pressure on adjacent plants and caused a domino effect
in “Koshihikari”. Young’s modulus, an indicator of the rigidity of the culm, was significantly higher in S1 than in “Koshihikari”.
In the upper culm, the starch content in S1 was 4.8 times the value in “Koshihikari”, and senescence was delayed in the upper
leaves of S1. These results suggest that the rigidity of the upper culm by the higher starch content (as a result of delayed
senescence in the upper leaves) may be responsible for the higher lodging resistance during a typhoon in rice.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
13.
Ryoo N Yu C Park CS Baik MY Park IM Cho MH Bhoo SH An G Hahn TR Jeon JS 《Plant cell reports》2007,26(7):1083-1095
To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM)
analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls,
and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence,
the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant
endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls.
This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition,
DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC),
the gelatinization temperatures of endosperm starch were found to be 1–5°C lower than those of the control. We propose a distinct
role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice. 相似文献
14.
The fungal species ofRhizopus oryzae 2062 has the capacity to carry out a single stage fermentation process for lactic acid production from potato starch wastewater.
Starch hydrolysis, reducing sugar accumulation, biomass formation, and lactic acid production were affected with variations
in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/L
at pH 6.0 and 30°C was favourable for starch fermentation, resulting in a lactic acid yield of 78.3%–85.5% associated with
1.5–2.0 g/L fungal biomass produced in 36 h of fermentation. 相似文献
15.
Arsenic (As) is a potential contaminant of groundwater as well as soil in many parts of the world. The effects of increasing
concentration of As (25 μm and 50 μm As2O3) in the medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism
i.e. α-amylase, β-amylase, starch phosphorylase and acid invertase were studied in germinating seeds of two rice cvs. Malviya-36 and Pant-12 during 0–120 h period. As toxicity in situ led to a marked decline in the activities of α-amylase, β-amylase in endosperms as well as embryoaxes of germinating rice
seeds. The activity of acid invertase increased in endosperms as well as embryoaxes whereas starch phosphorylase activity
declined in endosperms but increased in embryoaxes under As treatment. In endosperms a decline in starch mobilization was
observed under As toxicity, however under similar conditions the content of total soluble sugars increased in embryoaxes.
The observed inhibition in activities of amylolytic enzymes might contribute to delayed mobilization of endospermic starch
which could affect germination of seeds in As polluted environment, while the induced acid invertase activity and increased
sugar accumulation in embryoaxes could serve as a possible component for adaptation mechanism of rice seedlings grown under
As containing medium. 相似文献
16.
Experiments were carried out to investigate the contribution of ADP-glucose pyrophosphorylase and the plastid phosphoglucosemutase
to the control of starch synthesis. Mutants ofArabidopsis thaliana (L.) Heyhn. were constructed with 50% and 7% of the wild-type adenosine 5′-diphosphoglucose pyrophosphorylase (ADPGlc-PPase),
or 50% and null plastid phosphoglucomutase (PGM). The changes in the steady-state rates of sucrose synthesis, starch synthesis
and CO2 fixation were measured in saturating CO2 in low (75 μmol·m−2·s−1) and high (600 μmol·m−2·s−1) irradiance. In low irradiance, a 50% decrease of PGM had no significant effect on fluxes, while a 50% and 93% decrease of
ADPGlc-PPase led to a 23% and 74% inhibition of starch synthesis. Decreased ADPGlc-PPase led to an increase of hexose phosphates,
triose phosphates and fructose-1,6-bisphosphate. Fixation of CO2 was not inhibited because the inhibition of starch synthesis was matched by a stimulation of sucrose synthesis. In high irradiance,
a 50% decrease of PGM led to a 20% inhibition of starch synthesis. A 50% and 93% decrease of ADPGlc-PPase led to a 39% and
90% inhibition of starch synthesis. Sucrose synthesis was also inhibited, and the rate of photosynthesis was decreased. Decreased
ADPGlc-PPase led to an increase of hexose phosphates but triose phosphates and fructose-1,6-bisphosphate did not increase.
These results are used to estimate flux-control coefficients for these enzymes for starch synthesis. Firstly, the flux to
starch is only controlled by ADPGlc-PPase in low irradiance, but control is redistributed to other enzymes in the pathway
when a rapid flux is imposed, e.g. in high irradiance and CO2. Secondly, reducing the rate of starch synthesis by decreasing the activity of enzymes in this pathway does not always lead
to a compensating increase in the rate of sucrose synthesis. Thirdly, decreasing the activity of an enzyme by a factor of
two compared to the remainder of the pathway often leads to it exerting very considerable control. Fourthly, each enzyme starts
to exert considerable control when only a fraction of its Vmax activity is being utilised in vivo, for example the maximum flux at ADPGlc-PPase never exceeded 20% of the Vmax activity. The summation theory is also applied to check whether additional major control sites are required. In low irradiance,
the efficiency of light harvesting will exert considerable control over the rate of starch synthesis. 相似文献
17.
Haiyan Sun Xiangyang Ge Weiguo Zhang 《World journal of microbiology & biotechnology》2007,23(5):603-613
Penicillium sp. X−1, isolated from decayed raw corn, produced high level of raw-starch-digesting glucoamylase (RSDG) under solid state
fermentation (SSF). Maximum enzyme yield of 306.2 U g−1 dry mouldy bran (DMB) was obtained after 36 h of culture upon optimized production. The enzyme could hydrolyse both small
and large granule starches but did not adsorb on raw starch. The enzyme exhibited maximum activity at 65°C and pH 6.5, which
provided an opportunity of synergism with α-amylase. It significantly hydrolysed 15% (w/v) raw corn starch slurry in synergism
with the commercial α-amylase and a degree of hydrolysis of 92.4% was obtained after 2 h of incubation. 相似文献
18.
Michelin M Ruller R Ward RJ Moraes LA Jorge JA Terenzi HF Polizeli Mde L 《Journal of industrial microbiology & biotechnology》2008,35(1):17-25
An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated
as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and
pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C,
with a t
50 of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated
by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose,
p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis,
analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix.
The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase). 相似文献
19.
cDNAs of barley α-amylase andA. niger glucoamylase were cloned in oneE. coli-yeast shuttle plasmid resulting in the construction of expression secretion vector pMAG15. pMAG15 was transformed intoS. cerevisiae GRF18 by protoplast transformation. The barley α-amylase andA. niger glucoamylase were efficiently expressed under the control of promoter and terminator of yeast PGK gene and their own signal
sequence. Over 99% of the enzyme activity expressed was secreted to the medium. The recombinant yeast strain, S.cerevisiae GRF18 (pMAG15), hydrolyzes 99% of the starch in YPS medium containing 15% starch in 47 h. The glucose produced can be used
for the production of ethanol.
Project supported by the Guangdong Natural Science Foundation. 相似文献
20.
Tateno T Okada Y Tsuchidate T Tanaka T Fukuda H Kondo A 《Applied microbiology and biotechnology》2009,82(1):115-121
Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium glutamicum strain coexpressing Streptococcus bovis 148 α-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli–C. glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed
this vector into C. glutamicum CSS secreting AmyA. The engineered C. glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch
as the sole carbon source without pyridoxal-5’-phosphate, which is the coenzyme for CadA. C. glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after
21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert l-lysine to cadaverine, as there was no accumulation of l-lysine in the culture medium during fermentation. Thus, we demonstrated that C. glutamicum has great potential to produce cadaverine from biomass resources. 相似文献