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1.
Extracellular matrix provides an architectural structure and mechanical stability for aerobic granules. Distributions of cells
and extracellular polymeric substances (EPS), including proteins, α- and β-d-glucopyranose polysaccharides, in acetate-fed granules and phenol-fed granules were probed using a novel quadruple staining
scheme. In acetate-fed granules, protein and β-d-glucopyranose polysaccharides formed the core, whereas, the cells and α-d-glucopyranose polysaccharides accumulated in the granule outer layers. Based on these experimental findings, this study indicated
that different conclusions can be obtained regarding EPS distributions when granules were stained differently. The core of
phenol-fed granules, conversely, was formed principally by proteins; whereas, the cells and α- and β-d-glucopyranose polysaccharides were accumulated at an outer filamentous layer. Using a series of confocal laser scanning microscope
(CLSM) images whose threshold values were determined via Otsu’s scheme, the three-dimensional distributions of cells and EPS
were produced using a polygonal surface model. Structural information extracted can be applied in further development of comprehensive
granule models. 相似文献
2.
Mohammad Shafiei Abed-Ali Ziaee Mohammad Ali Amoozegar 《Journal of industrial microbiology & biotechnology》2011,38(2):275-281
A halophilic α-amylase produced by Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange, and Sephacryl S-200 gel
filtration chromatography. The purified amylase exhibited specific activity of 357 unit/mg protein that corresponds to twofold
purification. The molecular mass of the amylase was determined to be 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and gel filtration chromatography. The optimal pH and temperature for enzyme activity were 6.5 and 45°C, respectively.
The amylase was active over a wide range of salt concentrations (0–4 M) with maximum activity at 0.75–1 M NaCl. The α-amylase
activity was stimulated by Ca2+ and inhibited by ethylenediamine tetraacetic acid (EDTA), suggesting that this enzyme is a metalloenzyme. The purified enzyme
showed remarkable stability towards surfactants (Tween 20, Tween 80, and Triton X-100), and its activity was increased by
β-mercaptoethanol. The halophilic α-amylase was stable in the presence of various organic solvents such as benzene, chloroform,
toluene, and cyclohexane. These properties indicate wide potential applications of this α-amylase in starch-processing industries. 相似文献
3.
Hui-Mei Lin Yung-Ho Chang Jheng-Hua Lin Jay-lin Jane Ming-Jen Sheu Ting-Jang Lu 《Carbohydrate polymers》2006,66(4):528-536
Lotus (Nelumbo nucifera Gaertn.) rhizome starch granules have an elongated oval shape with the hilum located at one end. The morphologic characteristics were used as a direction anchor to study the heterogeneity of molecular organization of starch granules using microscopy before and after partial digestion by bacterial α-amylase (Bacillus sp.) The partially digested granule showed a single, big eroded hole at the end distant from the hilum. The enzyme-attacked end was revealed to be the loosely packed end and to be the weak point for enzyme hydrolysis. The α-amylase hydrolyzed the loosely packed central part of the granule faster than the densely packed periphery, and left an empty shell with a fish-bone-like tunnel inside. The periphery was more resistant to amylase hydrolysis and had strong birefringence. For the whole starch granule, the selectivity of α-amylase hydrolysis was low for the crystalline and amorphous regions and for amylose and amylopectin molecules. This study elucidated that the molecular organization of lotus rhizome starch granules was heterogeneous. 相似文献
4.
Chao-Hsun Yang Yu-Chun Huang Cheng-Yu Chen Chia-Ying Wen 《Journal of industrial microbiology & biotechnology》2010,37(4):401-406
A gene encoding the thermostable raw starch digesting α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression
resulted in high levels of extracellular amylase production, as high as 510 U/l in the Hinton flask culture broth. The purified
amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H, and this agrees with the predicted size based on the nucleotide sequence. About 75% of the original
activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and
60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 48-h treatment, the
DPw of raw sago starch obviously decreased from 830,945 to 378,732. The surface of starch granules was rough, and some granules
displayed deep cavities. 相似文献
5.
A halophilic isolate Thalassobacillus sp. LY18 producing extracellular amylase was isolated from the saline soil of Yuncheng Salt Lake, China. Production of the enzyme was synchronized with bacterial growth and reached a maximum level during the early stationary phase. The amylase was purified to homogeneity with a molecular mass of 31 kDa. Major products of soluble starch hydrolysis were maltose and maltotriose, indicating an α-amylase activity. Optimal enzyme activity was found to be at 70°C, pH 9.0, and 10 % NaCl. The α-amylase was highly stable over broad temperature (30–90°C), pH (6.0–12.0), and NaCl concentration (0–20 %) ranges, showing excellent thermostable, alkalistable, and halotolerant nature. The enzyme was stimulated by Ca2+, but greatly inhibited by EDTA, indicating it was a metalloenzyme. Complete inhibition by diethyl pyrocarbonate and β-mercaptoethanol revealed that histidine residue and disulfide bond were essential for enzyme catalysis. The surfactants tested had no significant effects on the amylase activity. Furthermore, it showed high activity and stability in the presence of water-insoluble organic solvents with log P ow ≥ 2.13. 相似文献
6.
J. Yamaguchi S. Itoh T. Saitoh A. Ikeda T. Tashiro Y. Nagato 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,98(1):32-38
β-Amylase deficiency in various cultivars of rice was examined at the molecular level. Using an antibody against β-amylase
purified from germinating seeds of rice, we were able to demonstrate the expression and organization of the β-amylase gene
in normal and deficient cultivars. Although β-amylase is a starch-hydrolyzing enzyme, as is α-amylase, the β-amylase protein/gene
is expressed differently from the α-amylase protein/gene; i.e. (1) β-amylase is synthesized only in aleurone cells, (2) the
enzyme production in the embryo-less half-seeds is not under hormonal control. We identified some cultivars of rice that are
deficient for β-amylase activity. We present new evidence that synthesis is blocked at the level of mRNA synthesis in the
deficient cultivars. The usefulness of β-amylase as a crop trait is also discussed.
Received: 8 May 1998 / Accepted: 5 June 1998 相似文献
7.
InPenaeus vannamei, α-amylase is the most important glucosidase and is present as at least two major isoenzymes which have been purified. In
order to obtain information on their structure, a hepatopancreas cDNA library constructed in phage lambda-Zap II (Strategene)
was screened using a synthetic oligonucleotide based on the amino acid sequence of a V8 staphylococcal protease peptide ofP. vannamei α-amylase. Three clones were selected: AMY SK 37 (EMBL sequence accession number: X 77318) is the most complete of the analyzed
clones and was completely sequenced. It contains the complete cDNA sequence coding for one of the major isoenzymes of shrimp
amylase. The deduced amino acid sequence shows the existence of a 511-residue-long pre-enzyme containing a highly hydrophobic
signal peptide of 16 amino acids. Northern hybridization of total RNA with the amylase cDNA confirms the size of the messenger
at around 1,600 bases. AMY SK 28, which contains the complete mature sequence of amylase, belonged to the same family characterized
by a common 3′ terminus and presented four amino acid changes. Some other variants of this family were also partially sequenced.
AMY SK 20 was found to encode a minor variant of the protein with a different 3′ terminus and 57 amino acid changes.
Phylogenetic analysis established with the conserved amino acid regions of the (β/α) eight-barrel domain and with the total
sequence ofP. vannamei showed close evolutionary relationships with mammals (59–63% identity) and with insect α-amylase (52–62% identity). The use
of conserved sequences increased the level of similarity but it did not alter the ordering of the groupings. Location of the
secondary structure elements confirmed the high level of sequence similarity of shrimp α-amylase with pig α-amylase.
Correspondence to: A. Van Wormhoudt 相似文献
8.
Zhi-Hua Feng Yuan-Shan Wang Yu-Guo Zheng 《Biotechnology and Bioprocess Engineering》2011,16(5):894-900
Alpha-amylase inhibitors are widely used by the pharmaceutical and agricultural industries, such as the treatment of diabetes
and obesity and insect controller. Here, we developed a colorimetric method to screen for α-amylase inhibitor producing strains or mutants with higher α-amylase inhibitor productivity. This method relies on absorbance changes at 402 nm that are due to the inhibition of α-amylase catalyzed hydrolysis of 2-Chloro-4-nitrophenyl-4-O-β-D-galactopyranosyl-maltoside by α-amylase inhibitors. The assay can be performed on a microtiter plate, making it simple and convenient. Using this method,
α-amylase inhibitor producing strains and mutants with higher α-amylase inhibitor productivity can be rapidly screened. One strain, ZJB-08196, with the highest α-amylase inhibition was isolated and identified as Actinoplanes utahensis, and one mutant with higher acarbose production was obtained by screening 3,000 variants using this method. 相似文献
9.
Role of β-amylase in starch metabolism during soybean seed development and germination 总被引:1,自引:0,他引:1
Differences in starch metabolism during seed development and germination of two soybean [ Glycine max (L.) Merrill] genotypes with normal seed β-amylase activity ['Williams' ( Sp 1b and 'Altona' ( Sp 1b )] and two soybean genotypes with undetectable seed β-amylase activity ['Chestnut' ( Sp 1au ) and 'Altona' ( Sp 1)] were investigated. Starch and soluble sugar profiles were essentially the same during seed development and germination. Total amylase activity of Williams and Altona ( Sp 1b ) peaked just prior to seed maturity and then dropped off slowly; whereas, the total amylase activity of Chestnut and Altona ( sp 1) was very low throughout seed development and germination. The differences in amylase activity between Altona ( Sp 1 b ) and Altona ( sp 1) was also seen in leaves. α-Amylase activity was similar in the four genotypes when β-amylase was inhibited with Hg2+ but was higher in the two genotypes with normal β-amylase activity when β-amylase was inhibited with heat plus Ca2+ . Low levels of starch phosphorylase activity were detected throughout seed development and germination, and the activity was similar in three of the genotypes and higher in Altona ( sp 1).
The protein, oil and oligosaccharide contents of mature seeds of the four genotypes were similar. Altona ( sp 1b ) and ( sp 1), which appear to be near isogenic lines, were not different in any morphological character or yield.
Altona ( Sp 1b ) showed greater hydrolysis of soybean seed starch than Altona ( sp 1), but the evidence indicates that the mutation resulting in greatly reduced or missing β-amylase activity has no effect on starch metabolism of developing and germinating soybean seeds. 相似文献
The protein, oil and oligosaccharide contents of mature seeds of the four genotypes were similar. Altona ( sp 1
Altona ( Sp 1
10.
Changes in α- and β-amylase activities in the cotyledons of germinating clover were examined. The activity of α-amylase decreased
during germination and growth of seedling, while β-amylase activity increased. These changes are different from those of germinating
seeds of many other plants, but similar to the germinating seeds of alfalfa, which belongs to the same tribe (Trifolieae)
as clover. 相似文献
11.
A moderately halophilic strain LY9 with high amylolytic activity was isolated from soil sample obtained from Yuncheng, China.
Biochemical and physiological characterization along with 16S rRNA sequence analysis placed the isolate in the genus Halobacillus. Amylase production started from the post-exponential phase of bacterial growth and reached a maximum level during the early-stationary
phase. The isolate LY9 was found to secrete the amylase, the production of which depended on the salinity of the growth medium.
Maximum amylase production was observed in the presence of 10% KCl or 10% NaCl. Maltose was the main product of soluble starch
hydrolysis, indicating a β-amylase activity. The enzyme showed optimal activity at 60°C, pH 8.0, and 10–12.5% of NaCl. It
was highly active over broad temperature (50–70°C), NaCl concentration (5.0–20.0%), and pH (4.0–12.0) ranges, indicating its
thermoactive and alkali-stable nature. However, activity dropped off dramatically at low NaCl concentrations, showing the
amylase was halophilic. Ca2+ was found to stimulate the β-amylase activity, whereas ethylenediaminetetraacetic acid (EDTA), phenylarsine oxide (PAO),
and diethyl pyrocarbonate (DEPC) strongly inhibited the enzyme, indicating it probably was a metalloenzyme with cysteine and
histidine residues located in its active site. Moreover, the enzyme exhibited remarkable stability towards sodium dodecyl
sulfate
(SDS) and Triton X-100. This is the first report of β-amylase production from moderate halophiles. The present study indicates
that the extracellular β-amylase of Halobacillus sp. LY9 may have considerable potential for industrial application owing to its properties. 相似文献
12.
Singh Lokendra Ram M. Sai Agarwal M.K. Alam S.I. 《World journal of microbiology & biotechnology》2000,16(7):625-630
Among 67 psychrotrophic bacterial isolates of Leh, India screened for production of hydrolytic enzymes at 10 °C, four belonging
to Aeromonas hydrophila were characterized and evaluated for biodegradation of night soil. All strains produced metalloproteases on a variety of
carbon and nitrogen sources. Strains LA1 and LA15 also produced α-amylase and PC5 both α- & β-amylase. No amylase was produced
by PN7, however it produced lipase. Casein and glucose induced maximum enzyme activity (protease and amylase) in LA15 and
PC5, respectively. In LA1, maximum induction of protease was observed with casein and of amylase with maltose. Corn oil/tributyrin
served as the best inducers for protease and lipase production by PN7. A. hydrophila strains were found to be psychrotrophic with optimum growth and enzyme activity at 20 and 37 °C, respectively. Maximum biodegradation
of night soil was observed by strain LA1 at 5–20 °C.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
13.
Polysaccharide components present in the pseudo-stem (scape) of M. paradisiaca were purified from acetone powder of the scape
by delignification followed by extraction with aqueous solvents into water soluble polysaccharide (WSP), EDTA-soluble polysaccharide
(EDTA-SP), alkali-soluble polysaccharide (ASP) and alkali-insoluble polysaccharide (AISP) fractions. Sugar compositional analysis
showed that WSP and EDTA-SP contained only D-Glc whereas ASP contained D-Glc, L-Ara and D-Xyl in ∼ 1:1:10 ratio, respectively,
and AISP' contained D-Glc, L-Ara and D-Xyl in ∼ 10:1:2 ratio, respectively. WSP was further purified by complexation with
iso-amylalcohol and characterized by specific rotation, IR spectroscopy, Iodine affinity, ferricyanide number, blue value,
hydrolysis with α-amylase and glucoamylase, and methylation linkage analysis, and shown to be a amylopectin type α-D-glucan.
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
14.
Romina Rodríguez-Sanoja N. Oviedo L. Escalante B. Ruiz S. Sánchez 《Journal of industrial microbiology & biotechnology》2009,36(3):341-346
Starch is degraded by amylases that frequently have a modular structure composed of a catalytic domain and at least one non-catalytic
domain that is involved in polysaccharide binding. The C-terminal domain from the Lactobacillus amylovorus α-amylase has an unusual architecture composed of five tandem starch-binding domains (SBDs). These domains belong to family
26 in the carbohydrate-binding modules (CBM) classification. It has been reported that members of this family have only one
site for starch binding, where aromatic amino acids perform the binding function. In SBDs, fold similarities are better conserved
than sequences; nevertheless, it is possible to identify in CBM26 members at least two aromatic residues highly conserved.
We attempt to explain polysaccharide recognition for the L. amylovorus α–amylase SBD through site-directed mutagenesis of aromatic amino acids. Three amino acids were identified as essential for
binding, two tyrosines and one tryptophan. Y18L and Y20L mutations were found to decrease the SBD binding capacity, but unexpectedly,
the mutation at W32L led to a total loss of affinity, either with linear or ramified substrates. The critical role of Trp
32 in substrate binding confirms the presence of just one binding site in each α-amylase SBD. 相似文献
15.
A new α-amylase from Rhizomucor sp. (RA) was studied in detail due to its very efficient hydrolysis of raw starch granules at low temperature (32 °C). RA contains a starch binding domain (SBD) connected to the core amylase catalytic domain by a O-glycosylated linker. The mode of degradation of native maize starch granules and, in particular, the changes in the starch structure during the hydrolysis, was monitored for hydrolysis of raw starch at concentrations varying between 0.1 and 31%. RA was compared to porcine pancreatic α-amylase (PPA), which has been widely studied either on resistant starch or as a model enzyme in solid starch hydrolysis studies. RA is particularly efficient on native maize starch and release glucose only. The hydrolysis rate reaches 75% for a 31% starch solution and is complete at 0.1% starch concentration. The final hydrolysis rate was dependent on both starch concentration and enzyme amount applied. RA is also very efficient in hydrolyzing the crystalline domains in the maize starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in the first stages of hydrolysis. This is in agreement with the observed preferential hydrolysis of amylopectin, the starch constituent that forms the backbone of the crystalline part of the granule. Amylose-lipid complexes present in most cereal starches are degraded in a second stage, yielding amylose fragments that then reassociate into B-type crystalline structures, forming the final resistant fraction. 相似文献
16.
17.
Michikatsu Sato Machiko Watanabe Hiroto Nagano Yoshiaki Yagi 《Biotechnology letters》1994,16(7):703-708
A simple and specific recovery method for α-cyclodextrin (α-CD) was developed by employing co-digestion of CD reaction mixtures
with CGTase fromBacillus ohbensis and α-glucosidase. The combination of CGTase fromB. ohbensis and α-glucosidase, such as α-amylase, β-amylase, or glucoamylase was examined for the selective degradation of β-and γ-CD
in the CD reaction mixture formed by CGTase fromB. macerans. The co-digestion of the CD mixture with Taka-amylase and the CGTase resulted in α-CD and maltodextrins, the combination
with β-amylase resulted in α-CD and maltose, and that with glucoamylase resulted in α-CD and glucose. The conditions of selective
degradation of β- and γ-CD by co-digestion with the CGTase and glucoamylase were optimized as follows: the incubation pH,
5.5; incubation temperature, 50°C; CGTase concentration, 15 u/g of substrate; glucoamylase, 10 u/g of substrate; substrate
concentration, 10% (w/v); the incubation time was fixed for 18 hr from the stand point of operation convenience.
Most part of the content was presented in poster session at the 7th International Cyclodextrin Symposium, Tokyo, April 1994. 相似文献
18.
α-Amylase activity during pullulan production and α-amylase gene analyses of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> 总被引:1,自引:0,他引:1
Manitchotpisit P Skory CD Leathers TD Lotrakul P Eveleigh DE Prasongsuk S Punnapayak H 《Journal of industrial microbiology & biotechnology》2011,38(9):1211-1218
19.
M. C. V. Egas M. S. da Costa Don A. Cowan Euclides M. V. Pires 《Extremophiles : life under extreme conditions》1998,2(1):23-32
An extracellular α-amylase produced by the thermophilic bacterium Thermus filiformis Ork A2 was purified from cell-free culture supernatant by ion exchange chromatography. The molecular mass was estimated to
be 60 000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was rich in both basic and hydrophobic
amino acids, presenting the following NH2-terminal amino acid sequence: Thr-Ala-Asp-Leu-Ile-Val-Lys-Ile-Asn-Phe. Amylolytic activity on soluble starch was optimal
at pH 5.5–6.0 and 95°C, and the enzyme was stable in the pH range of 4.0–8.0. Calcium enhanced thermostability at temperatures
above 80°C, increasing the half-life of activity to more than 8 h at 85°C, 80 min at 90°C, and 19 min at 95°C. Ethylenediaminetetraacetic
acid (EDTA) inhibited amylase activity, the inhibition being reversed by the addition of calcium or strontium ions. The α-amylase
was also inhibited by copper and mercuric ions, and p-chloromercuribenzoic acid, the latter being reversed in the presence of dithiothreitol. Dithiothreitol and β-mercaptoethanol
activated the enzyme. The α-amylase exhibited Michaelis-Menten kinetics for starch, with a K
m of 5.0 mg·ml−1 and k
cat/K
m of 5.2 × 105 ml·mg−1 s−1. Similar values were obtained for amylose, amylopectin, and glycogen. The hydrolysis pattern was similar for maltooligosaccharides
and polysaccharides, with maltose being the major hydrolysis product. Glucose and maltotriose were generated as secondary
products, although glucose was produced in high levels after a 6-h digestion. To our knowledge this is the first report of
the characterization of an α-amylase from a strain of the genus Thermus.
Received: June 2, 1997 / Accepted: September 16, 1997 相似文献
20.
Degradation of reserve starch in turions, perennation organs of the duckweed Spirodela polyrhiza , is induced by continuous red light (cR). Irradiation of the turions with this light results in the autophosphorylation of starch-associated glucan water dikinase (GWD). The ensuing phosphorylation of the starch by this enzyme was proposed to result in the enhanced association of starch-degrading enzymes to the starch granules and in the initiation of starch breakdown. The present results confirm that the irradiation of dark-adapted turions with cR results in phosphorylation of the starch, accompanying changes in the capacity of the granule starch to bind turion endogenous α-amylase, as well as changes in the starch degradation level. All three effects show very similar dependence on the time of irradiation, suggesting that they may be linked. The α-amylase is a plausible candidate for effecting starch breakdown initiation. However, the increased binding capacity of the starch granules for this enzyme is insufficient to account for the initiation of the starch breakdown as this capacity is already high prior to the irradiation. The decisive effect of cR irradiation on starch degradation may lie in enabling α-amylase to gain access to otherwise sequestered starch granules or in activating α-amylase bound to the granules. 相似文献