共查询到20条相似文献,搜索用时 15 毫秒
1.
Bacillus stearothermophilus Neopullulanase Selective Hydrolysis of Amylose to Maltose in the Presence of Amylopectin 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Hiroshi Kamasaka Kazuhisa Sugimoto Hiroki Takata Takahisa Nishimura Takashi Kuriki 《Applied microbiology》2002,68(4):1658-1664
The specificity of Bacillus stearothermophilus TRS40 neopullulanase toward amylose and amylopectin was analyzed. Although this neopullulanase completely hydrolyzed amylose to produce maltose as the main product, it scarcely hydrolyzed amylopectin. The molecular mass of amylopectin was decreased by only one order of magnitude, from approximately 108 to 107 Da. Furthermore, this neopullulanase selectively hydrolyzed amylose when starch was used as a substrate. This phenomenon, efficient hydrolysis of amylose but not amylopectin, was also observed with cyclomaltodextrinase from alkaliphilic Bacillus sp. strain A2-5a and maltogenic amylase from Bacillus licheniformis ATCC 27811. These three enzymes hydrolyzed cyclomaltodextrins and amylose much faster than pullulan. Other amylolytic enzymes, such as bacterial saccharifying α-amylase, bacterial liquefying α-amylase, β-amylase, and neopullulanase from Bacillus megaterium, did not exhibit this distinct substrate specificity at all, i.e., the preference of amylose to amylopectin. 相似文献
2.
Yoshiyuki Takasaki 《Bioscience, biotechnology, and biochemistry》2013,77(8):1523-1530
A β-amylase and a pullulanase produced by Bacillus cereus var. mycoides were purified by means of ammonium sulfate fractionation, adsorption on starch and celite and Sephadex G–100 column chromatography. The purified enzymes were homogeneous in disc electrophoresis.The β-amylase released only maltose from amylose, amylopectin, starch and glycogen, and the released maltose was in β-form. The pullulanase released maltose, maltotriose and maltotetraose from β-limit dextrin and maltotriose from pullulan, but not amylose-like substance from amylopectin.The optimum pHs of β-amylase and pullulanase were about 7 and 6~6.5, respectively. The optimum temperatures of the enzymes were about 50°C. The enzymes were inhibited by the sulfhydryl reagents such as mercuric chloride and p-chloromercuribenzoate, and the inhibitions with p-chloromercuribenzoate were restored by the addition of cysteine. The molecular weights of β-amylase and pullulanase were estimated to be 35,000±5,000 and 110,000±20,000, respectively. 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.
Masako Yamamoto Shoji Harada Takayuki Sano Tatsuya Yasunaga Nobuhide Tatsumoto 《Biopolymers》1984,23(10):2083-2096
Complex formation in the ternary system of amylose (degree of polymerization, DP, 1100), SDS, and iodine was studied statically by spectrophotometry and amperometric titration and kinetically by the pressure-jump method. It was clarified that (1) iodine (I3?) to some extent binds to amylose saturated with SDS to form an inclusion complex (ASI system); (2) the binding of SDS apparently transforms amylose of DP 1100 to that of much lower DP (less than 60) from the viewpoint of iodine binding; and (3) iodine binds to sites unoccupied by SDS in the center of the helical segment of amylose. Pressure-jump relaxation phenomenon was not observed in solutions in which iodine was dissolved prior to SDS (AIS system), but it was observed in the ASI system; it is ascribed to the association and dissociation of three molecules of iodine in the center of the amylose helix. Comparison of the rate constants in the ASI system with those in the amylose (DP 32) and iodine system indicates that iodine runs to and from the helical segment of amylose perpendicularly to the axial plane in the former, while it runs horizontally in the latter. We discuss the order of ligand mixing on the resulting structure of the ternary complexes of amylose, SDS, and iodine. 相似文献
5.
《Bioscience, biotechnology, and biochemistry》2013,77(6):1080-1086
To express the cloned β-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had β-amylase activity that produced β-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley β-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn’t change throughout the incubation. But Western blot analysis found that one β-amylase having a molecular weight of about 56,000 was synthesized. The recombinant β-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS–PAGE but showed heterogeneity on isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2–5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombiant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27–29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the Km values for maltohexaose and maltoheptaose. The pI of recombiant barley β-amylase calculated by Genetyx Version 9 based on the presumed amino acid sequence was 5.60, but the real pIs were 5.20–6.13. Therefore, some post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences in the pI and the Km values for maltohexaose and maltoheptaose between the barley and the recombinant β-amylases. 相似文献
6.
By means of amperometric mercurimetric titration, the –SH groups of native and oxidized sweet potato ²-amylase have been determined.Although eight titratable –SH groups were found in the native enzyme molecule, no distinction between essential and non-essential –SH groups was observed. A partially active enzyme after treatment with o-iodosobenzoate was crystallized by salting out with ammonium sulfate, and only an extensively oxidized one from water upon dialysis. The oxidized enzyme did not restore its activity upon treatment with sodium thioglycolate. Oxidation by iodine was also carried out from which it was presumed that the oxidation of the enzyme either by o-iodosobenzoate or by iodine does not proceed through the “ all or none ” mechanism, and also that the essentiality of –SH groups would rather be indirect. 相似文献
7.
Kyung-Min Park So-Young Jun Kyoung-Hwa Choi Kwan-Hwa Park Cheon-Seok Park Jaeho Cha 《Applied microbiology and biotechnology》2010,86(2):555-566
We cloned and expressed the gene for an intracellular α-amylase, designated AmyB, from the hyperthermophilic bacterium Thermotoga neapolitana in Escherichia coli. The putative intracellular amylolytic enzyme contained four regions that are highly conserved among glycoside hydrolase
family (GH) 13 α-amylases. AmyB exhibited maximum activity at pH 6.5 and 75°C, and its thermostability was slightly enhanced
by Ca2+. However, Ca2+ was not required for the activity of AmyB as EDTA had no effect on enzyme activity. AmyB hydrolyzed the typical substrates
for α-amylase, including soluble starch, amylose, amylopectin, and glycogen, to liberate maltose and minor amount of glucose.
The hydrolytic pattern of AmyB is most similar to those of maltogenic amylases (EC 3.2.1.133) among GH 13 α-amylases; however,
it can be distinguished by its inability to hydrolyze pullulan and β-cyclodextrin. AmyB enzymatic activity was negligible
when acarbose, a maltotetraose analog in which a maltose residue at the nonreducing end was replaced by acarviosine, was present,
indicating that AmyB cleaves maltose units from the nonreducing end of maltooligosaccharides. These results indicate that
AmyB is a new type exo-acting intracellular α-amylase possessing distinct characteristics that distinguish it from typical
α-amylase and cyclodextrin-/pullulan-hydrolyzing enzymes. 相似文献
8.
Michele Michelin Vivian M. Benassi Luiz Alberto B. Moraes João A. Jorge Maria de Lourdes T.M. Polizeli 《Carbohydrate research》2010,345(16):2348-2353
An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t50 of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The Km of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase). 相似文献
9.
《Bioscience, biotechnology, and biochemistry》2013,77(12):1933-1936
p-Nitrophenyl 65-O-β-D-galactopyraosyl-α-maltopentaoside (L6G5P) was synthesized by the sequential use of the transglycosylation and hydrolytic action of β-D-galactosidase from Bacillus circulans. The enzyme produced L6G5P (at a yield of 8.0% based on the amount of p-nitrophenyl α-maltopentaoside added) from lactose as the donor and p-nitrophenyl α-maltopentaoside as the acceptor. The frequency at which of human pancreatic α-amylase and salivary α-amylase catalyzed the cleavage of glycosidic linkages in L6G5P was calculated by analysis of the digests by high-pressure liquid chromatography. The modes of action of the two isozymes differed. Both hydrolyzed L6G5P and produced p-nitrophenyl α-maltoside and p-nitrophenyl α-D-glucopyranoside, but human pancreatic α-amylase produced more of the latter than human salivary α-amylase. Thus, L6G5P could be used to assay of the two enzymes differentially in serum. 相似文献
10.
Katsutada Takahashi 《Bioscience, biotechnology, and biochemistry》2013,77(7):629-633
In the latest work, a method to determine calorimetrically the α-glucosidic linkage contents in starches was introduced.The present work refers to the application of this method to the determination of the α-1,4 glucosidic linkage content in two kinds of sweet potato starch (i.e., Norin-1 and Okinawa-100) isolated at the two different stages of development.It has been found that the sample isolated on October 31 (1953) had a larger value for α-1,4 glucosidic linkage content than the sample isolated on August 6 (1953) for both the two sweet potato starches.Combined with the amperometric titration method the increase in α-1,4 glucosidic linkage content has been found to be due to both the increases in amylose content and in average unit chain length of amylopectin component. 相似文献
11.
Yoshiki Yamasaki Akira Tsuboi Yukio Suzuki 《Bioscience, biotechnology, and biochemistry》2013,77(11):2139-2148
Both of the two forms of glucoamylase (glucoamylases I and II) from the wheat bran culture of Mucor rouxianus hydrolyzed amylopectin, amylose, glycogen, soluble starch, maltotriose, and maltose, but did not act on isomaltose and isomaltotriose. Phenyl α-maltoside was hydrolyzed into glucose and phenyl α-glucoside by both glucoamylases. Maltose was hydrolyzed about one-fifth as rapidly as amylopectin. Both enzymes produced glucose from amylopectin, amylose, glycogen, soluble starch in the yields of almost complete hydrolysis. They hydrolyzed amylose with the inversion of configuration, producing the β-anomer of glucose. Glucoamylase II hydrolyzed raw starch at 3-fold higher rate than glucoamylase I. The former hydrolyzed rice starch almost completely into glucose, whereas the latter hydrolyzed it incompletely (nearly 50%). 相似文献
12.
An α-glucosidase and a glucoamylase have been isolated from fruit bodies of Lentinus edodes (Berk.) Sing., by a procedure including fractionation with ammonium sulfate, DEAE-cellulose column chromatography, and preparative gel electrofocusing. Both of them were homogeneous on gel electrofocusing and ultracentrifugation. The molecular weight of α-glucosidase and glucoamylase was 51,000 and 55,000, respectively. The α-glucosidase hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose, and soluble starch, but did not act on sucrose. The glucoamylase hydrolyzed maltose, maltotriose, phenyl α-maltoside, soluble starch, amylose, amylopectin, and glycogen, glucose being the sole product formed in the digests of these substrates. Both enzymes hydrolyzed phenyl a-maltoside into glucose and phenyl α-glucoside. The glucoamylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen, converting them almost completely into glucose. It was found that β-glucose was liberated from amylose by the action of glucoamylase, while α-glucose was produced by the α-glucosidase.Maltotriose was the main α-glucosyltransfer product formed from maltose by the α-glucosidase. 相似文献
13.
Summary The presence of phospholipids reduces the breakdown of amylose catalyzed by -amylase, phosphorylase and -amylase. The activities of the -amylases of sweet potato (Ipomoea batatas) and barley (Hordeum vulgare L.) disminish to less than 10% of the activity in the control without the phospholipids. When the amylose was complexed with phospholipids the activity of the -amylase of Bacillus subtilis was reduced to about 25% of the control value. A similar effect was observed for the amylases of Zea mays leaves. The phosphorylase effected almost no phosphorolysis of the complexed amylose, but starch synthesis from glucose-1-phosphate proceeded at a rate that was about 60% of that with pure amylose. The activity of the synthetase from bundle sheath cells of maize leaves was not influenced much by the presence of phospholipids, whereas the branching enzyme of maize endosperm did not produce any amylopectin from the complexed amylose. —These facts could explain the simultaneous deposition of amylose and amylopectin in the starch granules. Some of the newly formed glucan chains may be protected by formation of a complex with the phospholipids. This protected amylose can not undergo branching or breakdown, but it can be elongated owing to the activity of synthetase or phosphorylase. Amylopectin is formed from the chains that are not complexed. 相似文献
14.
Starch-utilizing mutants of Escherichia coli which can grow well on starch or amylose as the sole carbon source were isolated. The maximal viable cell number of the starch-utilizing mutants on the polysaccharide media reached the same level (4 × 109 cells/ml) as that with glucose medium after incubation for 24 hours at 37°C. The isolated mutants could produce more intracellular α-amylase than the wild-type strain, and the enzyme activity was detected in the extracellular fluid. Polyacrylamide gel electrophoresis showed that the intracellular and extracellular enzymes had similar electrophoretic mobilities. These observations suggested that the ability of growth on the polysaccharide media was due to the excreted α-amylase, which appeared to be identical with the intracellular enzyme. 相似文献
15.
Naomichi Baba Kazuyoshi Nishiyama Jun’ichi Oda Yuzo Inouye 《Bioscience, biotechnology, and biochemistry》2013,77(7):1441-1443
A gram positive bacterium (strain No. 109) isolated from soil as a producer of cyclodextrinase was identified as Bacillus coagulans. The cyclodextrinase from B. coagulans was purified to a homogeneous state by disc-electrophoresis after Streptomycin treatment, DEAE-Sephadex column chromatography, Ultrogel AcA44 gel filtration and hydroxyapatite column chromatography. The molecular weight of the enzyme was determined to be 6.2}104 by sodium dodecyl-sulfate gel electrophoresis. The isoelectric point of the enzyme was pH 5.0. The enzyme was most active at pH 6.2 and 50°C, and stable up to 45°C at pH 7.0 and in the range of pH 6.0 ~ 7.3 at 40°C on 2 hr incubation. This enzyme hydrolyzed linear maltooligosaccharides (such as maltotetraose (G4), maltopentaose (G5) and maltohexaose (G6)) and α-, β- and α-cyclodextrins (CDs) faster than maltotriose (G3) and short chain amylose ( 18), but did not hydrolyze maltose. The rates of hydrolysis for polysaccharides (such as starch, amylose and amylopectin) were below 1 % as compared to that for β-CD. The Km values for G3, G4, G5, G6, short chain amylose ( 18) and α, β- and γ-CD were 4.5, 4.0,2.3,1.5,1.5,10,2.8 and 0.47 mM, respectively. The products with this enzyme had the α-configulation. 相似文献
16.
《Bioscience, biotechnology, and biochemistry》2013,77(6):1152-1153
Random mutations were introduced into recombinant barley β-amylase by modified PCR to increase its thermostability. Two clones were obtained. One was found to have a change of Ser-351 to Pro and another, a change of Ala-376 to Ser, and 2.3°C and 1.0°C increases, respectively, in the thermostabilities compared with that of native recombinant β-amylase. 相似文献
17.
Starch isolated from mature Ginkgo biloba seeds and commercial normal maize starches were subjected to α-amylolysis and acid hydrolysis. Ginkgo starch was more resistant to pancreatic α-amylase hydrolysis than the normal maize starch. The chain length distribution of debranched amylopectin of the starches was analyzed by using high performance anion-exchange chromatography equipped with an amyloglucosidase reactor and a pulsed amperometric detector. The chain length distribution of ginkgo amylopectin showed higher amounts of both short and long chains compared to maize starch. Naegeli dextrins of the starches prepared by extensive acid hydrolysis over 12 days demonstrated that ginkgo starch was more susceptible than normal maize to acid hydrolysis. Ginkgo dextrins also demonstrate a lower concentration of singly branched chains than maize dextrins, and unlike maize dextrin, debranched ginkgo shows no multiple branched chains. The ginkgo starch displayed a C-type X-ray diffraction pattern, compared to an A-type pattern for maize. Ginkgo starch and maize starch contained 24.0 and 17.6% absolute amylose contents, respectively. 相似文献
18.
Anomeric forms of glucose and maltose produced from phenyl, p-nitrophenyl, p-tert-butylphenyl, p-ethylphenyl and p-chlorophenyl α-maltosides and maltopentaose by α- and β-amylases were determined quantitatively by a gas-liquid chromatographic method. All of the three kinds of α-amylases tested, B. subtilis saccharifying α-amylase, Taka-amylase A, and porcine pancreas α-amylase, were found to produce only α-maltose from the maltosides. Sweet potato and barley β-amylases produced β-maltose from maltopentaose.Saccharifying α-amylase from B. subtilis also released α-maltose from all the maltosides mentioned above, contrary to the report by Shibaoka et al. that the enzyme released β-maltose from maltosides other than phenyl α-maltoside: FEBS Lett., 16, 33 (1971); J. Biochem., 77, 1215 (1975). It appears unlikely that the α-amylase releases β-maltose, depending on the kind of substrate. 相似文献
19.
Various saccharides were hydrolyzed with the purified amyloglucosidase of Endornyces sp. IFO 0111.Glucose was the only reducing product in the digest of soluble starch. The amyloglucosidase could hydrolyze starch and amylose only incompletely though it had the ability to split α-d-(1→6) bonds and hydrolyzed amylopectin and glycogen to high extents.It hydrolyzed maito-oligosaccharides by stepwise removal of glucose units from the nonreducing end of the molecules. 相似文献
20.
Mizuho Kusuda Masaru Nagai Tae-Chul Hur Mitsuhiro Ueda Takao Terashita 《Mycoscience》2003,44(4):311-317
-Amylase from a still culture filtrate of Tricholoma matsutake, an ectomycorrhizal fungus, was isolated and characterized. The enzyme was purified to a homogeneous preparation with Toyopearl-DEAE, gel filtration, and Mono Q column chromatography. The -amylase was highly purified (3580 fold) with a recovery of 10.5% and showed a single protein band by SDS-PAGE. The enzyme was most active at pH 5.0–6.0 toward soluble starch and stable within the broad pH range 4.0–10.0. This -amylase was a relatively thermostable enzyme (optimum temperature, 60°C; thermal stability, 50°C). The molecular mass was 34kDa by size-exclusion chromatography and 46kDa by SDS-PAGE. This enzyme was not inhibited by the Hg2+ ion. Measurement of viscosity and TLC and HPLC analysis of the hydrolysates obtained from amylose showed that the amylase from T. matsutake is an endo-type (-amylase). Substrate specificity was tested using amylose with different polysaccharides. This -amylase readily hydrolyzed the -1,4 glucoside bond in soluble starch and amylose A (MW, 2900), but did not hydrolyze the -1,6 bond and cyclic polysaccharides such as - and -cyclodextrin. 相似文献