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1.
Yasuji Minoda Motoo Arai Koichi Yamada 《Bioscience, biotechnology, and biochemistry》2013,77(4):572-578
The amino acid compositions of the acid-stable α-amylase and the acid-unstable α-amylase obtained from Aspergillus niger were determined by automatic column chromatography. The amino acid composition of the acid-unstable α-amylase was very similar to that of the α-amylase of Aspergillus oryzae. The amino acid composition of the acid-stable α-amylase was also similar in most part, but differed from that of the acid-unstable α-amylase in the following features, (a) The lysine content was lower, (b) Although the totals of carboxyl and amide were almost equal, there were considerably more free carboxyl residues, (c) The serine content was higher, (d) The proline content was lower. These facts may be related to the lower isoelectric point (pH 3.44) of the acid-stable α-amylase.Amino-terminal amino acid analysis demonstrated one mole of amino-terminal leucine or isoleucine per mole of the acid-stable α-amylase and one mole of amino-terminal alanine per mole of the acid-unstable α-amylase. 相似文献
2.
Kenji Aoki Motoo Arai Yasuji Minoda Koichi Yamada 《Bioscience, biotechnology, and biochemistry》2013,77(12):1913-1920
The Acid-stable α-amylase and the acid-unstable α-amylase from Aspergillus niger contained one mole of sulfhydryl group per one mole of enzyme, which probably existed correlating with calcium atom that was essential for the amylase activity.Iodine reacted at acidic pH specifically with the sulfhydryl group of both enzymes and oxidized it to considerably high degree, since about 4 eq of iodine per mole of sulfhydryl group of both enzymes were consumed. The modification of the sulfhydryl group of the acid-stable α-amylase did not affect the amylase acitvity, while, that of the acid-unstable α-amylase reduced it to 70 per cents intact enzyme. It was difficult to carry out carboxy-methylation of the sulfhydryl group of the acid-stable α-amylase under mild conditions maintaining its activity, but that of the acid-unstable α-amylase was easily achieved.These facts suggested that some differences existed in the neighborhood of the sulfhydryl group of both enzymes, and that the sulfhydryl group of them was not the active site. 相似文献
3.
Motoo Arai Yasuji Minoda Koichi Yamada 《Bioscience, biotechnology, and biochemistry》2013,77(6):922-928
The acid-stable α-amylase or the acid-unstable α-amylase from Aspergillus niger contained 24 moles or 7 moles mannose and 4 moles or 1 mole hexosamine per mole of protein, respectively.The acid-stable α-amylase and the acid-unstable α-amylase contained calcium only, but not detectable amounts of other metals. Calcium contents of the both enzymes were converged to at least one gram atom per mole of enzyme by dialysis against acetate buffer. The last calcium could be removed under the suitable conditions by EDTA. Calcium removal by EDTA was accompanied by the loss of activity and by the little change of UV absorption spectra. The phenomenon caused by calcium removal were partially reversible. This last one atom of calcium seemed to be essential for the maintenance of active structure of α-amylase. 相似文献
4.
Kenji Aoki Motoo Arai Yasuji Minoda 《Bioscience, biotechnology, and biochemistry》2013,77(13):2479-2486
Monochlorotrifluoro-p-benzoquinone (CFQ) was used for investigating the state of the amino groups of acid-stable α-amylase and acid-unstable α-amylase. About half of the total amino groups in both enzyme molecules were reacted with the reagent. The unreactive amino groups seemed to exist in a different state from the reactive ones. Both enzymes whose amino groups were modified by CFQ still maintained the α-phenylmaltosidase activity in spite of losing or decreasing the amylase activity. These facts suggest that the amino groups of both enzymes were not in the active site but the modification of them caused steric hindrance.The pH-stability of the acid-unstable α-amylase whose one or two amino groups were modified with succinic anhydride or 2,4,6-trinitrobenzene-l-sulfonate (TNBS) increased on the acidic side and decreased on the alkaline side, but further modification of them led to decrease the stability on both sides. 相似文献
5.
6.
Takehiko Yamamoto Akimasa Nishida Juichiro Fukumoto 《Bioscience, biotechnology, and biochemistry》2013,77(10):994-1003
Inactivation of Bacillus subtilis’ α-amylase by heat was found to be reversible under a certain condition, and the factors affecting there were investigated, distinguishing into two groups: those influencing on the inactivation process by heat and those on the reactivation at the subsequent incubation after heating. Generally, the amylase heated in borate buffer solution was best in the reactivation degree. For reactivation of the heat-inactivated enzyme there was found an optimum in temperature, pH and concentration of enzyme, respectively. The reactivation was temporarily prevented by urea, but irreversibly inhibited by either calcium salts or calcium binding agents. In the reversible heat-inactivation of the enzyme was also found a reversible change in the absorption spectra as well as in the behavior of the enzyme toward proteinase. 相似文献
7.
The inhibition of α-amylase from human saliva by polyphenolic components of tea and its specificity was investigated in vitro. Four kinds of green tea catechins, and their isomers and four kinds of their dimeric compounds (theaflavins) produced oxidatively during black tea production were isolated. They were (?)-epicatechin (EC), (?)-epigallocatechin (EGC), (?)-epicatechin gallate (ECg), (?)-epigallocatechin gallate (EGCg), (?)-catechin (C), (?)-gallocatechin (GC), (?)-catechin gallate (Cg), (?)-gallocatechin gallate (GCg), theaflavin (TF1), theaflavin monogallates (TF2A and TF2B), and theaflavin digallate (TF3). Among the samples tested, EC, EGC, and their isomers did not have significant effects on the activity of α-amylase. All the other samples were potent inhibitors and the inhibitory effects were in the order of TF3>TF2A>TF2B>TFl>Cg> GCg > ECg > EGCg. The inhibitory patterns were noncompetitive except for TF3. 相似文献
8.
Marek Gabriško 《Journal of molecular evolution》2013,76(3):129-145
Although some α-glucosidases from the α-amylase family (glycoside hydrolase family GH13) have been studied extensively, their exact number, organization on the chromosome, and orthology/paralogy relationship were unknown. This was true even for important disease vectors where gut α-glucosidase is known to be receptor for the Bin toxin used to control the population of some mosquito species. In some cases orthologs from related species were studied intensively, while potentially important paralogs were omitted. We have, therefore, used a bioinformatics approach to identify all family GH13 α-glucosidases from the selected species from Metazoa (including three mosquito species: Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus) as well as from Fungi in an effort to characterize their arrangement on the chromosome and evolutionary relationships among orthologs and among paralogs. We also searched for pseudogenes and genes coding for enzymatically inactive proteins with a possible new function. We have found GH13 α-glucosidases mostly in Arthropoda and Fungi where they form gene families, as a result of multiple lineage-specific gene duplications. In mosquito species we have identified 14 α-glucosidase (Aglu) genes of which only five have been biochemically characterized so far, two are putative pseudogenes and the rest remains uncharacterized. We also revealed quite a complex evolutionary history of the eukaryotic α-glucosidases probably involving multiple losses of genes or horizontal gene transfer from bacteria. 相似文献
9.
Kazumasa Hitotsuyanagi Kunio Yamane Bunji Maruo 《Bioscience, biotechnology, and biochemistry》2013,77(11):2343-2349
The production of extracellular α-amylase in Bacillus subtilis is probably regulated by many genetic elements, such as amyR, tmrA7, pap, amyB and sacU. Additional genetic elements, C-108 and A-2 for production of the α-amylase were found in D-cycloserine and ampicillin resistant mutants (C108 and A2) of B. subtilis 6160, respectively. Strain C108 increased the production of α-amylase about 5 times and protease about 80 times compared to parental 6160 strain. Strain A2 showed a nearly 6-fold increased α-amylase production.These genetic elements displayed a synergistic effect with other genetic factors in production of extracellular α-amylase when these elements were transferred by DNA mediated transformation. By stepwise introduction of these and other genetic elements into B. subtilis 6160 by transformation and mutation, strains with higher α-amylase producing activity were obtained. The finally obtained strain, T2N26, produced about 1,500-2,000 times more α-amylase than parental 6160 strain. 相似文献
10.
Subsequent production of amylase is severely inhibited if barban [4-chloro-2-butynyl N-(3-chlorophenyl) carbamate] is added to embryo-free half seeds of barley within 4 to 5 hours after gibberellic acid treatment of these seeds. Thirty to 50 mg/L concentrations of barban are effective. Barban inhibition is non-competitive with respect to gibberellic acid. Addition of barban 7 hours or more after gibberellic acid treatment is almost without effect.The delay between gibberellic acid treatment and amylase formation tends to become shorter with more prolonged imbibition periods. Regardless of imbibition period, susceptibility to barban is lost within 7 hours after gibberellic acid treatment.Other herbicidally active phenylurethanes are also inhibitors, but none are as effective as barban. Phenethyl alcohol and 2 arylcarbamates can act as inhibitors. 相似文献
11.
Kashchenko N. I. Chirikova N. K. Olennikov D. N. 《Russian Journal of Bioorganic Chemistry》2018,44(7):876-886
Russian Journal of Bioorganic Chemistry - Spiraea L. belongs to a genus of deciduous-leaved shrubs in the Rosaceae family that is abundant in Eastern Siberia. The study of six species of Spiraea... 相似文献
12.
Lorenzo Guglielminetti Héctor Abel Busilacchi Amedeo Alpi 《Journal of plant research》2000,113(2):185-192
α -amylase as well as other enzymes involved in starch degradation under anoxia. Carbohydrates resulting from starch breakdown
allow maize caryopses to avoid sugar starvation. Most interestingly, this correlates well with the ability of maize caryopses
to sustain relatively prolonged anaerobiosis, in agreement with the hypothesis linking carbohydrate availability to anoxia
tolerance.
Received 25 September 1999/ Accepted in revised form 24 February 2000 相似文献
13.
Dominic W. S. Wong Sarah B. Batt Brian K. Tibbot George H. Robertson 《The protein journal》2000,19(5):373-377
Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α7-and α8-helices of the (α/β)8 domain. 相似文献
14.
Kenzo Tomomura Hideo Suzuki Norio Nakamura Kenichi Kuraya Osamu Tanabe 《Bioscience, biotechnology, and biochemistry》2013,77(1):1-6
In a previous paper it has been described that α-amylase formation in Aspergillus oryzae is stimulated by soluble starch, glycogen and maltose, whereas it is inhibited by glucose, which is added into a growing medium or a secondary incubation medium as the carbon source. The present paper reports that isomaltose and panose are the most effective inducers among a large number of sugars examined here, and suggests the importance of transglucosidase action demonstrated in view of α-amylase formation. The initial action of inducers in this system is also discussed. 相似文献
15.
The structural X-ray map of a pig pancreatic α-amylase crystal soaked (and flash-frozen) with a maltopentaose substrate showed a pattern of electron density corresponding to the binding of oligosaccharides at the active site and at three surface binding sites. The electron density region observed at the active site, filling subsites ?3 through ?1, was interpreted in terms of the process of enzyme-catalyzed hydrolysis undergone by maltopentaose. Because the expected conformational changes in the “flexible loop” that constitutes the surface edge of the active site were not observed, the movement of the loop may depend on aglycone site being filled. The crystal structure was refined at 2.01 å resolution to an R factor of 17.0% (R free factor of 19.8%). The final model consists of 3910 protein atoms, one calcium ion, two chloride ions, 103 oligosaccharide atoms, 761 atoms of water molecules, and 23 ethylene glycol atoms. 相似文献
16.
AmyL, an extracellular α-amylase from Bacillus licheniformis, is resistant to extracellular proteases secreted by Bacillus subtilis during growth. Nevertheless, when AmyL is produced and secreted by B. subtilis, it is subject to considerable cell-associated proteolysis. Cell-wall-bound proteins CWBP52 and CWBP23 are the processed products of the B. subtilis wprA gene. Although no activity has been ascribed to CWBP23, CWBP52 exhibits serine protease activity. Using a strain encoding an inducible wprA gene, we show that a product of wprA, most likely CWBP52, is involved in the posttranslocational stability of AmyL. A construct in which wprA is not expressed exhibits an increased yield of α-amylase. The potential role of wprA in protein secretion is discussed, together with implications for the use of B. subtilis and related bacteria as hosts for the secretion of heterologous proteins.The cell envelope of the gram-positive bacterium Bacillus subtilis consists of a single (cytoplasmic) membrane surrounded by a relatively thick cell wall consisting of similar proportions of peptidoglycan and covalently attached anionic polymers. The absence of an outer membrane means that there is no equivalent of the membrane-enclosed periplasm found in gram-negative bacteria. However, by virtue of its thickness and high density of negative charge, the cell wall may perform some of the roles of the periplasm in gram-positive bacteria.The absence of an outer membrane in gram-positive bacteria also simplifies the secretion pathway, and, consequently, B. subtilis and its close relatives have the potential to secrete proteins directly into the growth medium, at concentrations in excess of 5 grams per liter (4). Despite its extensive use in the production of commercially important Bacillus enzymes (e.g., α-amylases and alkaline proteases), attempts to exploit B. subtilis for the production of heterologous proteins at high concentrations have proved disappointing (8). One reason for this failure is the production and release into the culture medium of several extracellular proteases (24, 28, 37). Although native Bacillus proteins are generally resistant to these proteases, heterologous proteins are often rapidly degraded in their presence. As a result, strains of B. subtilis that are multiply deficient in extracellular proteases have been developed (11, 37). The more developed of these strains have less than 1% of the proteolytic activity of the wild type (37). To date, efforts have concentrated mainly on the proteases which reside in a truly extracellular location, while those which remain cell associated have been largely overlooked.Although strains deficient in extracellular proteases have improved the productivity of B. subtilis for the production of heterologous proteins, they have only partially overcome problems of unexpectedly low yields. We and others have recently shown (22, 31) that significant amounts of secretory protein are degraded within minutes of being synthesized. This degradation is observed even for Bacillus proteins that are highly resistant to proteases released into the culture medium, suggesting that a component of this degradation is cell associated.Margot and Karamata recently reported the identification of a cell-wall-associated protease encoded by the wprA gene (21). The primary product of this gene is a 96-kDa polypeptide that is processed into two previously identified cell wall proteins, namely, CWBP52 and CWBP23. The processing of the WprA precursor during secretion accompanies the targeting of CWBP52 and CWBP23 to the cell wall and is analagous to the processing of another B. subtilis cell-wall-bound protein, namely, WapA (5). The amino acid sequence of CWBP52 shows a high degree of similarity with serine proteases of the subtilisin family, and phenylmethylsulfonile fluoride (PMSF)-sensitive protease activity was detected in proteins extracted from the cell wall of a wprA+ strain, but not one in which this gene had been insertionally inactivated (21). In the absence of homology to proteins in the databases, the N-terminal CWBP23 moiety was presumed to function as a chaperone-like propeptide that is proteolytically processed on the trans side of the membrane. In this paper, we report on a potential role of products of wprA in the integrity of secretory proteins during late stages in the secretion pathway. We also discuss the potential of wprA mutants to increase the productivity of B. subtilis for secretory proteins. 相似文献
17.
The primary structures of three α-amylase inhibitors (TAI, DAI, and MAI-2) consisting of glycoprotein subunits α and β from the respective seeds of three cultivars of Phaseolus beans, Toramame (Phaseolus vulgaris L.), Daifukumame (Phaseolus vulgaris L.), and Murasakihanamame (Phaseolus coccineus L.) were determined by sequencing the peptide fragments derived from their enzymatic digestions. Major sugar chains of the inhibitors were also assessed by analyzing glycopeptides in the enzymatic digests. The subunits, α and β, were shown to be composed of 76 and 139 amino acid residues, respectively, in each inhibitor. The overall amino acid sequences of the inhibitors were slightly different from one another. Furthermore, the sequence of TAI was the same as that deduced from a cDNA clone encording α-amylase inhibitor-1 from the common bean (Phaseolus vulgaris L.). It was also revealed that there were two N-glycosylation sites in each α-subunit: PA-derivatives of the major N-glycans were estimated to be M6B at Asn(12) and M9A at Asn(65). Each β-subunit of TAI and MAI-2 had two N-glycosylation sites, while the β-subunit of DAI had only one site. The major N-glycans pyridylaminated were estimated to be M3X at Asn(63) in each β-subunit and M3FX at Asn(83) in β-subunits of TAI and MAI-2. 相似文献
18.
19.
Kenzo Tonomura Fusae Futai Osamu Tanabe 《Bioscience, biotechnology, and biochemistry》2013,77(7):434-441
It was previously reported that α-amylase accumulation is caused within the mycelium grown in a phosphate deficient medium and the concentration of anions or pH in a surrounding medium is responsible for its liberation. As it was subsequently found that α-amylase liberation from the mycelium of Aspergillus oryzae is stimulated by peptone, an attempt was made on purification of effective substances from it. The present paper describes on purification and properties of phosphopeptides found as an effective substance for α-amylase liberation, and discusses on the stimulation effect, comparing with the effects on pH and concentration of anions which were previously observed. 相似文献
20.
The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their
domain B, a distinct domain that protrudes from the regular catalytic (β/α)8-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed
by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional
structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different
forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in
the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase,
cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B
of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable
similarity with (β/α)8-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in
turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only
parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family.
Received: 4 December 1996 / Accepted: 13 March 1997 相似文献