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Andrea Brandolini - One of the best experts on this subject based on the ideXlab platform.
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polyphenol oxidase alpha amylase and beta amylase activities of triticum monococcum triticum turgidum and triticum aestivum a two year study
Journal of Cereal Science, 2013Co-Authors: Alyssa Hidalgo, Marta Brusco, L Plizzari, Andrea BrandoliniAbstract:Abstract Enzymatic activity often reduces the nutritional value of wheat flour during food manufacturing, causing compound degradation and/or heat damage. The choice of wheat varieties with low enzymatic activity could therefore help to preserve the nutritional quality of food. The aim of this research was to evaluate polyphenol oxidase, alpha-amylase and Beta-Amylase activities in whole meal flours of 59 accessions belonging to different wheat species and subspecies, cropped in two years. The extraction pH (7.0), reaction pH (5.5) and reaction temperature (45 °C) were determined by preliminary trials. The ANOVA highlighted significant differences for all enzymes among species/subspecies and, for amylases, between cropping years; however, the year influence was overwhelming only for alpha-amylase. Einkorn showed the highest polyphenol oxidase (362.1 ± 9.46 U/g DM) as well as the lowest alpha-amylase (0.20 ± 0.006 CU/g DM) and Beta-Amylase (12.0 ± 0.36 B3U/g DM) activities. The embryo/scutellum had the highest polyphenol oxidase and alpha-amylase values, followed by the bran and the endosperm; in contrast, Beta-Amylase was evenly distributed in the bran and the endosperm, and was absent in the embryo/scutellum.
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Polyphenol oxidase, alpha-amylase and Beta-Amylase activities of Triticum monococcum, Triticum turgidum and Triticum aestivum: A two-year study
Journal of Cereal Science, 2013Co-Authors: Alyssa Hidalgo, Marta Brusco, L Plizzari, Andrea BrandoliniAbstract:Abstract Enzymatic activity often reduces the nutritional value of wheat flour during food manufacturing, causing compound degradation and/or heat damage. The choice of wheat varieties with low enzymatic activity could therefore help to preserve the nutritional quality of food. The aim of this research was to evaluate polyphenol oxidase, alpha-amylase and Beta-Amylase activities in whole meal flours of 59 accessions belonging to different wheat species and subspecies, cropped in two years. The extraction pH (7.0), reaction pH (5.5) and reaction temperature (45 °C) were determined by preliminary trials. The ANOVA highlighted significant differences for all enzymes among species/subspecies and, for amylases, between cropping years; however, the year influence was overwhelming only for alpha-amylase. Einkorn showed the highest polyphenol oxidase (362.1 ± 9.46 U/g DM) as well as the lowest alpha-amylase (0.20 ± 0.006 CU/g DM) and Beta-Amylase (12.0 ± 0.36 B3U/g DM) activities. The embryo/scutellum had the highest polyphenol oxidase and alpha-amylase values, followed by the bran and the endosperm; in contrast, Beta-Amylase was evenly distributed in the bran and the endosperm, and was absent in the embryo/scutellum.
De Evans - One of the best experts on this subject based on the ideXlab platform.
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A single amino acid substitution that determines IEF band pattern of barley Beta-Amylase
Journal of Cereal Science, 2002Co-Authors: Peter Langridge, Sj Logue, De EvansAbstract:Abstract Barley beta -amylase exhibits two distinct band patterns after isoelectric focusing (IEF), termed Sd1 and Sd2. A comparison of the deduced amino acid sequences revealed five amino acid differences between the two types ofbeta -amylase. To investigate whether the two band patterns are due to these amino acid substitutions, four Sd2 mutants (Sd2-R115C, Sd2-D165E, Sd2-L347S and Sd2-V430A) were constructed by site-directed mutagenesis. The analysis of IEF band patterns of mutant and wild-type beta -amylases revealed that only the replacement of R115 with cysteine converted the Sd2 band pattern to the Sd1 one. The contribution of the R115C substitution to the IEF band pattern of barley beta -amylase was further confirmed by generating a Sd1 mutant (Sd1-C115R), where cysteine at this position was in turn replaced by arginine. As a result of this mutation, the Sd1 band pattern was converted into the Sd2 pattern. Calculation of the electrostatic potential and reducing agent treatment revealed that the R115C substitution altered both the net charge on the protein surface and intermolecular interactions by disulfide bonds, thereby altering the IEF band pattern of barley beta -amylase.
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Removal of the four C-terminal glycine-rich repeats enhances the thermostability and substrate binding affinity of barley Beta-Amylase.
Biochemistry, 2000Co-Authors: Jason Eglinton, De Evans, Sj Logue, Peter LangridgeAbstract:Barley Beta-Amylase undergoes proteolytic cleavage in the C-terminal region after germination. The implication of the cleavage in the enzyme's characteristics is unclear. With purified native Beta-Amylases from both mature barley grain and germinated barley, we found that the Beta-Amylase from germinated barley had significantly higher thermostability and substrate binding affinity for starch than that from mature barley grain. To better understand the effect of the proteolytic cleavage on the enzyme's thermostability and substrate binding affinity for starch, recombinant barley Beta-Amylases with specific deletions at the C-terminal tail were generated. The complete deletion of the four C-terminal glycine-rich repeats significantly increased the enzyme's thermostability, but an incomplete deletion with one repeat remaining did not change the thermostability. Although different C-terminal deletions affect the thermostability differently, they all increased the enzyme's affinity for starch. The possible reasons for the increased thermostability and substrate binding affinity, due to the removal of the four C-terminal glycine-rich repeats, are discussed in terms of the three-dimensional structure of Beta-Amylase.
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Comparative Enzyme Kinetics of Two Allelic Forms of Barley (Hordeum vulgare L.) Beta -amylase
Journal of Cereal Science, 2000Co-Authors: D.c. Stewart, Peter Langridge, Jason Eglinton, Sj Logue, De EvansAbstract:Abstract The barley ( Hordeum vulgare L.) varieties, Franklin and Schooner, contain two different allelic forms of beta -amylase (EC 3.2.1.2) encoded on chromosome 4H by the Bmy 1-Sd1 and Bmy 1-Sd2L alleles, respectively. The corresponding enzymes, referred to as Sd1 and Sd2L, were purified from both mature barley grain and germinated barley (green malt), and their physical and kinetic properties studied. Approximately 4 kDa were cleaved from both Sd1 and Sd2L beta -amylases after germination. The K m value for green malt beta -amylase was less than that of mature grain beta -amylase for both varieties when potato starch was used as a substrate, although V max was similar. This indicated that proteolysis after germination increased the affinity of beta -amylase for potato starch. No significant kinetic differences were observed between beta -amylase from mature grain and green malt of the two barley varieties when amylose (degree of polymerisation 100 and 18) and maltopentaose were used as substrates. Kinetic differences were also observed between the two allelic forms of beta -amylase. Sd1 beta -amylase from green malt exhibited a lower K m value for potato starch than Sd2L beta -amylase, demonstrating that at non-saturating starch concentrations Sd1 beta -amylase is better able to hydrolyse starch than Sd2L beta -amylase. As the degree of polymerisation of the substrates decreased from approximately 740 (potato starch) to 5 (maltopentaose), the K m values for beta -amylase increased, whereas V max values decreased. Maltose, the hydrolytic product of beta -amylase, was found to be a weak competitive inhibitor of both Sd1 and Sd2L green malt beta -amylases with respect to potato starch and amylose. Taken together the kinetic observations for bet a-amylase suggest that the allelic differences and C-terminal proteolysis might be exploited to improve the efficiency of starch hydrolysis during the mashing stage of the brewing process.
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Thermostability variation in alleles of barley Beta-Amylase
Journal of Cereal Science, 1998Co-Authors: Jason Eglinton, Peter Langridge, De EvansAbstract:Abstract Thermostability assays in conjunction with IEF and molecular mapping were used to identify three Beta-Amylase alleles (Bmyl-Sd1, -Sd2L, -Sd2H) in cultivated barley and an additional allele (Bmy1-Sd3) in an accession of wild barley Hordeum vulgare ssp. spontaneum. The four forms of Beta-Amylase exhibit different rates of thermal inactivation in barley extracts. This variation was shown to persist after the proteolytic processing of the enzyme that occurs during germination. Three forms of Beta-Amylase representing the range of thermostabilities were purified and shown to have T50 temperatures of 56·8°C for the Sd2L enzyme, 58·5°C for the Sd1 enzyme, and 60·8°C for the Sd3 Beta-Amylase from wild barley. Analysis of the relationship between Beta-Amylase thermostability and fermentability, i.e. the yield of fermentable sugars obtained from starch hydrolysis during brewing in 42 commercial malt samples suggests that increased thermostability results in more efficient starch degradation. Screening for specific Beta-Amylase alleles is proposed as a method for increasing fermentability in malting barley.
Alyssa Hidalgo - One of the best experts on this subject based on the ideXlab platform.
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polyphenol oxidase alpha amylase and beta amylase activities of triticum monococcum triticum turgidum and triticum aestivum a two year study
Journal of Cereal Science, 2013Co-Authors: Alyssa Hidalgo, Marta Brusco, L Plizzari, Andrea BrandoliniAbstract:Abstract Enzymatic activity often reduces the nutritional value of wheat flour during food manufacturing, causing compound degradation and/or heat damage. The choice of wheat varieties with low enzymatic activity could therefore help to preserve the nutritional quality of food. The aim of this research was to evaluate polyphenol oxidase, alpha-amylase and Beta-Amylase activities in whole meal flours of 59 accessions belonging to different wheat species and subspecies, cropped in two years. The extraction pH (7.0), reaction pH (5.5) and reaction temperature (45 °C) were determined by preliminary trials. The ANOVA highlighted significant differences for all enzymes among species/subspecies and, for amylases, between cropping years; however, the year influence was overwhelming only for alpha-amylase. Einkorn showed the highest polyphenol oxidase (362.1 ± 9.46 U/g DM) as well as the lowest alpha-amylase (0.20 ± 0.006 CU/g DM) and Beta-Amylase (12.0 ± 0.36 B3U/g DM) activities. The embryo/scutellum had the highest polyphenol oxidase and alpha-amylase values, followed by the bran and the endosperm; in contrast, Beta-Amylase was evenly distributed in the bran and the endosperm, and was absent in the embryo/scutellum.
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Polyphenol oxidase, alpha-amylase and Beta-Amylase activities of Triticum monococcum, Triticum turgidum and Triticum aestivum: A two-year study
Journal of Cereal Science, 2013Co-Authors: Alyssa Hidalgo, Marta Brusco, L Plizzari, Andrea BrandoliniAbstract:Abstract Enzymatic activity often reduces the nutritional value of wheat flour during food manufacturing, causing compound degradation and/or heat damage. The choice of wheat varieties with low enzymatic activity could therefore help to preserve the nutritional quality of food. The aim of this research was to evaluate polyphenol oxidase, alpha-amylase and Beta-Amylase activities in whole meal flours of 59 accessions belonging to different wheat species and subspecies, cropped in two years. The extraction pH (7.0), reaction pH (5.5) and reaction temperature (45 °C) were determined by preliminary trials. The ANOVA highlighted significant differences for all enzymes among species/subspecies and, for amylases, between cropping years; however, the year influence was overwhelming only for alpha-amylase. Einkorn showed the highest polyphenol oxidase (362.1 ± 9.46 U/g DM) as well as the lowest alpha-amylase (0.20 ± 0.006 CU/g DM) and Beta-Amylase (12.0 ± 0.36 B3U/g DM) activities. The embryo/scutellum had the highest polyphenol oxidase and alpha-amylase values, followed by the bran and the endosperm; in contrast, Beta-Amylase was evenly distributed in the bran and the endosperm, and was absent in the embryo/scutellum.
Peter Langridge - One of the best experts on this subject based on the ideXlab platform.
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mapping of barley hordeum vulgare l beta amylase alleles in which an amino acid substitution determines beta amylase isoenzyme type and the level of free beta amylase
Journal of Cereal Science, 2002Co-Authors: Peter Langridge, Xiaoqi Zhang, P E Eckstein, B G Rossnagel, R C M Lance, E Lefol, Bryan L Harvey, G J ScolesAbstract:The three beta -amylase genes (Bmy1, 2 and 3) in cultivated barley were mapped to chromosomes 4HL, 2HL And 4HL respectively using RFLP analysis. No recombinants between Bmy1 andBmy3 were detected among 264 DH lines. Polymorphism of the Sd1 and Sd2 isoenzymes of beta -amylase co-segregated with the Bmy loci on chromosome 4HL in a doubled-haploid population of the cross Chebec (Sd2)×Harrington (Sd1). This locus also explained 90·5% of the variation in the level of free enzyme between the two parents. Two cDNAs ofbeta -amylase were isolated by RT-PCR from the developing grains of Harrington (Sd1) and Galleon (Sd2). Alignment of the deduced amino acid sequences identified three amino-acid substitutions between the Sd2 and Sd1 forms of beta -amylase (Arg115 – Cys, Asp165 – Glu, and Val430 – Ala). Three allele-specific PCR primer pairs based on the three amino acid substitutions were used to amplify the beta -amylase genes in genomic DNA of sixteen barley cultivars/lines. Only the Arg115(Sd2)/Cys(Sd1) substitution was consistent with the isoenzyme form. This amino acid replacement reduced the pI of the Sd1 beta -amylase consistent with the fact that the Sd2 form is more basic than the Sd1 form when separated by IEF. The mutation from Arg115 to Cys in the Sd1 form also provides one more -SH group to form S-S-bridges. As bound beta -amylase is linked to the insoluble proteins of the endosperm and its inhibitor via disulphide bridges this could explain the higher level of binding exhibited by Sd1 vs Sd2. Thus a single amino acid substitution determines both the isoenzyme type and beta -amylase binding.
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A single amino acid substitution that determines IEF band pattern of barley Beta-Amylase
Journal of Cereal Science, 2002Co-Authors: Peter Langridge, Sj Logue, De EvansAbstract:Abstract Barley beta -amylase exhibits two distinct band patterns after isoelectric focusing (IEF), termed Sd1 and Sd2. A comparison of the deduced amino acid sequences revealed five amino acid differences between the two types ofbeta -amylase. To investigate whether the two band patterns are due to these amino acid substitutions, four Sd2 mutants (Sd2-R115C, Sd2-D165E, Sd2-L347S and Sd2-V430A) were constructed by site-directed mutagenesis. The analysis of IEF band patterns of mutant and wild-type beta -amylases revealed that only the replacement of R115 with cysteine converted the Sd2 band pattern to the Sd1 one. The contribution of the R115C substitution to the IEF band pattern of barley beta -amylase was further confirmed by generating a Sd1 mutant (Sd1-C115R), where cysteine at this position was in turn replaced by arginine. As a result of this mutation, the Sd1 band pattern was converted into the Sd2 pattern. Calculation of the electrostatic potential and reducing agent treatment revealed that the R115C substitution altered both the net charge on the protein surface and intermolecular interactions by disulfide bonds, thereby altering the IEF band pattern of barley beta -amylase.
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Removal of the four C-terminal glycine-rich repeats enhances the thermostability and substrate binding affinity of barley Beta-Amylase.
Biochemistry, 2000Co-Authors: Jason Eglinton, De Evans, Sj Logue, Peter LangridgeAbstract:Barley Beta-Amylase undergoes proteolytic cleavage in the C-terminal region after germination. The implication of the cleavage in the enzyme's characteristics is unclear. With purified native Beta-Amylases from both mature barley grain and germinated barley, we found that the Beta-Amylase from germinated barley had significantly higher thermostability and substrate binding affinity for starch than that from mature barley grain. To better understand the effect of the proteolytic cleavage on the enzyme's thermostability and substrate binding affinity for starch, recombinant barley Beta-Amylases with specific deletions at the C-terminal tail were generated. The complete deletion of the four C-terminal glycine-rich repeats significantly increased the enzyme's thermostability, but an incomplete deletion with one repeat remaining did not change the thermostability. Although different C-terminal deletions affect the thermostability differently, they all increased the enzyme's affinity for starch. The possible reasons for the increased thermostability and substrate binding affinity, due to the removal of the four C-terminal glycine-rich repeats, are discussed in terms of the three-dimensional structure of Beta-Amylase.
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Comparative Enzyme Kinetics of Two Allelic Forms of Barley (Hordeum vulgare L.) Beta -amylase
Journal of Cereal Science, 2000Co-Authors: D.c. Stewart, Peter Langridge, Jason Eglinton, Sj Logue, De EvansAbstract:Abstract The barley ( Hordeum vulgare L.) varieties, Franklin and Schooner, contain two different allelic forms of beta -amylase (EC 3.2.1.2) encoded on chromosome 4H by the Bmy 1-Sd1 and Bmy 1-Sd2L alleles, respectively. The corresponding enzymes, referred to as Sd1 and Sd2L, were purified from both mature barley grain and germinated barley (green malt), and their physical and kinetic properties studied. Approximately 4 kDa were cleaved from both Sd1 and Sd2L beta -amylases after germination. The K m value for green malt beta -amylase was less than that of mature grain beta -amylase for both varieties when potato starch was used as a substrate, although V max was similar. This indicated that proteolysis after germination increased the affinity of beta -amylase for potato starch. No significant kinetic differences were observed between beta -amylase from mature grain and green malt of the two barley varieties when amylose (degree of polymerisation 100 and 18) and maltopentaose were used as substrates. Kinetic differences were also observed between the two allelic forms of beta -amylase. Sd1 beta -amylase from green malt exhibited a lower K m value for potato starch than Sd2L beta -amylase, demonstrating that at non-saturating starch concentrations Sd1 beta -amylase is better able to hydrolyse starch than Sd2L beta -amylase. As the degree of polymerisation of the substrates decreased from approximately 740 (potato starch) to 5 (maltopentaose), the K m values for beta -amylase increased, whereas V max values decreased. Maltose, the hydrolytic product of beta -amylase, was found to be a weak competitive inhibitor of both Sd1 and Sd2L green malt beta -amylases with respect to potato starch and amylose. Taken together the kinetic observations for bet a-amylase suggest that the allelic differences and C-terminal proteolysis might be exploited to improve the efficiency of starch hydrolysis during the mashing stage of the brewing process.
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Thermostability variation in alleles of barley Beta-Amylase
Journal of Cereal Science, 1998Co-Authors: Jason Eglinton, Peter Langridge, De EvansAbstract:Abstract Thermostability assays in conjunction with IEF and molecular mapping were used to identify three Beta-Amylase alleles (Bmyl-Sd1, -Sd2L, -Sd2H) in cultivated barley and an additional allele (Bmy1-Sd3) in an accession of wild barley Hordeum vulgare ssp. spontaneum. The four forms of Beta-Amylase exhibit different rates of thermal inactivation in barley extracts. This variation was shown to persist after the proteolytic processing of the enzyme that occurs during germination. Three forms of Beta-Amylase representing the range of thermostabilities were purified and shown to have T50 temperatures of 56·8°C for the Sd2L enzyme, 58·5°C for the Sd1 enzyme, and 60·8°C for the Sd3 Beta-Amylase from wild barley. Analysis of the relationship between Beta-Amylase thermostability and fermentability, i.e. the yield of fermentable sugars obtained from starch hydrolysis during brewing in 42 commercial malt samples suggests that increased thermostability results in more efficient starch degradation. Screening for specific Beta-Amylase alleles is proposed as a method for increasing fermentability in malting barley.
Naohiro Yoshigi - One of the best experts on this subject based on the ideXlab platform.
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Role of the C-Terminal Region of β-Amylase from Barley
Journal of biochemistry, 1995Co-Authors: Naohiro Yoshigi, Hirohisa Sahara, Shohei KoshinoAbstract:To investigate the role of the C-terminal region of barley Beta-Amylase, plasmid delta 54 was constructed with an expression vector (pBETA92) of barley Beta-Amylase by site-directed mutagenesis. Escherichia coli JM109 harboring plasmid delta 54 was expected to express delta 54 Beta-Amylase in which 54 amino acid residues were deleted from the C-terminus. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. delta 54 Beta-Amylase gave a single activity band on isoelectric focusing (pI 6.85). delta 54 Beta-Amylase was purified from the cells by consecutive alpha-cyclodextrin/Sepharose 6B column chromatography. A comparison of the properties of the mutant enzyme with those of the original recombinant Beta-Amylase [Biosci. Biotech. Biochem. (1994) 58, 1080-1086] revealed two major differences. First, the original recombinant Beta-Amylase showed heterogeneity on isoelectric focusing, but delta 54 Beta-Amylase gave a single main band of protein (pI 6.85). Therefore, the isoelectrophoretic heterogeneity of the original recombinant Beta-Amylase was apparently due to its C-terminal region. Secondly, delta 54 Beta-Amylase lacked thermostability. Therefore, it was concluded that the C-terminal region was significantly involved in the thermostability of Beta-Amylase.
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Construction of a Plasmid Used for the Expression of a Sevenfold-Mutant Barley β-Amylase with Increased Thermostability in Escherichia coli and Properties of the Sevenfold-Mutant β-Amylase
Journal of biochemistry, 1995Co-Authors: Naohiro Yoshigi, Hirohisa Sahara, Yukio Okada, Hideo Maeba, Teruo TamakiAbstract:To increase the thermostability of Beta-Amylase, seven kinds of single-mutant plasmids were constructed with an expression vector of barley Beta-Amylase by mutagenesis. The remaining activity versus temperature curves were used to determine the temperatures (T50) at which 50% of the initial activity was lost during a 30-min heating period. These mutations increased the T50 values by amounts ranging from 0.8 to 3.2 degrees C. To express the sevenfold-mutant Beta-Amylase in Escherichia coli, plasmid pB927 was constructed. E. coli harboring plasmid pB927 produced sevenfold-mutant beta- amylase. The T50 value of purified sevenfold-mutant Beta-Amylase (69.0 degrees C) was higher than that of not only the original recombinant Beta-Amylase (57.4 degrees C) by 11.6 degrees C but also soybean Beta-Amylase (63.2 degrees C) by 5.8 degrees C. The intragenic amino acid replacements were found to have simple additive effects on the thermostability of Beta-Amylase. The sevenfold-mutant Beta-Amylase was found to be stable at pHs up to 12.5, while the original recombinant Beta-Amylase was unstable at pHs above 9.5. The data obtained from kinetics studies suggested that the sevenfold-mutant Beta-Amylase acquired enhanced thermostability, but its function as a Beta-Amylase remained unchanged.
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PCR cloning and sequencing of the Beta-Amylase cDNA from barley.
Journal of biochemistry, 1994Co-Authors: Naohiro Yoshigi, Yukio Yaizu-shi Okada, Hirohisa Sahara, Shohei KoshinoAbstract:Polymerase chain reaction (PCR) amplification of mRNA from developing barley (cultivar Haruna two-rows) endosperm was used to clone and sequence full-length cDNA encoding Beta-Amylase. The Beta-Amylase cDNA was 1,775 bp in length. The Beta-Amylase was deduced to be composed of 535 amino acid residues and its molecular weight was calculated to be 59,610. Kreis et al. reported that the Beta-Amylase cDNA from barley (cultivar Hiproly) was 1,754 bp in length and coded for a polypeptide of 535 amino acids [Eur. J. Biochem. (1987) 169, 517-525]. A comparison of the Beta-Amylase sequences from Haruna two-rows and Hiproly barleys revealed nine differences in the nucleotide sequence which resulted in three changes in the amino acid residues and 21 additional nucleotides at its 3'-end in the cultivar Haruna two-rows. The three changes were as follows: Ala-233, Ser-347, Met-527 (Haruna two-rows) and Val-233, Met-347, Ile-527 (Hiproly). Lundgard and Svensson pointed out that 23 amino acid residues of the peptide fragment derived from the COOH-terminal region of barley (cultivar Gula) Beta-Amylase were in agreement with the deduced amino acid sequence reported by Kreis et al., with the exception of a single position (Met-527 compared to Ile) [Carlsberg Res. Commun. (1986) 51, 487-491]. Our findings described above showed Met-527 is reasonable. In the cases of Beta-Amylases from soybean and sweet potato, the positions that corresponded to those at 233 and 347 in the amino acid sequence of Beta-Amylase from barley were Ala and Ser, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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Expression in Escherichia coli of cDNA Encoding Barley β-Amylase and Properties of Recombinant β-Amylase
Bioscience biotechnology and biochemistry, 1994Co-Authors: Naohiro Yoshigi, Hirohisa Sahara, Yukio Okada, Shohei KoshinoAbstract:To express the cloned Beta-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 Beta-Amylase activity that produced beta-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley Beta-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 Beta-Amylase having a molecular weight of about 56,000 was synthesized. The recombinant Beta-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 Beta-Amylase lacked four amino acids at positions 2-5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley Beta-Amylase. Therefore, the recombinant Beta-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 Beta-Amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley Beta-Amylase) at positions 27-29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant Beta-Amylase were almost the same as those of barley Beta-Amylase except for the pI and the Km values for maltohexaose and maltoheptaose.(ABSTRACT TRUNCATED AT 250 WORDS)