Production of thermotolerant β-amylase byBacillus circulans

An isolate from a Hong Kong soil sample which produced -amylase was identified as a thermotolerant strain ofBacillus circulans with a growth range of 35 to 55C. The -amylase was stable at 45°C for 30 min but lost half of its activity after 30 min at 50°C. Maximum specific activity of -amylase (36.2 units/mg protein) in the culture broth was detected after 36 h of cultivation at 45°C in a medium containing soluble starch, beef extract, coconut water and inorganic salts.     

Formation of amyloid fibrils from β-amylase

Fibril formation has been considered a significant feature of amyloid proteins. However, it has been proposed that fibril formation is a common property of many proteins under appropriate conditions. We studied the fibril formation of β-amylase, a non-amyloid protein rich in α-helical structure, because the secondary structure of β-amylase is similar to that of prions. With the conditions for the fibril formation of prions, β-amylase proteins were converted into amyloid fibrils. The features of β-amylase proteins and fibrils are compared to prion proteins and fibrils. Furthermore, the cause of neurotoxicity in amyloid diseases is discussed.     

Characterization of cDNA clones for rye endosperm β-amylase and analysis of β-amylase deficiency in rye mutant lines

Summary In order to characterize a -amylase deficiency in the endosperm of mutant rye lines, homologous cDNA probes were prepared. A rye cDNA library was constructed from a normal line and screened with a barley -amylase probe. Three partial cDNA clones specific for endosperm -amylase in rye were isolated and characterized. The largest of these clones was used to investigate the expression of endosperm -amylase in mutant and normal lines by Northern hybridization. These experiments, as well as in vitro translation experiments, demonstrate the absence of endosperm -amylase mRNA in mutant lines. Sequencing of three different cDNA clones revealed a single nucleotide difference, which suggests that two genes encoding endosperm -amylase genes might exist in rye. From Southern blots we anticipate that these two genes are tightly linked. Results of these experiments and previous data indicating that the mutation was located within the -amylase locus on chromosome 5 are consistent with the hypothesis that the mutation results from a deletion simultaneously affecting the two genes. However, due to extensive polymorphism within normal lines used as control, additional experiments will be required to further substantiate this conclusion. The deduced amino acid sequence reveals the occurrence of three short glycine-rich repeats containing 11 or 12 residues close to the carboxyl terminus of the protein. A comparison of the nucleotide sequence between rye and previously described barley cDNA clones revealed ca. 90% homology at the amino acid level, except in this C-terminal repeated part, where it drops to 45%.     

Construction of an industrial polyploid strain of Saccharomyces cerevisiae containing Saprolegnia ferax β-amylase gene and secreting β-amylase

An industrial polyploid strain of Saccharomyces cerevisiae containing Saprolegnia ferax -amylase gene was developed by using two yeast integrating plasmids. One plasmid was constructed that contains the geneticin resistance gene (Gt^r) as the selection marker and the ribosomal DNA (rDNA) portion that comprises the 18S rDNA as the recombination site. The other plasmid contains the aureobasidin A resistance gene (AUR1-C) as the selection marker and the chromosomal Ty sequence as the recombination site. The -amylase activity of one clone of Saccharomyces cerevisiae transformed sequentially with these two plasmids was approx. 9 times higher than that of Saprolegnia ferax. This type of integration was mitotically stable even after 100 generations of cell multiplication under non-selective conditions.     

Immunological studies on β-amylase of Bacillus cereus

Summary An antiserum against the -amylase from Bacillus cereus BQ10-S1 Spo II was prepared using rabbits. The antiserum obtained was confirmed to form a specific immunoprecipitate with the purified -amylase and showed a single band of protein with a molecular weight of 6.0x10⁴ on the nitrocellulose sheet by the Western-Blotting method. The antiserum showed a precipitin line with the -amylase from B. megaterium strain no. 32 by the Ouchterlony technique. However, the spur was formed on the Ouchterlony plate between the line of immunoprecipitin of the -amylase from B. cereus BQ10-S1 Spo II and that from B. megaterium strain no. 32. On the other hand, no immune reaction occurred with the -amylase from B. polymyxa no. 72 and those from higher plants such as soybean and barley. B. cereus BQ10-S1 Spo II was found to secret -amylase mainly from the mid to the late logarithnic phase of cell growth. With the use of antiserum, the amount of the -amylase secreted was estimated to be about 52 g/10⁹ cells, that of the parent strain (B. cereus BQ10-S1) about 14 g/10⁹ cells. These quantities of -amylase corresponded in each case with enzyme productivity of the two strains (about 1,100 U/ml and 270 U/ml).Abbreviations EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

The β-amylase polymorphism of winter common wheat grains

The polymorphism of winter common wheat with respect to β-amylase isoenzymes has been analyzed using electrophoresis in polyacrylamide gel (PAAG) buffered with a Tris-glycine system (pH 8.3). Seven β-amylase isoenzymes have been found in wheat cultivars and the breeding stock. Isoenzymes A, B, and C are the most frequent in Russian and Ukrainian cultivars (51.7 4.7, 30.7 3.8, and 11.9 2.5%, respectively). Two alleles of the β-Amy-D1 locus of the long arm of chromosome 4D have been identified. The substrate-enzyme affine effect can be used to locate the zones of activity of this enzyme by means of staining for proteins. It has been determined that β-amylase zymotypes may play a role in the aggregating capacity of the grain protein complex via the formation of S-S bonds.     

Subcellular distribution of β-amylase in developing barley endosperm

Protein bodies were isolated from maturing barley (Hordeum vulgare L. cv. Menuet) endosperm using sucrose density gradient centrifugation. Isolated protein bodies were not contaminated by other organelles such as mitochondria and microbodies, and contained a large amount of the barley storage protein, hordein, indicating that the protein bodies were prepared in high purity and were intact. Enzymatic and immunochemical analysis showed that β-amylase, which was previously thought to be localized in protein bodies, was not detected in the protein body fraction, but was found only in the cytosol fraction. The results show that β-amylase is synthesized as a cytosolic protein in maturing barley endosperm.     

A somaclonal variant of wheat with additional β-amylase isozymes

Summary The progeny of 149 plants regenerated from tissue culture of immature wheat (Triticum aestivum) embryos were screened for variation in their grain -amylase isozyme pattern. One regenerant was found which was heterozygous for a variant pattern characterized by the presence of at least five new isozyme bands, as well as an increased intensity in existing bands in two more positions. The F₂ of a homozygous variant crossed back to the parent segregated in an approximate 31 ratio but resolution of the gels was not sufficient to distinguish whether this represents a dominant or co-dominant single mutant gene. No chromosome abnormalities were evident in mitosis or meiosis of the homozygous variant or in the F₁ of the variant crossed back to the parent. No recombination has been seen between the variant bands and production of multiple bands from a single locus is consistent with the nature of the known -amylase loci. However, the variant bands were not evident in a survey of 111 diverse genotypes, nor were they present in developing grain of the parent cultivar. Therefore, this variant could represent a rare mutation leading to expression of a currently unexpressed locus.     

Genetic studies on the β-amylase isozymes of barley malt

From genetic studies on two -amylase forms (Sd1 and Sd2) observed in barley malts it is concluded that the Sd1 and Sd2 phenotypes are controlled by a pair of alleles acting without dominance. Gene dosage effects typical of characters determined by the endosperm were observed in F₁ hybrids, Preliminary studies on the distribution of Sd1 and Sd2 forms indicate that Sd1 is the rarer one; of thirty-one cultivars tested, twenty were Sd2, nine were typed as Sd1 and two cultivars had both Sd1 and Sd2 phenotypes. An analysis of joint segregations for Sd1, Sd2, the heat-stable -amylases and two esterase forms gave no evidence for linkage between these three loci.     

Unexpected mode of action of sweet potato β-amylase on maltooligomer substrates

β-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme catalyzing the hydrolysis of α(1,4) glycosidic linkages in polysaccharides and removes successively maltose units from the non-reducing ends. The enzyme belongs to glycoside hydrolase GH14 family and inverts the anomeric configuration of the hydrolysis product. Multiple attack or processivity is an important property of polymer active enzymes and there is still limited information about the processivity of carbohydrate active enzymes. Action pattern and kinetic measurements of sweet potato β-amylase were made on a series of aromatic chromophor group-containing substrates (degree of polymerization DP 3-13) using HPLC method. Measured catalytic efficiencies increased with increasing DP of the substrates. Processive cleavage was observed on all substrates except the shortest pentamer. The mean number of steps without dissociation of enzyme–product complex increases with DP of substrate and reached 3.3 in case of CNPG11 indicating that processivity on longer substrates was more significant. A unique transglycosylation was observed on those substrates, which suffer processive cleavage and the substrates were re-built by the enzyme. Our results are the first presentation of a transglycosylation during an inverting glycosidase catalyzed hydrolysis. The yield of transglycosylation was remarkable high as shown in the change of the CNPG11 quantity. The CNPG11 concentration was doubled (from 0.24 to 0.54 mM) in the early phase of the reaction.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Location of β-amylase sequences in wheat and its relatives

Summary A -amylase cDNA clone isolated from barley has been used to locate -amylase encoding sequences on wheat, rye, and Aegilops umbellulata chromosomes by hybridisation to restriction endonuclease digested DNA obtained from wheat aneuploid and wheat-alien addition lines. Structural genes were identified on homoeologous group 4 and 5 chromosomes, confirming the results of isozyme studies. In addition, a further set of structural genes was found on homoeologous group 2 chromosomes. It is proposed that there are two homoeoallelic series, -Amy-1 on group 4 or 5 chromosomes, and -Amy-2 on group 2 chromosomes. Evidence is presented that each locus contains one or two -amylase structural genes, and it is suggested that the large number of isozymes seen upon IEF are due to post-translational modifications.     

Allelic characteristics of the β-amylase gene of barley varieties in Ukraine

Allelic diversity in a set of 99 spring and winter barley varieties intended for different use (malting, cereal, and valuable) was studied. PCR analysis with the use of the β-amylase DNA marker showed that genotypes of different barley varieties may include different alleles of the β-amy1 gene.     

Multiple molecular forms of β-amylase in seeds and vegetative tissues of barley

The molecular forms of -amylase present in developing, mature, germinating and malted grains of barley (Hordeum vulgare L.), and in vegetative tissues, have been studied using Western-blot analyses and isoelectric focusing of isoenzymes. Five isoforms with different relative molecular masses (M_rs) could be recognised. The major isoform present in the mature grain, called isoform B, had an M_r of about 60 000. This was converted on malting or germination to two lower-M_r forms called C and D. Previous work (R. Lundgard and B. Svensson, 1986, Carlsberg Res. Commun. 51, 487–491) has shown that these result from partial proteolysis of isoform B. Isoenzyme analyses showed complex patterns of bands, with pIs between about 5.0 and 6.0. Two allelic types were present in the eight lines. A number of new bands with a range of pIs appeared during germination and malting.An isoform with the same M_r as D and a minor low-M_r isoform (E) were present in young developing whole caryopses (8–12 d after anthesis), but not in older developing endosperms (14–21 d after anthesis). Isoenzyme analyses also showed different patterns of bands in these two tissues, while hybrid-dot analyses indicated the presence of separate populations of mRNAs. It is suggested that the early endosperm isoforms (D and E) are green -amylases present in the pericarp and-or testa of the young caryopses.Roots but not shoots or leaves also contained an isoform with the same M_r as D, although the pattern of isoenzymes differed from that present in the seed tissues.The fifth isoform, A, was a diffuse high-M_r form present in small amounts in all seed and vegetative tissues, and may correspond to a constitutively expressed form.These multiple molecular forms of -amylase are discussed in relation to the recent report that -amylase is encoded by two structural loci, with a total copy number of two to three per haploid genome (Kreis et al, 1988, Genet. Res. Camb. 51, 13–16).Abbreviations M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

A protocol for optimal extraction and stabilization of barley β-amylase

Summary A semi-commercial scale protocol has been optimized for the extraction and clarification of barley -amylase preparation. -Amylase stability in the liquid state at 27 and 10 °C was examined as a function of storage period upon fortification with different categories of stabilizers. While the clarified -amylase preparation without stabilizer after 6 weeks storage at 27 °C and 16 weeks storage at 10 °C, respectively showed only 5 and 35% of the original activity, storage with 0.5% (w/v) thiourea at 27 °C or non-clarified preparation at 10 °C stabilized 80% of the original activity for 6 and 16 weeks, respectively.     

Genotyping single nucleotide polymorphisms for selection of barley β-amylase alleles

A high-throughput single nucleotide polymorphism (SNP) genotyping system was developed and used to select barley seedlings carrying superior alleles of β-amylase. In the malting process, β-amylase is a key enzyme involved in the degradation of starch. Four allelic forms of the enzyme are found in barley, each exhibiting a different rate of thermal inactivation, or thermostability. The level of thermostability influences starch degradation, which determines the yield of fermentable sugars for alcohol production during brewing. Control of the fermentability level is important for barley breeding programs to allow targeting quality profiles of new varieties to suit end-user requirements. Alignment of the cDNA sequences encoding the 4 enzyme forms revealed 6 SNPs (cSNPs). The 4 alleles could be identified unambiguously by codominantly genotyping 2 of the cSNPs using a duplex single nucleotide primer extension (SNuPE) assay. Two genotyping primers with their 3^′ ends directly flanking the selected SNPs were annealed to the amplified target sequences and extended by single dideoxynucleotides complementary to the polymorphic nucleotides. Extended primers were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). Making use of the inherent molecular weight difference between DNA bases, incorporated nucleotides were identified by the increase in mass of the extended primers. A cleaved amplified polymorphic sequence (CAPS) assay enabling broader classification of the alleles was also developed to facilitate the transfer of this marker to other laboratories. Plants carrying alternative β-amylase alleles were selected at the seedling stage for barley breeding.     

Purification and properties of β-amylase from Bacillus circulans S31

A thermotolerant -amylase was purified from Bacillus circulans S31 isolated from soil in Hong Kong. The purified enzyme has an M _r of 64 kDa and was stable at 50°C and pH 7.0 for 30 min. Its K _m for starch was 0.9 mg/ml with a V _max of 0.3 mg/min. It was not activated by any metal ion although sulphydrys reagents were inhibitory.H.S. Kwan, K.H. So and K.Y. Chan are with the Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong S.C. Cheng is with the Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic, Hung Hom, Hong Kong.