Cloning, molecular characterization and heterologous expression of AMY1, an α-amylase gene from Cryptococcus flavus

A Cryptococcus flavus gene ( AMY1 ) encoding an extracellular α-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) ¹⁴⁴DVVVNH¹⁴⁹, (II) ²³⁵GLRIDSLQQ²⁴³, (III) ²⁶³GEVFN²⁶⁷, (IV) ³²⁷FLENQD³³², placed the enzyme in the GH13 α-amylase family. Furthermore, sequence comparison suggests that the C. flavus α-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae . The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL⁻¹) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme ( c . 67 kDa), part of which was due to N-glycosylation.     

Nucleotide sequence of the extracellular α-amylase gene in the yeast Schwanniomyces occidentalis ATCC 26077

The Schwanniomyces occidentalis (formerly castellii) ATCC 26077 (CBS 2863) alpha-amylase (AMY 26077) gene was cloned in Saccharomyces cerevisiae and sequenced. An open-reading frame encoding the AMY consists of 1536 base pairs and contains 512 amino-acid residues, which is almost the same in size as the AMY of Sch. occidentalis ATCC 26076 and CCRC 21164. The amino-acid sequence of AMY 26077 differed from that of ATCC 26076 alpha-amylase (AMY 26076) at two residues and from that of CCRC 21164 alpha-amylase (AMY 21164) at three residues. Comparison of the AMY 26077 gene with its homologues from two other strains (Sch. alluvius CBS 1153 and Sch. persoonii CBS 2169) using several restriction enzymes revealed that the AMY 26077 was very similar to AMY CBS 1153 but different from that of CBS 2169.     

Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm).     

Analysis of the alpha-amylase gene of Schwanniomyces occidentalis and the secretion of its gene product in transformants of different yeast genera

We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases. The AMY l gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.     

Expression and secretion of Bacillus amyloliquefaciens alpha-amylase by using the yeast pheromone alpha-factor promoter and leader sequence in Saccharomyces cerevisiae.

Replacement of the regulatory and secretory signals of the alpha-amylase gene (AMY) from Bacillus amylolique-faciens with the complete yeast pheromone alpha-factor prepro region (MF alpha 1p) resulted in increased levels of extracellular alpha-amylase production in Saccharomyces cerevisiae. However, the removal of the (Glu-Ala)2 peptide from the MF alpha 1 spacer region (Lys-Arg-Glu-Ala-Glu-Ala) yielded decreased levels of extracellular alpha-amylase.     

Cloning, sequencing, and expression of the genes encoding an isocyclomaltooligosaccharide glucanotransferase and an alpha-amylase from a Bacillus circulans strain

The gene for a novel glucanotransferase, isocyclomaltooligosaccharide glucanotransferase (IgtY), involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}] from starch, was cloned from the genome of B. circulans AM7. The IgtY gene, designated igtY, consisted of 2,985 bp encoding a signal peptide of 35 amino acids and a mature protein of 960 amino acids with a calculated molecular mass of 102,071 Da. The deduced amino-acid sequence showed similarities to 6-alpha-maltosyltransferase, alpha-amylase, and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in this enzyme, indicating that this enzyme should be assigned to this family. The DNA sequence of 8,325-bp analyzed in this study contained two open reading frames (ORFs) downstream of igtY. The first ORF, designated igtZ, formed a gene cluster, igtYZ. The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme was purified. The enzyme acted on maltooligosaccharides that have a degree of polymerization (DP) of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme (IgtZ), which has a novel amino-acid sequence, should be assigned to alpha-amylase. It is notable that both IgtY and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to a family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.     

Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B. subtilis 2633.

An alpha-amylase gene of Bacillus subtilis (natto) IAM1212 was cloned in a lambda EMBL3 bacteriophage vector, and the nucleotide sequence was determined. An open reading frame encoding the alpha-amylase (AMY1212) consists of 1,431 base pairs and contains 477 amino acid residues, which is the same in size as the alpha-amylase (AMY2633) of B. subtilis 2633, an alpha-amylase-hyperproducing strain, and smaller than that of B. subtilis 168, Marburg strain. The amino acid sequence of AMY1212 is different from that of AMY2633 at five residues. Enzymatic properties of these two alpha-amylases were examined by introducing the cloned genes into an alpha-amylase-deficient strain, B. subtilis M15. It was revealed that products of soluble starch hydrolyzed by AMY1212 are maltose and maltotriose, while those of AMY2633 are glucose and maltose. From the detailed analyses with oligosaccharides as substrates, it was concluded that the difference in hydrolysis products of the two similar alpha-amylases should be ascribed to the different activity hydrolyzing low-molecular-weight substrates, especially maltotriose; AMY1212 slowly hydrolyzes maltotetraose and cannot hydrolyze maltotriose, while AMY2633 efficiently hydrolyzes maltotetraose and maltotriose. Further analyses with chimeric alpha-amylase molecules constructed from the cloned genes revealed that only one amino acid substitution is responsible for the differences in hydrolysis products.     

The human alpha-amylase multigene family consists of haplotypes with variable numbers of genes

Polymorphic amylase protein patterns have suggested the presence in the human genome of various haplotypes encoding these allozymes. To investigate the genomic organization of the human alpha-amylase genes, we isolated the pertinent genes from a cosmid library constructed of DNA from an individual expressing three different salivary amylase allozymes. From the restriction maps of the overlapping cosmids and a comparison of these maps with the restriction enzyme patterns of DNA from the donor and family members, we were able to identify two haplotypes consisting of very different numbers of salivary amylase genes. The short haplotype contains two pancreatic genes (AMY2A and AMY2B) and one salivary amylase gene (AMY1C), arranged in the order 2B-2A-1C, encompassing a total length of approximately 100 kb. The long haplotype spans about 300 kb and contains six additional genes arranged in two repeats, each one consisting of two salivary amylase genes (AMY1A and AMY1B) and a pseudogene lacking the first three exons (AMYP1). The order of the amylase genes within the repeat is 1A-1B-P1. All genes are in a head-to-tail orientation except AMY1B, which has the reverse orientation with respect to the other genes. Analysis of somatic cell hybrids confirmed the presence of these short and long haplotypes. Furthermore, we present evidence for the existence of additional haplotypes in the human population and propose a general model for the evolution of the human alpha-amylase multigene family. A general designation 2B-2A-(1A-1B-P)n-1C can describe these haplotypes, n being 0 and 2 for the short and the long haplotypes presented in this paper, respectively.     

Barley alpha-amylase/subtilisin inhibitor: structure, biophysics and protein engineering

Bifunctional alpha-amylase/subtilisin inhibitors have been implicated in plant defence and regulation of endogenous alpha-amylase action. The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits the barley alpha-amylase 2 (AMY2) and subtilisin-type serine proteases. BASI belongs to the Kunitz-type trypsin inhibitor family of the beta-trefoil fold proteins. Diverse approaches including site-directed mutagenesis, hybrid constructions, and crystallography have been used to characterise the structures and contact residues in the AMY2/BASI complex. The three-dimensional structure of the AMY2/BASI complex is characterised by a completely hydrated Ca2+ situated at the protein interface that connects the three catalytic carboxyl groups in AMY2 with side chains in BASI via water molecules. Using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), we have recently demonstrated Ca2+-modulated kinetics of the AMY2/BASI interaction and found that the complex formation involves minimal structural changes. The modulation of the interaction by calcium ions makes it unique among the currently known binding mechanisms of proteinaceous alpha-amylase inhibitors.     

On the mechanism of alpha-amylase.

Two inhibitors, acarbose and cyclodextrins (CD), were used to investigate the active site structure and function of barley alpha-amylase isozymes, AMY1 and AMY2. The hydrolysis of DP 4900-amylose, reduced (r) DP18-maltodextrin and maltoheptaose (catalysed by AMY1 and AMY2) was followed in the absence and in the presence of inhibitor. Without inhibitor, the highest activity was obtained with amylose, kcat/Km decreased 103-fold using rDP18-maltodextrin and 10(5) to 10(6)-fold using maltoheptaose as substrate. Acarbose is an uncompetitive inhibitor with inhibition constant (L1i) for amylose and maltodextrin in the micromolar range. Acarbose did not bind to the active site of the enzyme, but to a secondary site to give an abortive ESI complex. Only AMY2 has a second secondary binding site corresponding to an ESI2 complex. In contrast, acarbose is a mixed noncompetitive inhibitor of maltoheptaose hydrolysis. Consequently, in the presence of this oligosaccharide substrate, acarbose bound both to the active site and to a secondary binding site. alpha-CD inhibited the AMY1 and AMY2 catalysed hydrolysis of amylose, but was a very weak inhibitor compared to acarbose.beta- and gamma-CD are not inhibitors. These results are different from those obtained previously with PPA. However in AMY1, as already shown for amylases of animal and bacterial origin, in addition to the active site, one secondary carbohydrate binding site (s1) was necessary for activity whereas two secondary sites (s1 and s2) were required for the AMY2 activity. The first secondary site in both AMY1 and AMY2 was only functional when substrate was bound in the active site. This appears to be a general feature of the alpha-amylase family.     

Cloning and Expression of a Schwanniomyces occidentalis alpha-Amylase Gene in Saccharomyces cerevisiae

An alpha-amylase gene (AMY) was cloned from Schwanniomyces occidentalis CCRC 21164 into Saccharomyces cerevisiae AH22 by inserting Sau3AI-generated DNA fragments into the BamHI site of YEp16. The 5-kilobase insert was shown to direct the synthesis of alpha-amylase. After subclones containing various lengths of restricted fragments were screened, a 3.4-kilobase fragment of the donor strain DNA was found to be sufficient for alpha-amylase synthesis. The concentration of alpha-amylase in culture broth produced by the S. cerevisiae transformants was about 1.5 times higher than that of the gene donor strain. The secreted alpha-amylase was shown to be indistinguishable from that of Schwanniomyces occidentalis on the basis of molecular weight and enzyme properties.     

Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.     

Expression of cDNAs encoding barley alpha-amylase 1 and 2 in yeast and characterization of the secreted proteins

Amylolytic strains of the yeast, Saccharomyces cerevisiae, were constructed by transformation with expression plasmids containing cDNAs encoding either AMY1 (clone E) or AMY2 (clone pM/C). The alpha-amylases were efficiently secreted into the culture medium directed by their own signal peptides. When clone E without its 5'-noncoding region was expressed from the yeast PGK promoter, AMY1 was produced as 1% of total cell protein and was thus the major protein secreted, whereas a similar construct derived from pM/C produced much less AMY2. This level is the highest reported for a plant protein secreted by yeast as mediated by the endogenous signal peptide. Production of AMY1 increased 25-fold when the 5'-noncoding part of clone E which contains a 12-bp dG.dC homopolymer tail had been removed. Moreover, expression was one to two orders of magnitude higher when genes encoding AMY1 or AMY2 were inserted between promoter and terminator of the yeast PGK gene in comparison to expression directed from the ADC1 or GAL1 promoters. Recombinant AMY1 and AMY2 had the same Mr and N-terminal sequence as the corresponding barley malt enzymes. Furthermore, none of the enzymes were found to be N-glycosylated. Isoelectric focusing indicated that transformed yeast cells secreted one major form of AMY2 and four dominant forms of AMY1. One AMY1 form corresponded to one of the major forms found in malt while the others, having either low activity or unusually high pI, probably reflect inefficient/incorrect processing. Enzyme kinetic properties and pH activity-dependence of recombinant AMY2 were essentially identical to those of malt AMY2.(ABSTRACT TRUNCATED AT 250 WORDS)     

The missing link in the fungal L-arabinose catabolic pathway,identification of the L-xylulose reductase gene

The fungal L-arabinose pathway consists of five enzymes, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, and xylulokinase. All the genes encoding the enzymes of this pathway are known except for that of L-xylulose reductase (EC 1.1.1.10). We identified a gene encoding this enzyme from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). The gene was named lxr1. It was overexpressed in the yeast Saccharomyces cerevisiae, and the enzyme activity was confirmed in a yeast cell extract. Overexpression of all enzymes of the L-arabinose pathway in S. cerevisiae led to growth of S. cerevisiae on L-arabinose; i.e., we could show that the pathway is active in a heterologous host. The lxr1 gene encoded a protein with 266 amino acids and a calculated molecular mass of 28 428 Da. The LXRI protein is an NADPH-specific reductase. It has activity with L-xylulose, D-xylulose, D-fructose, and L-sorbose. The highest affinity is toward L-xylulose (K(m) = 16 mM). In the reverse direction, we found activity with xylitol, D-arabinitol, D-mannitol, and D-sorbitol. It requires a bivalent cation for activity. It belongs to the protein family of short chain dehydrogenases. The enzyme is catalytically similar and homologous in sequence to a D-mannitol:NADP 2-dehydrogenase (EC 1.1.1.138).     

Specific inhibition of barley alpha-amylase 2 by barley alpha-amylase/subtilisin inhibitor depends on charge interactions and can be conferred to isozyme 1 by mutation.

alpha-Amylase 2 (AMY2) and alpha-amylase/subtilisin inhibitor (BASI) from barley bind with Ki = 0.22 nM. AMY2 is a (beta/alpha)8-barrel enzyme and the segment Leu116-Phe143 in domain B (Val89-Ile152), protruding at beta-strand 3 of the (beta/alpha)8-barrel, was shown using isozyme hybrids to be crucial for the specificity of the inhibitor for AMY2. In the AMY2-BASI crystal structure [F. Vallée, A. Kadziola, Y. Bourne, M. Juy, K. W. Rodenburg, B. Svensson & R. Haser (1998) Structure 6, 649-659] Arg128AMY2 forms a hydrogen bond with Ser77BASI, while Asp142AMY2 makes a salt-bridge with Lys140BASI. These two enzyme residues are substituted by glutamine and asparagine, respectively, to assess their contribution in binding of the inhibitor. These mutations were performed in the well-expressed, inhibitor-sensitive hybrid barley alpha-amylase 1 (AMY1)-(1-90)/AMY2-(90-403) with Ki = 0.33 nM, because of poor production of AMY2 in yeast. In addition Arg128, only found in AMY2, was introduced into an AMY1 context by the mutation T129R/K130P in the inhibitor-insensitive hybrid AMY1-(1-161)/AMY2-(161-403). The binding energy was reduced by 2.7-3.0 kcal.mol-1 as determined from Ki after the mutations R128Q and D142N. This corresponds to loss of a charged interaction between the protein molecules. In contrast, sensitivity to the inhibitor was gained (Ki = 7 microM) by the mutation T129R/K130P in the insensitive isozyme hybrid. Charge screening raised Ki 14-20-fold for this latter mutant, AMY2, and the sensitive isozyme hybrid, but only twofold for the R128Q and D142N mutants. Thus electrostatic stabilization was effectively introduced and lost in the different mutant enzyme-inhibitor complexes and rational engineering using an inhibitor recognition motif to confer binding to the inhibitor mimicking the natural AMY2-BASI complex.     

Genetic coadaptation of the amylase gene system in Drosophila melanogaster: evidence for the selective advantage of the lowest AMY activity and of its epistatic genetic background

In natural populations of Drosophila melanogaster, an amylase isozyme with the lowest alpha-amylase activity (AMY(1,1)) is predominant. To evaluate the selective significance of AMY(1,1) and its regulatory factor(s), we examined selection experiments in laboratory populations on two distinct food environments. After 300 generations, AMY(1,1) became predominant (89%) in a glucose (a product of AMY)-rich environment, while an isozyme with higher alpha-amylase activity, AMY(1,6), became predominant (83%) in a starch (substrate)-rich environment. We found that the identical alleles of the amylase (Amy) gene, which encodes each of AMY(1,1) and AMY(1,6), were shared between the two populations in the different food environments, employing the nucleotide sequencing of the duplicated Amy genes. Nevertheless, AMY(1,6) homozygotes selected in the starch-rich environment had a twofold higher AMY enzyme activity than those selected in the glucose-rich environment, suggesting a coadaptation of the coding region and its regulatory factor(s) on the genetic background. Such a difference in AMY enzyme activity was not detected between AMY(1,1) homozygotes, suggesting that the effect of the genetic background is epistatic. Our results indicate that natural selection is working on the Amy gene system as a whole for flies to adapt to the various food environments of local populations.