Characteristics of two forms of alpha-amylases and structural implication.

Complete (Ba-L) and truncated (Ba-S) forms of alpha-amylases from Bacillus subtilis X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined. The amino acid sequence deduced from the nucleotide sequence of the alpha-amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region. The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same alpha-amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of B. subtilis X-23 cells. Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L. An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as alpha-amylase.     

Secreted amylolytic enzymes from Schwanniomyces occidentalis: purification by fast protein liquid chromatography (FPLC) and preliminary characterization

Amylolytic enzyme preparations are used extensively for the liquefaction and saccharification of starch in the production of ethanol and SCP (single cell protein). We report the first purification of two amylolytic enzymes from the yeast Schwanniomyces occidentalis using fast protein liquid chromatography (FPLC) in a two step process: size exclusion (Superose 12) followed by anion exchange (Mono Q). The procedure is amenable to direct scale up processes. The enzymes glucoamylase (E.C. 3.2.1.2) and alpha-amylase (E.C. 3.2.1.1) were found in the cell free supernatant of S. occidentalis when grown on a variety of carbon sources. The enzymes are substrate induced and catabolite repressed. Both amylolytic enzymes were purified from three separate culture broths containing either starch, maltose or cellobiose and their physical properties compared. Native molecular masses of glucoamylase and alpha-amylase were determined to be 122,000 +/- 28,000 daltons and 47,000 +/- 11,000 daltons, respectively, while subunit size was approximated at 143,000 +/- 2,000 daltons and 54,500 +/- 1,000 daltons, respectively. Both proteins are N-glycosylated with carbohydrate representing 10-15% of the total mass. The correlation of native mass and denatured subunit structure, while not identical due to slight aberrant behavior on gels and columns as a result of glycosylation, suggest that both proteins exist as monomeric polypeptides. Isoelectric points for both proteins under native conditions could not be determined since alpha-amylase failed to enter native polyacrylamide gels. However, a pI for glucoamylase of 6.2 +/- 0.2 (native) and a pI for alpha-amylase of 6.3 +/- 0.3 (in 6M urea) were determined. Glucoamylase and alpha-amylase specific activities (for the homogeneous proteins) were determined to be 48-67 x 10(3) units/mg and 214-457 x 10(3) units/mg respectively. We could find no apparent differences in either glucoamylase or alpha-amylase proteins obtained from three separate cultures which had been grown on different carbon sources. The purification method we have utilized is easily scaled up to larger protein concentrations, and provides a rapid procedure for analyzing and purifying these amylolytic enzymes.     

Starch-binding domain affects catalysis in two Lactobacillus alpha-amylases

A new starch-binding domain (SBD) was recently described in alpha-amylases from three lactobacilli (Lactobacillus amylovorus, Lactobacillus plantarum, and Lactobacillus manihotivorans). Usually, the SBD is formed by 100 amino acids, but the SBD sequences of the mentioned lactobacillus alpha-amylases consist of almost 500 amino acids that are organized in tandem repeats. The three lactobacillus amylase genes share more than 98% sequence identity. In spite of this identity, the SBD structures seem to be quite different. To investigate whether the observed differences in the SBDs have an effect on the hydrolytic capability of the enzymes, a kinetic study of L. amylovorus and L. plantarum amylases was developed, with both enzymes acting on several starch sources in granular and gelatinized forms. Results showed that the amylolytic capacities of these enzymes are quite different; the L. amylovorus alpha-amylase is, on average, 10 times more efficient than the L. plantarum enzyme in hydrolyzing all the tested polymeric starches, with only a minor difference in the adsorption capacities.     

A study of amylolytic system of Schwanniomyces castelii

The amylolytic system of Schwanniomyces castellii cultured on a yeast extract starch medium consists of 3 enzymes: an alpha-amylase (molecular weight 40,000), glucoamylase I (molecular weight 90,000), and glucoamylase II (molecular weight 45,000). The properties of the enzymes and the action of enzyme inhibitors were determined.     

Significance of HPr in catabolite repression of alpha-amylase.

CcpA and HPr are presently the only two proteins implicated in Bacillus subtilis global carbon source catabolite repression, and the ptsH1 mutation in the gene for the HPr protein was reported to relieve catabolite repression of several genes. However, alpha-amylase synthesis by B. subtilis SA003 containing the ptsH1 mutation was repressed by glucose. Our results suggest HPr(Ser-P) may be involved in but is not required for catabolite repression of alpha-amylase, indicating that HPr(Ser-P) is not the sole signaling molecule for CcpA-mediated catabolite repression in B. subtilis.     

Numerical taxonomy of alpha-amylase producing Bacillus species

A total of 134 alpha-amylase producing Bacillus isolates and 21 reference strains were divided into 12 groups according to their similarities (% SSM). Phenotypic characteristics determined by the API 20E and API 50CHB galleries, other biochemical tests and morphological characteristics were used for the numerical analysis. The API Computer Service identified 45% of the isolates. The amylase yields of 16 alpha-amylase hyperproducing (AHP) isolates were compared with those of seven amylolytic reference and type strains. The AHP isolates were related to Bacillus subtilis, B. licheniformis and 'B. amyloliquefaciens'.     

Temperature impacts the multiple attack action of amylases

The action pattern of several amylases was studied at 35, 50, and 70 degrees C using potato amylose, a soluble (Red Starch) and insoluble (cross-linked amylose) chromophoric substrate. With potato amylose as substrate, Bacillus stearothermophilus alpha-amylase (BStA) and porcine pancreatic alpha-amylase displayed a high degree of multiple attack (DMA, i.e., the number of bonds broken during the lifetime of an enzyme-substrate complex minus one), the fungal alpha-amylase from Aspergillus oryzae a low DMA, and the alpha-amylases from B. licheniformis, Thermoactinomyces vulgaris, B. amyloliquifaciens, and B. subtilis an intermediate DMA. These data are discussed in relation to structural properties of the enzymes. The level of multiple attack (LMA), based on the relation between the drop in iodine binding of amylose and the increase in total reducing value, proved to be a good alternative for DMA measurements. The LMA of the endo-amylases increased with temperature to a degree depending on the amylase. In contrast, BStA showed a decreased LMA when temperature was raised. Furthermore, different enzymes had different activities on Red Starch and cross-linked amylose. Hence, next to the temperature, the action pattern of alpha-amylases is influenced by structural parameters of the starch substrate.     

Isolation of Mutants Defective in α-Amylase from Bacillus subtilis: Genetic Analyses

The rate of alpha-amylase (EC 3.2.1.1) synthesis in Bacillus subtilis is regulated by a gene, amyR, located near a structural gene, amyE, for the enzyme. To construct a fine map of the amyR-amyE region, we isolated 28 mutants defective in alpha-amylase activity. Eleven mutants out of 28 showed no alpha-amylase activity, whereas the other 17 showed less alpha-amylase activity than the parent. Out of 17 partially positive alpha-amylase mutants, 10 produced temperature-sensitive enzymes, and 4 produced immunologically altered enzymes, two of which are concurrently temperature-sensitive, and 5 produced smaller amounts of alpha-amylases which are indistinguishable from normal enzyme in their temperature sensitivity and immunological properties. Two out of 11 alpha-amylase-negative mutants produced material that cross-reacted with anti-amylase serum, and 3 mutants carried suppressible mutations by the suppressor described by Okubo. Mapping data indicate that all 28 mutation sites are located in the amyE region, and none of the groups of the mutants mentioned above contains lesions that are clustered in a single region of amyE. The amyR gene seems most likely to adjoin the terminal region of amyE.     

Starch-hydrolyzing enzymes from thermophilic archaea and bacteria

Extremophlic microorganisms have developed a variety of molecular strategies in order to survive in harsh conditions. For the utilization of natural polymeric substrates such as starch, a number of extremophiles, belonging to different taxonomic groups, produce amylolytic enzymes. This class of enzyme is important not only for the study of biocatalysis and protein stability at extreme conditions but also for the many biotechnological opportunities they offer. In this review, we report on the different molecular properties of thermostable archaeal and bacterial enzymes including alpha-amylase, alpha-glucosidase, glucoamylase, pullulanase, and cyclodextrin glycosyltransferase. Comparison of the primary sequence of the pyrococcal pullulanase with other members of the glucosyl hydrolase family revealed that significant differences are responsible for the mode of action of these enzymes.     

Relationship between morological variability of Bacillus subtilis R-623 and biosynthesis of hydrolytic enzymes

By synthetic sorbent chromatography the influence of Bacillus subtilis R-623 morphology on the qualitative and quantitative composition of alpha-amylase and proteases was studied. It was found that morphological variants of natural variability of Bac. subtilis R-623, alpha-amylase producer, differed in their cultural, morphological and physiological properties as well as in the amount of hydrolytic enzymes synthesized per unit of the cultural medium. Cells of P variant produced the highest quantity of alpha-amylase (314 U/lm) and cells of P and M variants synthesized the greatest amount of proteases (4.3 and 4.0 U/ml, respectively). Quantitative variations of alpha-amylase and proteases were measured in the cultural medium of morphological variants of Bac. subtilis R-623 during cultivation. Qualitative composition of those enzymes was determined when their content in the cultural medium was at the highest level. R variant synthesized alpha-amylase and protease, P and S variants alpha-amylase and two proteases, and P and S variants alpha-amylase and two proteases, and M variant one protease.     

Nucleotide sequence of the neopullulanase gene from Bacillus stearothermophilus

The gene (nplT) for a new type of pullulan-hydrolysing enzyme, neopullulanase, from Bacillus stearothermophilus TRS40 was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1764 bases and 588 amino acid residues (Mr 69144). Although the thermostable neopullulanase contained eight cysteine residues, they did not provide conformational stability by disulphide bonds. A comparison was made of the amino acid sequences of alpha-amylase, neopullulanase, isoamylase, pullulanase and cyclodextrin glucanotransferase. All the enzymes examined contained four highly conserved regions which probably constitute the active centres of the enzymes. The amino acid residues required for the specificity of neopullulanase are compared with those of alpha-amylase and other amylolytic enzymes.     

Enzymatic analysis of an amylolytic enzyme from the hyperthermophilic archaeon Pyrococcus furiosus reveals its novel catalytic properties as both an alpha-amylase and a cyclodextrin-hydrolyzing enzyme

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90 degrees C. The substrate specificity of PFTA suggests that it possesses characteristics of both alpha-amylase and cyclodextrin-hydrolyzing enzyme. Like typical alpha-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and beta-cyclodextrin, which are resistant to alpha-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent alpha-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both alpha-amylase and cyclodextrin-hydrolyzing enzyme.     

Phylogenomic Relationships between Amylolytic Enzymes from 85 Strains of Fungi

Fungal amylolytic enzymes, including α-amylase, gluocoamylase and α-glucosidase, have been extensively exploited in diverse industrial applications such as high fructose syrup production, paper making, food processing and ethanol production. In this paper, amylolytic genes of 85 strains of fungi from the phyla Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota were annotated on the genomic scale according to the classification of glycoside hydrolase (GH) from the Carbohydrate-Active enZymes (CAZy) Database. Comparisons of gene abundance in the fungi suggested that the repertoire of amylolytic genes adapted to their respective lifestyles. Amylolytic enzymes in family GH13 were divided into four distinct clades identified as heterologous α- amylases, eukaryotic α-amylases, bacterial and fungal α-amylases and GH13 α-glucosidases. Family GH15 had two branches, one for gluocoamylases, and the other with currently unknown function. GH31 α-glucosidases showed diverse branches consisting of neutral α-glucosidases, lysosomal acid α-glucosidases and a new clade phylogenetically related to the bacterial counterparts. Distribution of starch-binding domains in above fungal amylolytic enzymes was related to the enzyme source and phylogeny. Finally, likely scenarios for the evolution of amylolytic enzymes in fungi based on phylogenetic analyses were proposed. Our results provide new insights into evolutionary relationships among subgroups of fungal amylolytic enzymes and fungal evolutionary adaptation to ecological conditions.     

Action of Bacillus subtilis alpha-amylase on native wheat starch

Native starch granules from wheat have been subjected to enzymatic depolymerization with an alpha-amylase from Bacillus subtilis. Crystallites made from short-chain amylose and residues from mild acid hydrolysis have been also tested. Electron microscopy, particle size analysis, DSC, and x-ray diffractometry reveal that enzymatic degradation occurs granule by granule. Gel permeation chromatography shows off the macromolecular nature of the remaining material. In contrast, acid erodes simultaneously all the granules, leading to a splitting into small particles. Crystalline fractions are completely degraded by alpha-amylase. These results support evidence for an active disentanglement of chains, carried out by the different subsites of alpha-amylase molecules. A simple mathematical treatment is proposed to explain the results of the kinetics.     

双启动子对重组溶源性枯草杆菌中外源蛋白表达的增强作用

探讨了双启动子对基于溶源性噬菌体构建的重组枯草杆菌中外源蛋白表达的影响。分别将不含或含有本身启动子的α-淀粉酶基因(来源于Bacillus amyloliquefaciens)和青霉素酰化酶基因(来源于Bacillus megaterium)克隆到溶源性枯草杆菌中,得到重组菌B.subtilisAMY1,B.subtilisAMY2,B.subtilisPA1以及B.subtilisPA2。由于同源重组,所克隆的片段整合到溶源性枯草杆菌中的噬菌体基因组上,并处于噬菌体强启动子的下游。在重组菌AMY1和PA1中,在热诱导的情况下外源基因的转录只受到噬菌体启动子的作用,而在重组菌AMY2和PA2中,在热诱导下外源基因的转录同时受到噬菌体启动子和基因本身所带启动子的作用。双启动子的应用使重组α-淀粉酶的表达量提高了133%,使重组青霉素酰化酶的表达量提高了113%。     

Establishment of an experimental system allowing immobilization of proteins on the surface of Bacillus subtilis cells

Gram-positive bacteria code for one or more enzymes termed sortases which catalyze the covalent anchoring of substrate proteins on their cell wall. They recognize an amino acid sequence designated sorting motif, present close to the C-terminal end of the substrate proteins, cleave within this motif and catalyze anchoring of the polypeptide chain to the peptide crossbridge linking the peptidoglycan strands in a transpeptidation reaction. Bacillus subtilis has been reported to code for two different sortases but the sorting sequences recognized by them are yet unknown. To be able to immobilize proteins on the surface of B. subtilis cells, we introduced the srtA gene coding for sortase A of Listeria monocytogenes with the known sorting motif (LPXTG) into B. subtilis. L. monocytogenes and B. subtilis share the same peptide crossbridge. Next, we fused the coding region of an alpha-amylase gene to the C-terminal region of Staphylococcus aureus fibronectin binding protein B containing the sorting motif. Covalent linkage could be proven by treatment of the cells with lysozyme and by immunofluorescence microscopy. Up to 240,000 molecules of alpha-amylase could be immobilized per cell, 24 times more than previously reported for other bacterial species. To study the influence of the distance between the sorting motif and the C-terminus of alpha-amylase on the activity of the enzyme, the length of the spacer was varied. It turned out that the highest activity was measured with a spacer length of 123 amino acid residues.