Protein thermostabilization by proline substitutions

Many recent approaches involving site-directed mutants have succeeded in increasing the thermostability of proteins. It is well known that replacements with proline residues reduce the conformational degrees of freedom in the main polypeptide chain and thus can increase protein thermostabilization. We have studied protein thermostabilization by introducing proline substitutions in the homologous oligo-1,6-glucosidases from various Bacillus strains which grow within different temperature ranges. As a consequence, the `proline rule' was proposed for protein thermostabilization. The principle of this rule is that an increase in the frequency of proline occurrence at β-turns and/or an increase in the total number of hydrophobic residues can enhance protein thermostability. We have generated several lines of evidence supporting the theory from the comparative analysis of oligo-1,6-glucosidases in their primary and secondary structures and molecular properties, the X-ray crystal structure analysis of the Bacillus cereus oligo-1,6-glucosidase, and the enhancement in thermostability of the oligo-1,6-glucosidase by cumulative replacements with prolines. As a new finding from the studies, two specific sites (second positions at β-turns and N1 positions of α-helices) were found to be the most critical to protein thermostabilization dependent on several structural prerequisites for proline substitution.     

Proline residues responsible for thermostability occur with high frequency in the loop regions of an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006.

The gene encoding for an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) was sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (1686 base pairs) corresponded to a protein of 562 amino acid residues with a Mr of 66,502. Its predicted amino acid composition, Mr, and N-terminal sequence of 12 residues were consistent with those determined for B. thermoglucosidasius oligo-1,6-glucosidase. The deduced sequence of the enzyme was 72% homologous to that of a thermolabile oligo-1,6-glucosidase (558 residues) from Bacillus cereus ATCC7064 (mesophile). B. cereus oligo-1,6-glucosidase contained 19 prolines. Eighteen of these were conserved at the equivalent positions of B. thermoglucosidasius oligo-1,6-glucosidase. This enzyme contained 14 extra prolines besides the conservative prolines. The majority of extra prolines was replaced by polar or charged residues (Glu, Thr, or Lys) in B. cereus oligo-1,6-glucosidase. The extra prolines were responsible for the difference in thermostability between these two enzymes. We suggested that 11 of the extra prolines in B. thermoglucosidasius oligo-1,6-glucosidase occur in beta-turns or in coils within the loops binding adjacent secondary structures.     

Purification and characterization of Bacillus coagulans oligo-1,6-glucosidase

A p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing oligo-1,6-glucosidase of Bacillus coagulans ATCC 7050 (facultative thermophile) was purified to homogeneity. The relative molecular mass, Stokes radius, sedimentation coefficient at 20 degrees C in water, molecular absorption coefficient at 280 nm and pH 6.8, and isoelectric point were estimated as 60 000, 3.29 nm, 4.8 X 10(-13) s, 1.34 X 10(5) M-1 cm-1, and 4.3, respectively. The amino-terminal amino acid was threonine. There was no common antigenic group between the enzyme and each of its homologous counterparts from Bacillus cereus ATCC 7064 (mesophile) and Bacillus thermoglucosidasius KP 1006 (obligate thermophile). These oligo-1,6-glucosidases strongly resembled one another in their amino acid composition, except that the proline content increased with the elevation of thermostability in the order, mesophile----facultative thermophile----obligate thermophile enzymes.     

Primary structure of the oligo-1,6-glucosidase of Bacillus cereus ATCC7064 deduced from the nucleotide sequence of the cloned gene

The gene coding for Bacillus cereus ATCC7064 (mesophile) oligo-1,6-glucosidase was cloned within a 2.8-kb SalI-EcoRI fragment of DNA, using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. E. coli C600 bearing the hybrid plasmid pBCE4 accumulated oligo-1,6-glucosidase in the cytoplasm. The cloned enzyme coincided absolutely with B. cereus oligo-1,6-glucosidase in its Mr (65,000), in its electrophoretic behavior on a polyacrylamide gel with or without sodium dodecyl sulfate, in its isoelectric point (4.5), in the temperature dependence of its stability and activity, and in its antigenic determinants. The nucleotide sequence of B. cereus oligo-1,6-glucosidase gene and its flanking regions was determined with both complementary strands of DNA (each 2838 nucleotides). The gene consisted of an open reading frame of 1674 bp commencing with a ATG start codon and followed by a TAA stop codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 558 amino acid residues with a Mr of 66,010. The amino acid composition and Mr were comparable with those of B. cereus oligo-1,6-glucosidase. The predicted N-terminal sequence of 10 amino acid residues agreed completely with that of the cloned ligo-1,6-glucosidase. The deduced amino acid sequence of B. cereus oligo-1,6-glucosidase was 72% and 42% similar to those from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) oligo-1,6-glucosidase and from Saccharomyces carlsbergensis CB11 alpha-glucosidase, respectively. Predictions of protein secondary structures along with amino acid sequence alignments demonstrated that B. cereus oligo-1,6-glucosidase may take the similar (alpha/beta)8-barrel super-secondary structure, a barrel of eight parallel beta-strands surrounded by eight alpha-helices, in its N-terminal active site domain as S. carlsbergensis alpha-glucosidase and Aspergillus oryzae alpha-amylase.     

枯草芽孢杆菌寡聚—1，6—葡萄糖苷酶基因的克隆及其在大肠杆菌中的表达

本研究用鸟枪法构建了枯草芽孢杆菌（Bacillus subtilis)HB002的基因组文库,经平板法筛选得到了六株能水解合成底物对－硝基苯－α-D－葡萄糖吡喃糖苷的阳性克隆,经鉴定均含克隆了寡聚－1,6－葡萄糖苷酶基因的重组质粒（命名为pHBM001-pHBM006)。选择pHBM003,对其插入片段测序分析,此片段内有一编码561个氨基酸的开放阅读框,该 蛋白质的计算分子量为65.985kD。HB002的寡聚－1,6－葡萄糖苷酶的氨基酸序列与Bacillus sp.和凝结芽孢杆菌（Bacillus coagulans)的寡聚－1,6－葡萄糖苷酶的氨基酸序列一致性分别为81％、67％,相似性分别为89％、79％。从pHBM003中扩增出寡聚－1,6－葡萄糖苷酶基因,克隆到pBV220上,转化大肠杆菌（Escherichia coli)DH5α,得到三个能水解对－硝基苯－α－D－葡萄糖吡喃糖苷的阳性克隆HBM003－1～HBM003－3,将此三个菌株热诱导表达,SDS－PAGE电泳可检测到特异表达的蛋白质,其中HBM003－1、HBM003－2表达的蛋白约66kD,为完整的寡聚－1,6－葡萄糖苷酶,而HBM003－3表达的蛋白质偏小;表达的蛋白质均有寡聚－1,6－葡萄糖苷酶活性。     

A strong correlation between the increase in number of proline residues and the rise in thermostability of five Bacillus oligo-1,6-glucosidases

Summary A p-nitrophenyl-α-d-glucopyranoside-hydrolysing oligo-1,6-glucosidase (dextrin 6-α-d-glucanohydrolase, EC 3.2.1.10) of Bacillus sp. KP 1071 capable of growing at 30°–66°C was purified to homogeneity. The molecular weight was estimated to be 62,000. The amino-terminal amino acid was methionine. The enzyme shared its antigenic groups in part with its homologous counterpart from Bacillus thermoglucosidasius KP 1006 (obligate thermophile), but did not at all with any one of oligo-1,6-glucosidases from Bacillus cereus ATCC 7064 (mesophile), Bacillus coagulans ATCC 7050 (facultative thermophile) and Bacillus flavocaldarius KP 1288 (extreme thermophile). A comparison of amino acid composition showed that the proline content increased greatly in a linearity with the rise in thermostability in the order, mesophile → facultative thermophile → KP 1071 → obligate thermophile → extreme thermophile enzymes. Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Kyoto, April 3, 1986     

Identification of catalytic and substrate-binding site residues in Bacillus cereus ATCC7064 oligo-1,6-glucosidase.

Three active site residues (Asp199, Glu255, Asp329) and two substrate-binding site residues (His103, His328) of oligo-1,6-glucosidase (EC 3.2.1.10) from Bacillus cereus ATCC7064 were identified by site-directed mutagenesis. These residues were deduced from the X-ray crystallographic analysis and the comparison of the primary structure of the oligo-1,6-glucosidase with those of Saccharomyces carlsbergensis alpha-glucosidase, Aspergillus oryzae alpha-amylase and pig pancreatic alpha-amylase which act on alpha-1,4-glucosidic linkages. The distances between these putative residues of B. cereus oligo-1,6-glucosidase calculated from the X-ray analysis data closely resemble those of A. oryzae alpha-amylase and pig pancreatic alpha-amylase. A single mutation of Asp199-->Asn, Glu255-->Gln, or Asp329-->Asn resulted in drastic reduction in activity, confirming that three residues are crucial for the reaction process of alpha-1,6-glucosidic bond cleavage. Thus, it is identified that the basic mechanism of oligo-1,6-glucosidase for the hydrolysis of alpha-1,6-glucosidic linkage is essentially the same as those of other amylolytic enzymes belonging to Family 13 (alpha-amylase family). On the other hand, mutations of histidine residues His103 and His328 resulted in pronounced dissimilarity in catalytic function. The mutation His328-->Asn caused the essential loss in activity, while the mutation His103-->Asn yielded a mutant enzyme that retained 59% of the k0/Km of that for the wild-type enzyme. Since mutants of other alpha-amylases acting on alpha-1,4-glucosidic bond linkage lost most of their activity by the site-directed mutagenesis at their equivalent residues to His103 and His328, the retaining of activity by His103-->Asn mutation in B. cereus oligo-1,6-glucosidase revealed the distinguished role of His103 for the hydrolysis of alpha-1,6-glucosidic bond linkage.     

Identification of Catalytic and Substrate-binding Site Residues in Bacillus cereus ATCC7064 Oligo-1,6-glucosidase

Three active site residues (Asp199, Glu255, Asp329) and two substrate-binding site residues (His103, His328) of oligo-1,6-glucosidase (EC 3.2.1.10) from Bacillus cereus ATCC7064 were identified by site-directed mutagenesis. These residues were deduced from the X-ray crystallographic analysis and the comparison of the primary structure of the oligo-1,6-glucosidase with those of Saccharomyces carlsbergensis α-glucosidase, Aspergillus oryzae α-amylase and pig pancreatic α-amylase which act on α-1,4-glucosidic linkages. The distances between these putative residues of B. cereus oligo-1,6-glucosidase calculated from the X-ray analysis data closely resemble those of A. oryzae α-amylase and pig pancreatic α-amylase. A single mutation of Asp199→Asn, Glu255→Gln, or Asp329→Asn resulted in drastic reduction in activity, confirming that three residues are crucial for the reaction process of α-1,6-glucosidic bond cleavage. Thus, it is identified that the basic mechanism of oligo-1,6-glucosidase for the hydrolysis of α-1,6-glucosidic linkage is essentially the same as those of other amylolytic enzymes belonging to Family 13 (α-amylase family). On the other hand, mutations of histidine residues His103 and His328 resulted in pronounced dissimilarity in catalytic function. The mutation His328→Asn caused the essential loss in activity, while the mutation His103→Asn yielded a mutant enzyme that retained 59% of the κ₀/K_m of that for the wild-type enzyme. Since mutants of other α-amylases acting on α-1,4-glucosidic bond linkage lost most of their activity by the site-directed mutagenesis at their equivalent residues to His103 and His328, the retaining of activity by Hisl03→Asn mutation in B. cereus oligo-1,6-glucosidase revealed the distinguished role of His103 for the hydrolysis of α-1,6-glucosidic bond linkage.     

Cloning and expression in Escherichia coli of an extremely thermostable oligo-1,6-glucosidase gene from Bacillus thermoglucosidasius.

The gene for an extremely thermostable oligo-1,6-glucosidase (dextrin-6-alpha-D-glucanohydrolase; EC 3.2.1.10) of obligately thermophilic Bacillus thermoglucosidasius KP1006 was cloned within a 4.2-kilobase HindIII-PvuII fragment of DNA by using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. The gene was transcribed, presumably from its own promoter, in E. coli. E. coli with the hybrid plasmid accumulated oligo-1,6-glucosidase mainly in the cytoplasm. The level of enzyme production was comparable to that observed for B. thermoglucosidasius. The enzyme coincided absolutely with the B. thermoglucosidasius enzyme in its molecular weight (60,000), in its electrophoretic behavior on denaturing and nondenaturing polyacrylamide gels, in the temperature dependency of its stability and activity, and in its antigenic determinants.     

Structural elements in dextran glucosidase responsible for high specificity to long chain substrate

Dextran glucosidase from Streptococcus mutans (SMDG) and Bacillus oligo-1,6-glucosidases, members of glycoside hydrolase family 13 enzymes, have the high sequence similarity. Each of them is specific to alpha-1,6-glucosidic linkage at the non-reducing end of substrate to liberate glucose. The activities toward long isomaltooligosaccharides were different in both enzymes, in which SMDG and oligo-1,6-glucosidase showed high and low activities, respectively. We determined the structural elements essential for high activity toward long-chain substrate. From conformational comparison between SMDG and B. cereus oligo-1,6-glucosidase (three-dimensional structure has been solved), Trp238 and short beta-->alpha loop 4 of SMDG were considered to contribute to the high activity to long-chain substrate. W238A had similar kcat/Km value for isomaltotriose to that for isomaltose, suggesting that the affinity of subsite +2 was decreased by Trp238 replacement. Trp238 mutants as well as the chimeric enzyme having longer beta-->alpha loop 4 of B. subtilis oligo-1,6-glucosidase showed lower preference for long-chain substrates, indicating that both Trp238 and short beta-->alpha loop 4 were important for high activity to long-chain substrates.     

Overproduction, purification and crystallization of Bacillus cereus oligo-1,6-glucosidase

The gene coding for oligo-1,6-glucosidase from Bacillus cereus ATCC7064 has been overexpressed in Escherichia coli MV1184 cells under the control of the lac promoter in the genetically engineered plasmid pBCE4-2. Oligo-1,6-glucosidase was purified in large quantities and was crystallized at 25 degrees C by using a hanging drop vapor diffusion method with 53% saturated ammonium sulfate. The crystals have the shape of hexagonal bipyramids and belong to the space group P6(2) or P6(4) with lattice constants of a = b = 106.1 A, c = 120.0 A and gamma = 120 degrees.     

Identification of a helix-turn-helix motif of Bacillus thermoglucosidasius HrcA essential for binding to the CIRCE element and thermostability of the HrcA-CIRCE complex,indicating a role as a thermosensor

In the heat shock response of bacillary cells, HrcA repressor proteins negatively control the expression of the major heat shock genes, the groE and dnaK operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element. Studies on two critical but yet unresolved issues related to the structure and function of HrcA were performed using mainly the HrcA from the obligate thermophile Bacillus thermoglucosidasius KP1006. These two critical issues are (i) identifying the region at which HrcA binds to the CIRCE element and (ii) determining whether HrcA can play the role of a thermosensor. We identified the position of a helix-turn-helix (HTH) motif in B. thermoglucosidasius HrcA, which is typical of DNA-binding proteins, and indicated that two residues in the HTH motif are crucial for the binding of HrcA to the CIRCE element. Furthermore, we compared the thermostabilities of the HrcA-CIRCE complexes derived from Bacillus subtilis and B. thermoglucosidasius, which grow at vastly different ranges of temperature. The thermostability profiles of their HrcA-CIRCE complexes were quite consistent with the difference in the growth temperatures of B. thermoglucosidasius and B. subtilis and, thus, suggested that HrcA can function as a thermosensor to detect temperature changes in cells.     

Thermostabilization by proline substitution in an alkaline, liquefying alpha-amylase from Bacillus sp. strain KSM-1378.

alpha-Amylase (LAMY) from alkaliphilic Bacillus sp. strain KSM-1378 is a novel semi-alkaline enzyme which has 5-fold higher specific activity than that of a Bacillus licheniformis enzyme. The Arg124 in LAMY was replaced with proline by site-directed mutagenesis to increase thermostability of the enzyme. The wild-type and engineered LAMYs were very similar with respect to specific activity, kinetic values, pH-activity curve, and degree of inhibition by chelating reagents. Thermostability and structure stiffness of LAMYs as measured by fluorescence were increased by the proline substitution. The change of Arg124 to proline is assumed to stabilize the loop region involving amino acid residues from 122 to 134. This is the first report that thermostability of an alpha-amylase is improved by proline substitution.     

Deciphering the molecular basis of the broad substrate specificity of alpha-glucosidase from Bacillus sp. SAM1606

The alpha-glucosidase of Bacillus sp. strain SAM1606 is a member of glycosyl hydrolase family 13, and shows an extraordinarily broad substrate specificity and is one of very few alpha-glucosidases that can efficiently hydrolyze the alpha-1,1-glucosidic linkage of alpha,alpha'-trehalose (trehalose). Phylogenetic analysis of family-13 enzymes suggests that SAM1606 alpha-glucosidase may be evolutionally derived from an alpha-1,6-specific ancestor, oligo-1,6-glucosidase (O16G). Indeed, replacement of Pro(273*) and Thr(342*) of B. cereus O16G by glycine and asparagine (the corresponding residues in the SAM1606 enzyme), respectively, was found to cause 192-fold enhancement of the relative catalytic efficiency for trehalose, suggesting that O16G may easily "evolved" into an enzyme with an extended substrate specificity by substitution of a limited number of amino acids, including that at position 273* (an asterisk indicates the amino-acid numbering of the SAM1606 sequence). To probe the role of the amino acid at position 273* of alpha-glucosidase in determination of the substrate specificity, the amino acid at position 273 of SAM1606 alpha-glucosidase was replaced by all other naturally occurring amino acids, and the resultant mutants were kinetically characterized. The results showed that substitution of bulky residues (e.g., isoleucine and methionine) for glycine at this position resulted in large increases in the K(m) values for trehalose and maltose, whereas the affinity to isomaltose was only minimally affected by such an amino-acid substitution at this position. Three-dimensional structural models of the enzyme-substrate complexes of the wild-type and mutant SAM1606 alpha-glucosidases were built to explore the mechanism responsible for these observations. It is proposed that substitution by glycine at position 273* could eliminate steric hindrance around subsite +1 that originally occurred in parental O16G and is, at least in part, responsible for the acquired broad substrate specificity of SAM1606 alpha-glucosidase.     

Molecular cloning and characterization of an enzyme hydrolyzing p-nitrophenyl alpha-D-glucoside from Bacillus stearothermophilus SA0301

Bacillus stearothermophilus SA0301 produces an extracellular oligo-1,6-glucosidase (bsO16G) that also hydrolyzes p-nitrophenyl alpha-D-glucoside (Tonozuka et al., J. Appl. Glycosci., 45, 397-400 (1998)). We cloned a gene for an enzyme hydrolyzing p-nitrophenyl alpha-D-glucoside, which was different from the one mentioned above, from B. stearothermophilus SA0301. The k(0)/K(m) values of bsO16G for isomaltotriose and isomaltose were 13.2 and 1.39 s(-1).mM(-1) respectively, while the newly cloned enzyme did not hydrolyze isomaltotriose, and the k(0)/K(m) value for isomaltose was 0.81 s(-1).mM(-1). The primary structure of the cloned enzyme more closely resembled those of trehalose-6-phosphate hydrolases than those of oligo-1,6-glucosidases, and the cloned enzyme hydrolyzed trehalose 6-phosphate. An open reading frame encoding a protein homologous to the trehalose-specific IIBC component of the phopshotransferase system was also found upstream of the gene for this enzyme.     

Bacillus stearothermophilus ATCC12016 α-glucosidase specific for α-1,4 bonds of maltosaccharides and α-glucans shows high amino acid sequence similarities to seven α-d-glucohydrolases with different substrate specificity

We have sequenced the gene encoding Bacillus stearothermophilus ATCC12016 -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.20) specific for non-reducing terminal -1,4 bonds of maltosaccharides and -glucans. The amino acid sequence of the enzyme deduced from the nucleotide sequence of the gene (1665 base pairs) consisted of 555 residues with a molecular mass of 65233. The enzyme showed 40%–57% sequence similarities to -d-glucohydrolases with very different substrate specificity, such as Bacillus cereus ATCC7064 oligo-1,6-glucosidase, Bacillus thermoglucosidasius KP1006 oligo-1,6-glucosidase, Saccharomyces carlsbergensis CB11 -glucosidase, Bacillus sp. F5 -glucosidase, Streptococcus mutans (Ingbritt strain) dextran glucosidase, Bacillus sp. SAM1606 -glucosidase and Escherichia coli ECL116 trehalose-6-phosphate hydrolase. All these enzymes had sequences equivalent to secondary elements revealed in B. cereus oligo-1,6-glucosidase by X-ray crystallography. We have suggested that the B.stearothermophilus enzyme adopts the same polypeptide folding, i.e. an (/)₈-barrel in the N-terminal active-site domain, as the B.cereus enzyme and other -glucohydrolases.     

Molecular characterization of the Thermomonospora curvata aglA gene encoding a thermotolerant alpha-1,4-glucosidase

The cloning, sequencing and structural characterization of a gene encoding a thermostable alpha-1,4-glucosidase from Thermomonospora curvata is described. DNA sequence analysis revealed four open reading frames designated aglA, aglR, aglE and aglF. The aglA gene encodes a thermostable alpha-1,4-glucosidase from T. curvata and is situated between two genes, aglR and aglE. Genes aglA, aglE and aglF are transcribed in the same direction, while aglR is transcribed in the opposite direction. By comparing the amino acid sequence of the alpha-1,4-glucosidase from T. curvata with other alpha-glucanases, it appears that the enzyme is a member of the alpha-amylase family. The proteins of this family have an (alpha/beta)8 barrel super secondary structure. The topology of the alpha-1,4-glucosidase was predicted by computer-assisted analysis. The topology of the secondary structures of the alpha-1,4-glucosidase resembles the structure of barley alpha-amylase, but the primary structure resembles most closely the oligo-1,6-glucosidase from Bacillus cereus. Putative catalytic residues (D221, E281 and D343) and calcium binding residues (N116, E179, D191, H224 or G225) are proposed.     

Structural elucidation of the nonclassical secondary cell wall polysaccharide from Bacillus cereus ATCC 10987. Comparison with the polysaccharides from Bacillus anthracis and B. cereus type strain ATCC 14579 reveals both unique and common structural features

Nonclassical secondary cell wall polysaccharides constitute a major cell wall structure in the Bacillus cereus group of bacteria. The structure of the secondary cell wall polysaccharide from Bacillus cereus ATCC 10987, a strain that is closely related to Bacillus anthracis, was determined. This polysaccharide was released from the cell wall with aqueous hydrogen fluoride (HF) and purified by gel filtration chromatography. The purified polysaccharide, HF-PS, was characterized by glycosyl composition and linkage analyses, mass spectrometry, and one- and two-dimensional NMR analysis. The results showed that the B. cereus ATCC 10987 HF-PS has a repeating oligosaccharide consisting of a -->6)-alpha-GalNAc-(1-->4)-beta-ManNAc-(1-->4)-beta-GlcNAc-(1--> trisaccharide that is substituted with beta-Gal at O3 of the alpha-GalNAc residue and nonstoichiometrically acetylated at O3 of the N-acetylmannosamine (ManNAc) residue. Comparison of this structure with that of the B. anthracis HF-PS and with structural data obtained for the HF-PS from B. cereus type strain ATCC 14579 revealed that each HF-PS had the same general structural theme consisting of three HexNAc and one Hex residues. A common structural feature in the HF-PSs from B. cereus ATCC 10987 and B. anthracis was the presence of a repeating unit consisting of a HexNAc(3) trisaccharide backbone in which two of the three HexNAc residues are GlcNAc and ManNAc and the third can be either GlcNAc or GalNAc. The implications of these results with regard to the possible functions of the HF-PSs are discussed.     

Molecular cloning of an alpha-glucosidase-like gene from Penicillium minioluteum and structure prediction of its gene product

The dexC cDNA, which is expressed in dextran-containing medium by the filamentous fungus Penicillium minioluteum, was cloned and sequence characterized. The cDNA sequence comprises 1859 bp plus a poly (A) tail, coding for a predicted protein of 597 amino acids. The genomic counterpart was isolated by PCR, finding three introns in its sequence. The dexC gene was located by Southern blot in the same 9-kb fragment that the previously isolated dextranase-encoding gene (dexA). Sequence analysis revealed that the deduced DexC protein belongs to glycosyl hydrolase family 13, showing a high sequence identity (58%) with Aspergillus parasiticus alpha-1,6-glucosidase. In addition, the high sequence identity (51%) between DexC protein and oligo-1,6-glucosidase of Bacillus cereus, with three-dimensional (3D) structure determined, leads us to proposed a 3D model for the structural core of DexC protein.     

A hyperthermostable pullulanase produced by an extreme thermophile,Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability

Summary A cell-associated pullalanase (-dextrin 6-glucanohydrolase, EC 3.2.1.41) of an extreme thermophile, Bacillus flavocaldarius KP 1228, was purified to homogeneity. The molecular weight and isoelectric point were estimated to be about 55 000 and 7.0, respectively. The N-terminal sequence was Ala-Try-Tyr-Glu-Gly-Ala-Phe-Phe-Tyr-Gln-Ile-Phe-Pro-Asp-Tyr-Phe-Phe-Tyr-Ala-Gly-. The enzyme was most active at pH 6.3. The activities for 5% pullulan and 5% soluble starch were maximal at 75–80° C and at 80–85° C, respectively. The enzyme was stable up to 90° C for 10 min at pH 6.8. The enzyme had no antigenic determinants shared with pullulanases from the mesophiles Klebsiella pneumoniae and B. acidopullulyticus NCIB 11647. A comparison of amino acid composition demonstrated that the proline content increased greatly in a linear fashion with the rise in thermostability in the order K. pneumoniae B. acidopullulyticus B. flavocaldarius enzymes, as found with Bacillus oligo-1,6-glucosidases.Presented in part at the Annual Meeting of the Agricultural Chemical Society of Japan at Tokyo, April 2, 1987 (Abstracts, p 91)Offprint requests to: Y. Suzuki