蜡样芽胞杆菌GXBC-3三个普鲁兰酶基因的表达及其酶学特性

探索获得优良的新型普鲁兰酶基因,丰富普鲁兰酶理论,对实现普鲁兰酶国产化具有重要意义。分析GenBank数据库中蜡样芽胞杆菌假定Ⅰ型、Ⅱ型普鲁兰酶基因序列,从实验室保藏的蜡样芽胞杆菌Bacilluscereus GXBC-3中克隆得到3个普鲁兰酶基因pulA、pulB、pulC,并分别导入大肠杆菌进行胞内诱导表达。纯化重组酶酶学性质研究表明重组酶PulA能水解α-l,6-和α-l,4-糖苷键,为Ⅱ型普鲁兰酶,以普鲁兰糖为底物时,最适反应温度及pH分别为40℃和6.5,比活力为32.89 U/mg;以可溶性淀粉为底物时,最适反应温度及pH分别为50℃和7.0,比活力为25.71 U/mg。重组酶PulB和PulC二者均只能水解α-l,6-糖苷键,为I型普鲁兰酶,以普鲁兰糖为底物时,其最适反应温度及pH分别为45℃、7.0和45℃、6.5,比活力分别为228.54 U/mg和229.65 U/mg。     

Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface.

The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned in Escherichia coli, and the nucleotide sequence was determined. The reading frame of amyB consisted of 5,586 bp encoding an exceptionally large enzyme of 205,991 Da. Sequence analysis revealed a composite structure of the pullulanase consisting of catalytic and noncatalytic domains. The N-terminal half of the protein contained a leader peptide of 35 amino acid residues and the catalytic domain, which included the four consensus regions of amylases. Comparison of the consensus regions of several pullulanases suggested that enzymes like pullulanase type II from T. thermosulfurigenes EM1 which hydrolyze alpha-1,4- and alpha-1,6-glycosidic linkages have specific amino acid sequences in the consensus regions. These are different from those of pullulanases type I which only cleave alpha-1,6 linkages. The C-terminal half, which is not necessary for enzymatic function, consisted of at least two different segments. One segment of about 70 kDa contained two copies of a fibronectin type III-like domain and was followed by a linker region rich in glycine, serine, and threonine residues. At the C terminus, we found three repeats of about 50 amino acids which are also present at the N-termini of surface layer (S-layer) proteins of, e.g., Thermus thermophilus and Acetogenium kivui. Since the pullulanase of T. thermosulfurigenes EM1 is known to be cell bound, our results suggest that this segment serves as an S-layer anchor to keep the pullulanase attached to the cell surface. Thus, a general model for the attachment of extracellular enzymes to the cell surface is proposed which assigns the S-layer a new function and might be widespread among bacteria with S-layers. The triplicated S-layer-like segment is present in several enzymes of different bacteria. Upstream of amyB, another open reading frame, coding for a hypothetical protein of 35.6 kDa, was identified. No significant similarity to other sequences available in DNA and protein data bases was found.     

A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in Bacillus subtilis

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85 degrees C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80 degrees C for 4 h and exhibited a half-life of 50 min at 85 degrees C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed alpha-1,6 glycosidic linkages of pullulan, producing maltotriose, and also alpha-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.     

Pullulanase type I from Fervidobacterium pennavorans Ven5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinant enzyme

The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80 degrees C and had a half-life of 2 h at 80 degrees C. The rPulAs hydrolyzed alpha-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, alpha-beta-limited dextrin. Interestingly, amylose, which contains only alpha-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.     

Cloning, sequencing, and characterization of a heat- and alkali-stable type I pullulanase from Anaerobranca gottschalkii

The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70 degrees C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg'). The N-terminal sequence of purified rPulAg' was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg' were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, beta-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg' was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from A. gottschalkii is the first thermoalkalistable type I pullulanase that has been described.     

Expression and biochemical characterization of a novel type I pullulanase from <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Objectives

To identify novel pullulanases from microorganisms and to investigate their biochemical characterizations.

Results

A novel pullulanase gene (BmPul) from Bacillus megaterium WW1210 was cloned and heterologously expressed in Escherichia coli. The gene has an ORF of 2814 bp encoding 937 amino acids. The recombinant pullulanase (BmPul) was purified to homogeneity and biochemically characterized. BmPul has an MW of approx. 112 kDa as indicated by SDS-PAGE. Optimum conditions were at 55 °C and pH 6.5. The enzyme was stable below 40 °C and from pH 6.5?8.5. The Km values of BmPul towards pullulan and amylopectin were 3.3 and 3.6 mg/ml, respectively. BmPul hydrolyzed pullulan to yield mainly maltotriose, indicating that it should be a type I pullulanase.

Conclusions

A novel type I pullulanase from Bacillus megaterium was identified, heterologously expressed and biochemically characterized. Its properties makes this enzyme as a good candidate for the food industry.

     

Biosynthesis and secretion of pullulanase, a lipoprotein from Klebsiella aerogenes

We constructed, by site-directed mutagenesis, a mutant pullulanase gene in which the cysteine residue in a pentapeptide sequence, Leu16-Leu-Ser-Gly-Cys20 within the NH2-terminal region of pullulanase from Klebsiella aerogenes, is replaced by serine (Ser20). The modification, processing, and subcellular localization of the mutant pullulanase were studied. Labeling studies with [3H]palmitate and immunoprecipitation with mouse antiserum raised against pullulanase showed that the wild form of both the extracellular and intracellular pullulanases contained lipids, whereas the mutant enzyme was not modified with lipids. Only the Cys20 was modified with glyceryl lipids. The bulk of the mutant pullulanase was located in the periplasm, but a portion of the unmodified, mutant pullulanase was secreted into the medium. Mutant pullulanases from the extracellular and the periplasm were purified and their NH2-terminal sequences were determined. Both the mutant pullulanases were cleaved between residues of Ser13 and Leu14 which is 6-amino acid residues upstream of the lipid modified pullulanase cleavage site. This new cleavage was resistant to globomycin, an inhibitor of the prolipoprotein signal peptidase of Escherichia coli. These results indicate that the pentapeptide sequence plays an important role in maturation and translocation of pullulanase in K. aerogenes. However, the modification of pullulanase with lipids seems to be not essential for export of the enzyme across the outer membrane.     

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis.

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.     

A novel type I thermostable pullulanase isolated from a thermophilic starch enrichment culture

In this paper we report identification, cloning and characterization of a novel thermostable pullulanase type I. Pullulanase AmyA1 was detected in a sample of extracellular proteins of thermophilic enrichment culture, growing on starch. The zone of enzymatic activity in zymogram was aligned with the corresponding band on the equivalent gel without substrate. The band was excised from SDS/polyacrylamide gel and subjected to liquid chromatography/mass spectrometry (LC/MS) analysis. LC/MS-based analysis identified thermostable pullulanases, homologues to type I pullulanases of Geobacillus thermodenitrificans NG80-2 and Geobacillus sp. G11MC16. Nucleotide sequences of these two pullulanases were used for design of primers for PCR with DNA from enrichment culture, leaded to 2181 bp PCR product, coding a 726 amino acids protein, named pullulanase AmyA1. Molecular weight of AmyA1 was calculated to be 81.7 kDa. AmyA1 was cloned and expressed in Escherichia coli. Recombinant pullulanase was purified by two chromatographic separation steps. Pullulanase AmyA1 was active against pullulan, glycogen and soluble starch. It was active in the temperature range of 4–95°C, optimum temperature was determined to be 60°C. The highest activity of the recombinant pullulanase was observed at pH 6. Divalent cations Mg²⁺ and Mn²⁺ as well as dithiothreitol, Brij 35 and Brij 58 had a stimulating effect on the enzymatic activity. Pullulanase AmyA1 was stable during incubation in the presence of 4 M urea. After removal of the His-tag, addition of Ca²⁺ stimulated activity of the enzyme suggesting the native pullulanase activity to be dependent on Ca²⁺. Thermostability of AmyA1 was not enhanced by the addition of Ca²⁺.     

Purification and Characterization of Thermostable Pullulanase from Bacillus stearothermophilus and Molecular Cloning and Expression of the Gene in Bacillus subtilis

A thermostable pullulanase (alpha-dextrin 6-glucanohydrolase [EC 3.2.1.41]) from a newly isolated Bacillus stearothermophilus strain (TRS128) was purified and characterized. The enzyme hydrolyzed (1-->6)-alpha-d-glucosidic linkages of pullulan to produce maltotriose, and the optimum temperature was 65 degrees C. About 90% of the enzyme activity was retained after treatment at 65 degrees C for 60 min. By using pTB522 as a vector plasmid, the pullulanase gene was cloned and expressed in Bacillus subtilis.     

Advances and challenges in the production of extracellular thermoduric pullulanases by wild-type and recombinant microorganisms: a review

Thermoduric pullulanases, acting as starch-debranching enzymes, are required in many industrial applications, mainly in the production of concentrated glucose, maltose, and fructose syrups. To date, however, a single pullulanase, from Bacillus acidopullulyticus, is available on the market for industrial purposes. This review is an investigation of the major advances as well as the major challenges being faced with regard to optimization of the production of extracellular thermoduric pullulanases either by their original hosts or by recombinant organisms. The critical aspects linked to industrial pullulanase production, which should always be considered, are emphasized, including those parameters influencing solubility, thermostability, and catalytic efficiency of the enzyme. This review provides new insights for improving the production of extracellular thermoduric pullulanases in the hope that such information may facilitate their commercial utilization and potentially be applied to the development of other industrially relevant enzymes.     

Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from Anaerobranca gottschalkii

The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg′). The N-terminal sequence of purified rPulAg′ was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg′ were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, β-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg′ was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from A. gottschalkii is the first thermoalkalistable type I pullulanase that has been described.     

Crystal structure of beta-amylase from Bacillus cereus var. mycoides at 2.2 A resolution.

The crystal structure of beta-amylase from Bacillus cereus var. mycoides was determined by the multiple isomorphous replacement method. The structure was refined to a final R-factor of 0.186 for 102,807 independent reflections with F/sigma(F) > or = 2.0 at 2.2 A resolution with root-mean-square deviations from ideality in bond lengths, and bond angles of 0.014 A and 3.00 degrees, respectively. The asymmetric unit comprises four molecules exhibiting a dimer-of-dimers structure. The enzyme, however, acts as a monomer in solution. The beta-amylase molecule folds into three domains; the first one is the N-terminal catalytic domain with a (beta/alpha)8 barrel, the second one is the excursion part from the first one, and the third one is the C-terminal domain with two almost anti-parallel beta-sheets. The active site cleft, including two putative catalytic residues (Glu172 and Glu367), is located on the carboxyl side of the central beta-sheet in the (beta/alpha)8 barrel, as in most amylases. The active site structure of the enzyme resembles that of soybean beta-amylase with slight differences. One calcium ion is bound per molecule far from the active site. The C-terminal domain has a fold similar to the raw starch binding domains of cyclodextrin glycosyltransferase and glucoamylase.     

A catalytic mechanism revealed by the crystal structures of the imidazolonepropionase from Bacillus subtilis

Imidazolonepropionase (EC 3.5.2.7) catalyzes the third step in the universal histidine degradation pathway, hydrolyzing the carbon-nitrogen bonds in 4-imidazolone-5-propionic acid to yield N-formimino-l-glutamic acid. Here we report the crystal structures of the Bacillus subtilis imidazolonepropionase and its complex at 2.0-A resolution with substrate analog imidazole-4-acetic acid sodium (I4AA). The structure of the native enzyme contains two domains, a TIM (triose-phosphate isomerase) barrel domain with two insertions and a small beta-sandwich domain. The TIM barrel domain is quite similar to the members of the alpha/beta barrel metallo-dependent hydrolase superfamily, especially to Escherichia coli cytosine deaminase. A metal ion was found in the central cavity of the TIM barrel and was tightly coordinated to residues His-80, His-82, His-249, Asp-324, and a water molecule. X-ray fluorescence scan analysis confirmed that the bound metal ion was a zinc ion. An acetate ion, 6 A away from the zinc ion, was also found in the potential active site. In the complex structure with I4AA, a substrate analog, I4AA replaced the acetate ion and contacted with Arg-89, Try-102, Tyr-152, His-185, and Glu-252, further defining and confirming the active site. The detailed structural studies allowed us to propose a zinc-activated nucleophilic attack mechanism for the hydrolysis reaction catalyzed by the enzyme.     

Role of lipid modification on a starch-debranching enzyme, Klebsieila pullulanase: comparison of properties of lipid-modified and unmodified pullulanases

Klebsiella pullulanase is a lipoprotein synthesized as a precursor with a signal peptide, which is processed by lipoprotein signal peptidase. To clarify the role of lipid modification of pullulanase, we purified lipid-modified wild-type and the unmodified (mutant) pullulanases and compared their properties. The Km and Vmax values of both pullulanases for pullulan were the same. The optimal pH and temperature, the stabilities over pH and temperature ranges, the specificity of substrates, and the patterns of inhibition of the lipid-modified and unmodified pullulanases were also the same. However, we found that the wild-type pullulanase formed trimers whereas the unmodified enzyme did not, and that the migrations of the two enzymes on sodium dodecyl sulphate/electrophoresis were different when the samples were applied on the gel without heating. The results presented in this paper and in previous work show that the correct processing and translocation of pullulanase in K. aerogenes require modification of lipid. However, the enzymatic properties and physical stabilities of pullulanase were not affected by the lipid modification.     

The Type II Pullulanase of Thermococcus hydrothermalis: Molecular Characterization of the Gene and Expression of the Catalytic Domain

The gene encoding a hyperthermostable type II pullulanase produced by Thermococcus hydrothermalis (Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence of three open reading frames, one of which (apuA) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a domain bearing S-layer homology-like motifs, a Thr-rich region, and a potential C-terminal transmembrane domain. The presence of these noncatalytic domains suggests that Th-Apu may be anchored to the cell surface and be O glycosylated.     

Functional and structural studies of pullulanase from Anoxybacillus sp. LM18‐11

Pullulanase is a debranching enzyme that specifically hydrolyzes the α‐1,6 glycosidic linkage of α‐glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18‐11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and K_m for pullulan of PulA are 750 U mg^?1 and 16.4 μmol L^?1, respectively. PulA has a half‐life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75–2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β‐strand and the third α‐helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate‐binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain. Proteins 2014; 82:1685–1693. © 2013 Wiley Periodicals, Inc.     

Domain evolution in the GH13 pullulanase subfamily with focus on the carbohydrate-binding module family 48

Glycoside hydrolase (GH) family 13 comprises about 30 different specificities. Four of them have been proposed to form the GH13 pullulanase subfamily: pullulanase, isoamylase, maltooligosyl trehalohydrolase and branching enzyme forming the seven CAZy GH13 subfamilies: GH13 8-GH13 14. Recently, a new family of carbohydrate-binding modules (CBMs), the family CBM48 has been established containing the putative starch-binding domains from the pullulanase subfamily, the β-subunit of AMP-activated protein kinase and some other GH13 enzymes with pullulanase and/or α-amylase-pullulanase specificity. Since all of these enzymes are multidomain proteins and the structure for at least one representative of each enzyme specificity has already been determined, the main goal of the present study was to elucidate domain evolution within this GH13 pullulanase subfamily (84 real enzymes) focusing on the CBM48 module. With regard to CBM48 positioning in the amino acid sequence, the N-terminal end of a protein appears to be a predominant position. This is especially true for isoamylases and maltooligosyl trehalohydrolases. Secondary structure-based alignment of CBM modules from CBM48, CBM20 and CBM21 revealed that several residues known as consensus for CBM20 and CBM21 could also be identified in CBM48, but only branching enzymes possess the aromatic residues that correspond with the two tryptophans forming the evolutionary conserved starch-binding site 1 in CBM20. The evolutionary trees constructed for the individual domains, complete alignment, and the conserved sequence regions of the α-amylase family were found to be comparable to each other (except for the C-domain tree) with two basic parts: (i) branching enzymes and maltooligosyl trehalohydrolases; and (ii) pullulanases and isoamylases. Taxonomy was respected only within clusters with pure specificity, i.e. the evolution of CBM48 reflects the evolution of specificities rather than evolution of species. This is a feature different from the one observed for the starch-binding domain of the family CBM20 where the starch-binding domain evolution reflects the evolution of species.     

Production of novel pullulanases at high concentrations by two newly isolated thermophilic clostridia

Two thermophilic bacteria, which are capable of growing on starch at 60-70 degrees C under anaerobic conditions, were isolated from a sugar refinery in Uelzen and from Solar lake in Israel. On the basis of their physiological characteristics they were identified as Clostridium thermohydrosulfuricum Uel 1 and C. thermohydrosulfuricum Sol 1, respectively. The product pattern of glucose polymer hydrolysis showed that both strains secreted enzymes that possess amylolytic and pullulytic activities. The major product formed was maltose. In addition, alpha-glucosidase activity could be detected in the supernatants of Uel 1 strain. Compared to most anaerobes investigated these isolates secreted extremely high concentrations of pullulanases in batch culture. Up to 85% of the total enzyme synthesized was detected in the culture fluid. Unlike the pullulanases of type I, which can only attack the alpha-1,6-glycosidic linkages, the pullulanases of both clostridial strains were also capable of hydrolyzing alpha-1,4-linkages. The enzyme system of both bacteria was found to be highly thermoactive; optimal activity was detected at pH 5.0 and 85 degrees C. Even at 95 degrees C and without the addition of metal ions still 15% to 25% of enzymatic activity was detectable.     

Functional Characterization of a Newly Identified Group B Streptococcus Pullulanase Eliciting Antibodies Able to Prevent Alpha-Glucans Degradation

Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade α-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of α-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of α-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections.