Thermophilus HB8 Research Articles

Inactivation and unfolding of protein tyrosine phosphatase from Thermus thermophilus HB27 during urea and guanidine hydrochloride denaturation.

The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase.

Lysine Propionylation Is a Prevalent Post-translational Modification in Thermus thermophilus

Recent studies of protein post-translational modifications revealed that various types of lysine acylation occur in eukaryotic and bacterial proteins. Lysine propionylation, a newly discovered type of acylation, occurs in several proteins, including some histones. In this study, we identified 361 propionylation sites in 183 mid-exponential phase and late stationary phase proteins from Thermus thermophilus HB8, an extremely thermophilic eubacterium. Functional classification of the propionylproteins revealed that the number of propionylation sites in metabolic enzymes increased in late stationary phase, irrespective of protein abundance. The propionylation sites on proteins expressed in mid-exponential and late stationary phases partially overlapped. Furthermore, amino acid frequencies in the vicinity of propionylation sites differed, not only between the two growth phases but also relative to acetylation sites. In addition, 33.8% of mid-exponential phase-specific and 80.0% of late stationary phase-specific propionylations (n ≥ 2) implied that specific mechanisms regulate propionylation in the cell. Moreover, the limited degree of overlap between lysine propionylation (36.8%) and acetylation (49.2%) sites in 67 proteins that were both acetylated and propionylated strongly suggested that the two acylation reactions are regulated separately by specific enzymes and may serve different functions. Finally, we also found that eight propionylation sites overlapped with acetylation sites critical for protein functions such as Schiff-base formation and ligand binding.

A novel six-coordinated ferric ion binding mode of TtFbpA

Iron is an essential element for the growth and survival of nearly all living organisms. However, it is difficult for most organisms to get enough iron from the environment, because of the extremely low solubility of ferric ion. One of the strategies for iron acquisition is to use the ATP-binding cassette (ABC) transport system. In Gram-negative bacteria, a typical iron uptake ABC transporter consists of a ferric ion-binding protein (Fbp) located in periplasm (FbpA), two transmembrane proteins that form a pathway for ferric ions (FbpB), and two peripheral ATP-binding proteins located at the cytoplasm side of the inner membrane (FbpC). TtFbpA is a ferric ion-binding protein of a putative iron uptake ABC transporter from Thermus thermophilus HB8. Here we report the crystal structures of the apo-form and ferric ion-bound form of TtFbpA at 1.8-Å and 1.7-Å resolutions, respectively [1]. The crystal structure of the ferric ion-bound TtFbpA shows that a ferric ion binds to a specific site of TtFbpA to form a six-coordinated complex by three tyrosine residues, two bicarbonates and a water molecule, revealing a novel mode of coordination to a ferric ion. Another crystal structure of ferric ion-bound TtFbpA reported earlier showed the bound ferric ion is five-coordinated by three tyrosine residues and a carbonate bound in the bidentate manner [2]. The different modes of the coordination would probably result from the different pHs used for crystallization: pH 5.5 (six-coordinated) vs. pH 7.5 (five-coordinated). The Gram negative bacterium T. thermophilus HB8 can live in a wide pH range of 3.4–9.6. We propose that TtFbpA, a periplasmic protein of T. thermophilus HB8, can act as a ferric ion-binding protein over the wide pH range by taking at least two different coordination manners to a ferric ion depending on pH. This is the first example of a periplasmic ferric iron-binding protein that can coordinate a ferric ion via multiple types of coordination complex formation.

Genetic analysis of lipolytic activities in Thermus thermophilus HB27

The extremely thermophilic bacterium Thermus thermophilus HB27 displays lipolytic activity for the hydrolysis of triglycerides. In this study we performed a mutational in vivo analysis of esterases and lipases that confer growth on tributyrin. We interrupted 10 ORFs suspected to encode lipolytic enzymes. Two chromosomal loci were identified that resulted in reduced hydrolysis capabilities against tributyrin and various para-nitrophenyl acyl esters. By implementation of a convenient new one-step method which abstains from the use of selectable markers, a mutant strain with multiple scar-less deletions was constructed by sequentially deleting ORFs TT_C1787, TT_C0340, TT_C0341 and TT_C0904. The quadruple deletion mutant of T. thermophilus exhibited significantly lower lipolytic activity (approximately 25% residual activity compared to wild type strain) over a broad range of fatty acyl esters and had lost the ability to grow on agar plates containing tributyrin as the sole carbon source. Furthermore, we were able to determine the impact of each gene disruption on the lipolytic activity profile in this model organism and show that the esterase activity in T. thermophilus HB27 is due to a concerted action of several hydrolases having different substrate preferences and activities. The esterase-less T. thermophilus multi-deletion mutant from this study can be used as a screening and expression host for esterase genes from thermophiles or metagenomes.

Correction for Shinkai et al., Identification of Promoters Recognized by RNA Polymerase-σ E Holoenzyme from Thermus thermophilus HB8

Volume 189, no. 23, pages [8758-8764][1], 2007. We corrected ([Fig. 4B][2] and ([C][2] of this article, to clarify that these figures were created by the assembly of several images. The raw data used for the creation of these figures are shown in Fig. S1 in the supplemental material. ![Figure][3]</

Environmental factors affecting the expression of type IV pilus genes as well as piliation of Thermus thermophilus.

The thermophilic bacterium Thermus thermophilus HB27 is known for its highly efficient natural transformation system, which has become a model system to study the structure and function of DNA transporter in thermophilic bacteria. The DNA transporter is functionally linked to type IV pili (T4P), which are essential for twitching motility and adhesion to solid surfaces. However, the pilus structures themselves are dispensable for natural transformation. Here, we report that the cellular mRNA levels of the major structural subunit of the T4P, PilA4, are regulated by environmental factors. Growth of T. thermophilus in minimal medium or low temperature (55 °C) leads to a significant increase in pilA4 transcripts. In contrast, the transcript levels of the minor pilin pilA1 as well as other T4P genes are nearly unaffected. The elevated pilA4 mRNA levels are accompanied by an increase in piliation of the cells but not by elevated natural transformation frequencies. Hyperpiliation leads to increased adhesion to plastic surfaces. The increased cell-surface interactions are suggested to represent an adaptive response to temperature stress and may be advantageous for survival of T. thermophilus.

Metabolic network reconstruction, growth characterization and 13C-metabolic flux analysis of the extremophile Thermus thermophilus HB8

Thermus thermophilus is an extremely thermophilic bacterium with significant biotechnological potential. In this work, we have characterized aerobic growth characteristics of T. thermophilus HB8 at temperatures between 50 and 85°C, constructed a metabolic network model of its central carbon metabolism and validated the model using 13C-metabolic flux analysis (13C–MFA). First, cells were grown in batch cultures in custom constructed mini-bioreactors at different temperatures to determine optimal growth conditions. The optimal temperature for T. thermophilus grown on defined medium with glucose was 81°C. The maximum growth rate was 0.25h−1. Between 50 and 81°C the growth rate increased by 7-fold and the temperature dependence was described well by an Arrhenius model with an activation energy of 47kJ/mol. Next, we performed a 13C-labeling experiment with [1,2-13C] glucose as the tracer and calculated intracellular metabolic fluxes using 13C–MFA. The results provided support for the constructed network model and highlighted several interesting characteristics of T. thermophilus metabolism. We found that T. thermophilus largely uses glycolysis and TCA cycle to produce biosynthetic precursors, ATP and reducing equivalents needed for cells growth. Consistent with its proposed metabolic network model, we did not detect any oxidative pentose phosphate pathway flux or Entner-Doudoroff pathway activity. The biomass precursors erythrose-4-phosphate and ribose-5-phosphate were produced via the non-oxidative pentose phosphate pathway, and largely via transketolase, with little contribution from transaldolase. The high biomass yield on glucose that was measured experimentally was also confirmed independently by 13C–MFA. The results presented here provide a solid foundation for future studies of T. thermophilus and its metabolic engineering applications.

Specificity and Catalytic Mechanism in Family 5 Uracil DNA Glycosylase

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O2 of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb relies on multiple catalytic residues to facilitate its excision of hypoxanthine and xanthine. This study underscores the structural and functional diversity in the UDG superfamily.

The role of ribonucleases in regulating global mRNA levels in the model organism Thermus thermophilus HB8.

BackgroundRNA metabolism, including RNA synthesis and RNA degradation, is one of the most conserved biological systems and has been intensively studied; however, the degradation network of ribonucleases (RNases) and RNA substrates is not fully understood.ResultsThe genome of the extreme thermophile, Thermus thermophilus HB8 includes 15 genes that encode RNases or putative RNases. Using DNA microarray analyses, we examined the effects of disruption of each RNase on mRNA abundance. Disruption of the genes encoding RNase J, RecJ-like protein and RNase P could not be isolated, indicating that these RNases are essential for cell viability. Disruption of the TTHA0252 gene, which was not previously considered to be involved in mRNA degradation, affected mRNA abundance, as did disruption of the putative RNases, YbeY and PhoH-like proteins, suggesting that they have RNase activity. The effects on mRNA abundance of disruption of several RNase genes were dependent on the phase of cell growth. Disruption of the RNase Y and RNase HII genes affected mRNA levels only during the log phase, whereas disruption of the PhoH-like gene affected mRNA levels only during the stationary phase. Moreover, disruption of the RNase R and PNPase genes had a greater impact on mRNA abundance during the stationary phase than the log phase, whereas the opposite was true for the TTHA0252 gene disruptant. Similar changes in mRNA levels were observed after disruption of YbeY or PhoH-like genes. The changes in mRNA levels in the bacterial Argonaute disruptant were similar to those in the RNase HI and RNase HII gene disruptants, suggesting that bacterial Argonaute is a functional homolog of RNase H.ConclusionThis study suggests that T. thermophilus HB8 has 13 functional RNases and that each RNase has a different function in the cell. The putative RNases, TTHA0252, YbeY and PhoH-like proteins, are suggested to have RNase activity and to be involved in mRNA degradation. In addition, PhoH-like and YbeY proteins may act cooperatively in the stationary phase. This study also suggests that endo-RNases function mainly during the log phase, whereas exo-RNases function mainly during the stationary phase. RNase HI and RNase HII may have similar substrate selectivity.

Functional and structural characterization of protein disulfide oxidoreductase from Thermus thermophilus HB27.

The paper reports the characterization of a protein disulfide oxidoreductase (PDO) from the thermophilic Gram negative bacterium Thermus thermophilus HB27, identified as TTC0486 by genome analysis and named TtPDO. PDO members are involved in the oxidative folding, redox balance and detoxification of peroxides in thermophilic prokaryotes. Ttpdo was cloned and expressed in E. coli and the recombinant purified protein was assayed for the dithiol-reductase activity using insulin as substrate and compared with other PDOs characterized so far. In the thermophilic archaeon Sulfolobus solfataricus PDOs work as thiol-reductases constituting a peculiar redox couple with Thioredoxin reductase (SsTr). To get insight into the role of TtPDO, a hybrid redox couple with SsTr, homologous to putative Trs of T. thermophilus, was assayed. The results showed that SsTr was able to reduce TtPDO in a concentration dependent manner with a calculated K M of 34.72 μM, suggesting the existence of a new redox system also in thermophilic bacteria. In addition, structural characterization of TtPDO by light scattering and circular dichroism revealed the monomeric structure and the high thermostability of the protein. The analysis of the genomic environment suggested a possible clustering of Ttpdo with TTC0487 and TTC0488 (tlpA). Accordingly, transcriptional analysis showed that Ttpdo is transcribed as polycistronic messenger. Primer extension analysis allowed the determination of its 5'end and the identification of the promoter region.

Cloning, expression, purification and characterization of an oligomeric His-tagged thermophilic esterase from Thermus thermophilus HB27

The esterase E34Tt (YP_004875.1) from Thermus thermophilus HB27 was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The gene sequence was subcloned into a T-vector, released with the restriction enzymes BamHI and HindIII, ligated to a pET-21d(+) vector, and transferred to E. coli BL21 (DE3) cells. Inducer concentration (isopropyl β-d-1-thiogalactopyranoside, IPTG) and cultivation time before and after induction were optimized. Best results were obtained by adding 0.25mM IPTG after 8h of cultivation and maintaining the induction during 4 extra hours. Most of the enzyme (94%) remained membrane-associated and had to be extracted with a detergent. From the membrane crude extract, the His-tagged E34Tt was purified as a dimer (71.8kDa) in a single purification step by using metal affinity chromatography. The Rosso's model was used to optimize the reaction conditions. E34Tt-His6 was active in a wide temperature (19.7–79.4°C) and pH range (4.0–9.3), and maximal activity was determined at pH 6.3 and 58.2°C, which is 10–18°C higher than the optimal reaction temperature of the previously reported variants expressed in mesophilic yeasts. E34Tt-His6 preferentially hydrolyzed esters with ten carbon atoms, and was highly thermostable (half-life of 107.9min at 85°C), suggesting that E34Tt-His6 has potential for industrial applications.

Thermus thermophilus HB27锰过氧化氢酶在大肠杆菌中的表达及酶学性质分析

碱性嗜热过氧化氢酶是一种重要的纺织用酶.根据大肠杆菌密码子使用偏爱性,对Thermus thermophilus HB27来源的含锰过氧化氢酶基因进行密码子优化,将优化后的基因连接至表达载体pET28a（＋）上,转化到E.coli BL21（DE3）中进行诱导表达.结果表明在含有14mmol/L Mn2＋浓度的培养液中以0.2 mmol/L的IPTG 42℃条件下诱导2h的情况下,菌体破碎上清液中的酶活力可达25 U/ml.利用Ni亲和层析柱对该Mn-CAT进行纯化,酶学性质研究表明：此酶的最适温度为70℃,最适pH为pH 10.0,在80℃保温2h,酶活力不损失;pH9.0～11.0的环境中放置2h后,酶活仅损失约10％,此酶具有良好的工业开发潜力.

Structural insights of post-translational modification sites in the proteome of Thermus thermophilus

Phosphorylation and acetylation are the most prevalent post-translational modifications (PTMs) detected in not only eukaryotes but also bacteria. We performed phosphoproteome and acetylome analyses of proteins from an extremely thermophilic eubacterium Thermus thermophilus HB8, and identified numerous phosphorylation and acetylation sites. To facilitate the elucidation of the structural aspects of these PTM events, we mapped the PTM sites on the known tertiary structures for the respective proteins and their homologs. Wu et al. (Mol Cell Proteomics 12:2701-2713, 2013) recently reported phosphoproteome analysis of proteins from T. thermophilus HB27. Therefore, we assessed the structural characteristics of these phosphorylation and acetylation sites on the tertiary structures of the identified proteins or their homologs. Our study revealed that many of the identified phosphosites are in close proximity to bound ligands, i.e., the numbers of 'nearby' and 'peripheral' phosphorylation sites represent 56 % (48/86 sites) of total identified phosphorylation sites. In addition, approximately 60 % of all phosphosites exhibited <10 % accessible surface area of their side chains, suggesting some structural rearrangement is required for phosphoryl transfer by kinases. Our findings also indicate that phosphorylation of a residue occurs more frequently at a flexible region of the protein, whereas lysine acetylation occurs more frequently in an ordered structure.

Biochemical and Structural Analysis of RraA Proteins To Decipher Their Relationships with 4-Hydroxy-4-methyl-2-oxoglutarate/4-Carboxy-4-hydroxy-2-oxoadipate Aldolases

4-Hydroxy-4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolases are class II (divalent metal ion dependent) pyruvate aldolases from the meta cleavage pathways of protocatechuate and gallate. The enzyme from Pseudomonas putida F1 is structurally similar to a group of proteins termed regulators of RNase E activity A (RraA) that bind to the regulatory domain of RNase E and inhibit the ribonuclease activity in certain bacteria. Analysis of homologous RraA-like proteins from varying species revealed that they share sequence conservation within the active site of HMG/CHA aldolase. In particular, the P. putida F1 HMG/CHA aldolase has a D-X20-R-D motif, whereas a G-X20-R-D-X2-E/D motif is observed in the structures of the RraA-like proteins from Thermus thermophilus HB8 (TtRraA) and Saccharomyces cerevisiae S288C (Yer010Cp) that may support metal binding. TtRraA and Yer010Cp were found to contain HMG aldolase and oxaloacetate decarboxylase activities. Similar to the P. putida F1 HMG/CHA aldolase, both TtRraA and Yer010Cp enzymes required divalent metal ions for activity and were competitively inhibited by oxalate, a pyruvate enolate analogue, suggesting a common mechanism among the enzymes. The RraA from Escherichia coli (EcRraA) lacked detectable C-C lyase activity. Upon restoration of the G-X20-R-D-X2-E/D motif, by site-specific mutagenesis, the EcRraA variant was able to catalyze oxaloacetate decarboxylation. Sequence analysis of RraA-like gene products found across all the domains of life revealed conservation of the metal binding motifs that can likely support a divalent metal ion-dependent enzyme reaction either in addition to or in place of the putative RraA function.

Genome-scale metabolic network reconstruction and in silico flux analysis of the thermophilic bacterium Thermus thermophilus HB27.

BackgroundThermus thermophilus, an extremely thermophilic bacterium, has been widely recognized as a model organism for studying how microbes can survive and adapt under high temperature environment. However, the thermotolerant mechanisms and cellular metabolism still remains mostly unravelled. Thus, it is highly required to consider systems biological approaches where T. thermophilus metabolic network model can be employed together with high throughput experimental data for elucidating its physiological characteristics under such harsh conditions.ResultsWe reconstructed a genome-scale metabolic model of T. thermophilus, iTT548, the first ever large-scale network of a thermophilic bacterium, accounting for 548 unique genes, 796 reactions and 635 unique metabolites. Our initial comparative analysis of the model with Escherichia coli has revealed several distinctive metabolic reactions, mainly in amino acid metabolism and carotenoid biosynthesis, producing relevant compounds to retain the cellular membrane for withstanding high temperature. Constraints-based flux analysis was, then, applied to simulate the metabolic state in glucose minimal and amino acid rich media. Remarkably, resulting growth predictions were highly consistent with the experimental observations. The subsequent comparative flux analysis under different environmental conditions highlighted that the cells consumed branched chain amino acids preferably and utilized them directly in the relevant anabolic pathways for the fatty acid synthesis. Finally, gene essentiality study was also conducted via single gene deletion analysis, to identify the conditional essential genes in glucose minimal and complex media.ConclusionsThe reconstructed genome-scale metabolic model elucidates the phenotypes of T. thermophilus, thus allowing us to gain valuable insights into its cellular metabolism through in silico simulations. The information obtained from such analysis would not only shed light on the understanding of physiology of thermophiles but also helps us to devise metabolic engineering strategies to develop T. thermophilus as a thermostable microbial cell factory.

Are ionic liquids extremophiles cell wall breakers? Esther Gutiérrez, M. Ángeles Sanromán, Ana Rodríguez, Francisco J. Deive * Department of Chemical Engineering, University of Vigo, 36310, Vigo, Spain * Corresponding author: Tel.: +34986818723; E-mail address: deive@uvigo.es

Are ionic liquids extremophiles cell wall breakers? Esther Gutiérrez, M. Ángeles Sanromán, Ana Rodríguez, Francisco J. Deive * Department of Chemical Engineering, University of Vigo, 36310, Vigo, Spain * Corresponding author: Tel.: +34986818723; E-mail address: deive@uvigo.es

Improved immobilization of laccase on a glassy carbon electrode by oriented covalent attachment

A laccase from Thermus thermophilus HB27 was reported to be potentially useful in the design of a temperature controlled biofuel cell. For enhancing its application in different thermal conditions, we engineered a laccase-oriented immobilized electrode. A site-directed mutant N323C of the laccase was constructed. A photometric assay was employed in order to compare the catalytic properties of wild-type laccase and mutant. The mutant was attached to a glass carbon electrode by covalent cross-linking. The electrochemical properties of the immobilized laccase were investigated by cyclic voltammetry. This immobilization allowed the active electrode to function at temperatures up to 95?C. The thermal and pH dependence profiles were similar to those of the soluble enzyme investigated by spectrophotometry.

Genetic incorporation of unnatural amino acids biosynthesized from α-keto acids by an aminotransferase

Four unnatural amino acids (UAAs) were synthesized from their corresponding α-keto acids by using glutamine:phenylpyruvate aminotransferase (GlnAT) from Thermus thermophilus HB8. The enzyme efficiently catalyzed the conversion of the unnatural substrates, with little decrease in its activity compared to that shown toward its natural substrates. The α-keto acids were converted into the corresponding amino acids in cells and then directly incorporated into a protein with high efficiency and fidelity. Because α-keto acids have no chiral center and are synthetically readily accessible, this method will further enhance the genetic incorporation technology and may present a new approach to the preparation of optically pure UAAs. Considering the substrate promiscuity of the GlnAT observed in the study, this method can be applied to the genetic incorporation of other UAAs derived from Tyr or Phe, which constitute approximately 50% of the successfully incorporated UAAs. In addition, this study provides an impetus for the development of an autonomous unnatural organism that can synthesize and incorporate UAAs.

A novel mode of ferric ion coordination by the periplasmic ferric ion-binding subunit FbpA of an ABC-type iron transporter fromThermus thermophilusHB8

Crystal structures of FbpA, the periplasmic ferric ion-binding protein of an iron-uptake ABC transporter, from Thermus thermophilus HB8 (TtFbpA) have been solved in apo and ferric ion-bound forms at 1.8 and 1.7 Å resolution, respectively. The latter crystal structure shows that the bound ferric ion forms a novel six-coordinated complex with three tyrosine side chains, two bicarbonates and a water molecule in the metal-binding site. The results of gel-filtration chromatography and dynamic light scattering show that TtFbpA exists as a monomer in solution regardless of ferric ion binding and that TtFbpA adopts a more compact conformation in the ferric ion-bound state than in the apo state in solution.

Cloning, overexpression and characterization of a thermostable pullulanase from Thermus thermophilus HB27

A gene encoding a special type of pullulanase from Thermus thermophilus HB27 (TTHpu) was cloned. It has an open reading frame of 1428bp encoding a mature protein with a molecular mass of 52kDa. The gene was expressed in Escherichia coli using pHsh and pET28a vectors. The pHsh expression system produced a 3.6-fold higher recombinant pullulanase than pET28a. The recombinant TTHpu was purified to homogeneity by heat treatment and Ni–NTA affinity chromatography. The purified TTHpu exhibited highest activity at pH 6.5 and 70°C. More than 90% activity was retained after incubation at 60–70°C for 2h and the half-life was 2h at 80°C. The stability of the enzyme was in a pH range from 6.0 to 8.0. Manganese at 5mM enhanced its activity up to 298%. The Km and Vmax for the enzyme activity on pullulan were 0.0031mgmL−1 and 23.8μmolmin−1, respectively. Unlike the most of pullulan-hydrolyzing enzymes described to date, this enzyme can attack α-1,6- and α-1,4-glycosidic linkages in pullulan, and produce a mixture of maltotriose, maltose and glucose. The enzyme could be further employed for industrial saccharification of starch.