Cloning and expression of the gene for thermostable pullulanase from Clostridium thermohydrosulfuricum in Escherichia coli

Using a pUC19-based genomic library of the anaerobic thermophilic bacterium C. thermohydrosulfuricum a DNA fragment that confers pullulanase activity to E. coli cells has been identified. Subcloning and restriction mapping procedures was carried out and the primary structure of the 5'-region of the pullulanase gene (pul) was determined. The pul enzyme was shown to be a protein with molecular weight of approximately 60,000. It was found that both pullulanase and glucoamylase activities resides in pullulanase. The intracellular distribution of pullulanase was studied. An E. coli strain that produces large amounts of thermostable pullulanase has been constructed.     

嗜热枯草芽孢杆菌普鲁兰酶基因的表达与重组酶的性质

克隆嗜热枯草芽孢杆菌WY-34普鲁兰酶基因并在大肠杆菌中进行表达,对重组酶进行纯化和酶学性质研究,根据枯草芽孢杆菌的普鲁兰酶蛋白序列,设计PCR引物对WY-34的普鲁兰酶基因进行克隆及异源表达.对表达蛋白的最适pH、pH稳定性及最适温度、温度稳定性等特性进行研究,并测定重组普鲁兰酶的底物特异性.将普鲁兰酶基因pluA克隆及分析序列后,发现基因长度为2.2 kb,编码718个氨基酸,在大肠杆菌中异源表达.通过Ni-IDA亲和层析一步纯化得到比活力为93.2 U/mg的纯酶,SDS-PAGE和凝胶层析测定的分子量分别为76.2 kD和74.3 kD.酶学性质研究表明,该酶的最适温度为40℃,在温度不高于45℃条件下稳定;最适pH为6.0,同一温度下pH 6.0-9.0范围内处理30 min可以保持80％以上的酶活力,此酶对普鲁兰糖有很强的底物特异性.此重组普鲁兰酶的酶学性质表明此酶具有一定的工业化应用价值.     

Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana

The gene encoding a type I pullulanase from the hyperthermophilic anaerobic bacterium Thermotoga neapolitana (pulA) was cloned in Escherichia coli and sequenced. The pulA gene from T. neapolitana showed 91.5% pairwise amino acid identity with pulA from Thermotoga maritima and contained the four regions conserved in all amylolytic enzymes. pulA encodes a protein of 843 amino acids with a 19-residue signal peptide. The pulA gene was subcloned and overexpressed in E. coli under the control of the T7 promoter. The purified recombinant enzyme (rPulA) produced a 93-kDa protein with pullulanase activity. rPulA was optimally active at pH 5-7 and 80°C and had a half-life of 88 min at 80°C. rPulA hydrolyzed pullulan, producing maltotriose, and hydrolytic activities were also detected with amylopectin, starch, and glycogen, but not with amylose. This substrate specificity is typical of a type I pullulanase. Thin layer chromatography of the reaction products in the reaction with pullulan and aesculin showed that the enzyme had transglycosylation activity. Analysis of the transfer product using NMR and isoamylase treatment revealed it to be α-maltotriosyl-(1,6)-aesculin, suggesting that the enzyme transferred the maltotriosyl residue of pullulan to aesculin by forming α-1,6-glucosidic linkages. Our findings suggest that the pullulanase from T. neapolitana is the first thermostable type I pullulanase which has α-1,6-transferring activity.     

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.     

Molecular cloning of a glucoamylase gene from a thermophilic Clostridium and kinetics of the cloned enzyme.

Clostridium sp. G0005 produces a cell-bound glucoamylase (CGA). The gene encoding CGA has been sequenced. The deduced amino acid sequence begins with a putative 21-residue signal sequence for secretion of bacterial lipoproteins, which suggests that a putative CGA precursor is modified and secreted like other bacterial lipoproteins in Clostridium sp. G0005, and that the modified residue is important in the cell-bound form of mature CGA. Comparison of the amino acid sequence of the CGA precursor with known eukaryotic enzymes showed several regions of high similarity in spite of low similarity throughout the overall primary structure. CGA is the first bacterial glucoamylase to be cloned. The CGA gene was expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5' non-coding region and the N-terminal coding region of the gene were replaced with the lac promoter. Kinetic studies of the cloned enzyme purified from E. coli were performed with a set of linear malto-oligosaccharides as substrates, and the subsite affinity was calculated from the kinetic parameters. CGA had typical kinetic properties for a glucoamylase, but this bacterial enzyme had higher isomaltose-hydrolyzing activity than other eukaryotic glucoamylases.     

Magnetite-alginate beads for purification of some starch degrading enzymes

Starch degrading enzymes, viz., β-amylase, glucoamylase, and pullulanase, were purified using magnetite-alginate beads. In each case, the enzyme activity was eluted by using 1.0 M maltose. β-Amylase (sweet potato), glucoamylase (Aspergillus niger), and pullulanase (Bacillus acidopullulyticus) from their crude preparations were purified 37-, 31-, and 49-fold with 86, 87, and 95% activity recovery, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed single band in each case.     

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.     

Macroaffinity ligand-facilitated three-phase partitioning for purification of glucoamylase and pullulanase using alginate

Starch-degrading enzymes glucoamylase (from Aspergillus niger), and pullulanase (from Bacillus acidopullulyticus) were purified using alginates (polysaccharides consisting of mannuronic acids and guluronic acids) by a recently developed technique called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this process, a crude preparation of the enzyme was mixed with alginate. On addition of appropriate amounts of ammonium sulfate and t-butanol, the alginate bound enzyme appeared as an interfacial precipitate between the lower aqueous and the upper t-butanol phase. Enzyme activity from this interfacial precipitate was recovered using 1M maltose. Glucoamylase and pullulanase were purified 20- and 38-fold with 83% and 89% activity recovery, respectively. Both the purified preparations showed a single band on SDS-PAGE.     

General Biochemical Characterization of Thermostable Pullulanase and Glucoamylase from Clostridium thermohydrosulfuricum

Cell extracts of Clostridium thermohydrosulfuricum, an anaerobic bacterium which ferments starch into ethanol at 65°C, contained both pullulanase and glucoamylase activities. The general physiochemical and catalytic properties of these enzyme activities were compared. Pullulanase and glucoamylase activities were stable and optimally active at 85 and 75°C, respectively. The pH optima for activity and pH stability ranges were, respectively, 5.5 to 6 and 4.5 to 5.5 for pullulanase and 4 to 6 and 5 to 6 for glucoamylase. The apparent [S]_0.5v and V_max for pullulanase activity on pullulan were 0.33 mg/ml and 2.6 U/mg of protein. The apparent [S]_0.5v and V_max for glucoamylase activity on starch were of 0.41 mg/ml and 0.31 U/mg of protein. These enzymes were active and stable in the presence of air or 10% (vol/vol) ethanol. These enzyme activities allowed the organism to actively degrade raw starch into glucose in the absence of significant α-amylase activity.     

General Biochemical Characterization of Thermostable Extracellular beta-Amylase from Clostridium thermosulfurogenes

Clostridium thermosulfurogenes, an anaerobic bacterium which ferments starch into ethanol at 62 degrees C, produced an active extracellular amylase and contained intracellular glucoamylase but not pullulanase activity. The extracellular amylase was purified 2.4-fold, and its general physicochemical and catalytic properties were examined. The extracellular amylase was characterized as a beta-amylase (1,4-alpha-d-glucan maltohydrolase) based on demonstration of exocleavage activity and the production of maltose with a beta-anomeric configuration from starch. The beta-amylase activity was stable and optimally active at 80 and 75 degrees C, respectively. The pH optimum for activity and the pH stability range was 5.5 to 6 and 3.5 to 6.5, respectively. The apparent [S](0.5V) and V(max) for beta-amylase activity on starch was 1 mg/ml and 60 U/mg of protein. Similar to described beta-amylase, the enzyme was inhibited by p-chloromercuribenzoate, Cu, and Hg; however, alpha- and beta-cyclodextrins were not competitive inhibitors. The beta-amylase was active and stable in the presence of air or 10% (vol/vol) ethanol. The beta-amylase and glucoamylase activities enabled the organism to actively ferment raw starch in the absence of significant pullulanase or alpha-amylase activity.     

Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris

AIMS: Chaetomium thermophilum is a soil-borne thermophilic fungus whose molecular biology is poorly understood. Only a few genes have been cloned from the Chaetomium genus. This study attempted to clone, to sequence and to express a thermostable glucoamylase gene of C. thermophilum. METHODS AND RESULTS: First strand cDNA was prepared from total RNA isolated from C. thermophilum and the glucoamylase gene amplified by using PCR. Degenerate primers based on the N-terminal sequences of the purified glucoamylase according to our previous works and a cDNA fragment encoding the glucoamylase gene was obtained through RT-PCR. Using RACE-PCR, full-length cDNA of glucoamylase gene was cloned from C. thermophilum. The full-length cDNA of the glucoamylase was 2016 bp and contained a 1797-bp open reading frame encoding a protein glucoamylase precursor of 599 amino acid residues. The amino-acid sequence from 31 to 45 corresponded to the N-terminal sequence of the purified protein. The first 30 amino acids were presumed to be a signal peptide. The alignment results of the putative amino acid sequence showed the catalytic domain of the glucoamylase was high homology with the catalytic domains of the other glucoamylases. The C. thermophilum glucoamylase gene was expressed in Pichia pastoris, and the glucoamylase was secreted into the culture medium by the yeast in a functionally active form. The recombinant glucoamylase purified was a glycoprotein with a size of about 66 kDa, and exhibited optimum catalytic activity at pH 4.5-5.0 and 65 degrees C. The enzyme was stable at 60 degrees C, the enzyme activity kept 80% after 60 min incubation at 70 degrees C. The half-life was 40 and 10 min under incubation at 80 and 90 degrees C respectively. CONCLUSIONS: A new thermostable glucoamylase gene of C. thermophilum was cloned, sequenced, overexpressed successfully in P. pastoris. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its thermostability and overexpression, this glucoamylase enzyme offers an interesting potential in saccharification steps in both starch enzymatic conversion and in alcohol production.     

Enhancement of starch conversion efficiency with free and immobilized pullulanase and alpha-1,4-glucosidase

Glucoamylase and pullulanase were immobilized on reconstituted bovine-hide collagen membranes using the covalent azide linkage method. A pretanning step was incorporated into the immobilization procedure to enable the support matrix to resist proteolytic activity while accommodating an operating temperature of 50 degrees C. The immobilized glucoamylase and pullulanase activities were 0.91 and 0.022 mg dextrose equivalent (DE) min(-1) cm(-2) of membrane, respectively. Immobilized glucoamylase had a half-life of 50 days while the immobilized pullulanase had a half-life of 7 days. This is a considerably improved stability over that reported by other researchers. The enzymes were studied in their free and immobilized forms on a variety of starch substrates including waxy maize, a material which contains 80% alpha-1-6-glucosidic linkages. Substrate concentrations ranged from 1% to a typical commercial concentration of 30%. Conversion efficiencies of 90-92% DE were obtained with free and immobilized glucoamylase preparations. Conversion enhancements of 4-5 mg of DE above this level were obtained by the use of pullulanase in its free or immobilized forms. Close examination of free pullulanase stability as a function of pH indicated improved thermal stability at higher pH values. At 50 degrees C and pH 5.0, the free enzyme was inactivated after 24 h. At pH 7.0, the enzyme still possessed one-half its activity after 72 h. Studies were conducted in both batch and continuous total recycle reactors. All experiments were conducted at 50 degrees C. Experiments conducted with coimmobilized enzymes proved quite promising. Levels of conversion equivalent to those obtained with the individually immobilized enzymes were realized.     

Characteristics of thermostable pullulanase from Bacillus stearothermophilus and the nucleotide sequence of the gene

Thermostable pullulanase was purified to homogeneity on sodium dodecyl sulfate-polyacrylamide gel from the culture supernatant of Bacillus stearothermophilus TRS128. However, multiformity of the pullulanase was suggested by activity staining on a pullulan-reactive red plate. The thermostability of the enzyme was tested. In the presence of Ca²⁺, the optimum temperature of the pullulanase was 75°C, and nearly 100% of the enzyme activity was retained even after treatment at 68°C for 60 min. Since the thermostable pullulanase gene (pulT) has been cloned, the nucleotide sequence was determined. Although the DNA sequence revealed only one large open reading frame, two possible pairs of SD sequence and initiation codon were found in the frame. To analyze the regulatory region, several mutations (deletion, insertion and substitution of nucleotides) were introduced in the flanking region of pulT, using site-directed mutagenesis. A putative promoter, SD sequence and initiation codon were inferred. The pulT gene was composed of 1974 bases and 658 amino acid residues (molecular weight 75,375). The deduced amino acid sequence of the thermostable pullulanase exhibited a fairly low homology with that of the thermolabile pullulanase from Klebsiella aerogenes. However, four consensus sequences containing catalytic and/or substrate binding sites for amylolytic enzymes were also found in the thermostable pullulanase and the thermolabile enzyme.     

Cloning and sequence analysis of the gene for glucodextranase from Arthrobacter globiformis T-3044 and expression in Escherichia coli cells

The gld gene for glucodextranase from Arthrobacter globiformis T-3044 was cloned by using a combination of gene walking and probe methods and expressed on the recombinant plasmid pGD8, which was constructed with pUC118, in Escherichia coli cells. The enzyme gene consisted of a unique open reading frame of 3,153 bp. The comparison of the DNA sequence data with the N-terminal and 6 internal amino acid sequences of the purified enzyme secreted from A. globiformis T-3044 suggested the enzyme was translated from mRNA as a secretory precursor with a signal peptide of 28 amino acids residues. The deduced amino acids sequence of the mature enzyme contained 1,023 residues, resulting in a polypeptide with a molecular mass of 107,475 daltons. The deduced sequence showed about 38% identity to that of the glucoamylase from Clostridium sp. G0005. The glucodextranase activity of transformant harboring pGD8 was about 40 mU/ml at 30 degrees C for a 16-h culture. Although the GDase that was produced from the transformant was shorter than authentic GDase by 2 amino acid residues at the N-terminal end side, its enzymatic properties were almost same as the authentic one. Two kinds of genes, dex1 and dex2, for endo-dextranases from A. globiformis T-3044 were also cloned into Escherichia coli cells. The N-terminal of the purified endo-dextranase from A. globiformis T-3044 agreed with the deduced amino acid sequence, after the 33rd alanine residue, of only the dex1 gene for edo-dextranase. This result suggests that the endo-dextranase is translated from mRNA as a secretory precursor with a signal peptide of 32 amino acids residues. The deduced sequence of endo-dextranase 1 and endo-dextranase 2 showed about 93% and 65% identity with that of known endo-dextranase from Arthrobacter sp. CB-8, respectively.     

Nucleotide sequence of the glucoamylase gene GLU1 in the yeast Saccharomycopsis fibuligera.

The complete nucleotide sequence of the glucoamylase gene GLU1 from the yeast Saccharomycopsis fibuligera has been determined. The GLU1 DNA hybridized to a polyadenylated RNA of 2.1 kilobases. A single open reading frame codes for a 519-amino-acid protein which contains four potential N-glycosylation sites. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. Glucoamylase was purified from a culture fluid of the yeast Saccharomyces cerevisiae which had been transformed with a plasmid carrying GLU1. The molecular weight of the protein was 57,000 by both gel filtration and acrylamide gel electrophoresis. The protein was glycosylated with asparagine-linked glycosides whose molecular weight was 2,000. The amino-terminal sequence of the protein began from the 28th amino acid residue from the first methionine of the putative precursor. The amino acid composition of the purified protein matched the predicted amino acid composition. These results confirmed that GLU1 encodes glucoamylase. A comparison of the amino acid sequence of glucoamylases from several fungi and yeast shows five highly conserved regions. One homology region is absent from the yeast enzyme and so may not be essential to glucoamylase function.     

Collagen-like sequences stabilize homotrimers of a bacterial hydrolase.

Pullulanase from Klebsiella pneumoniae strain FG9 has an unusual N-terminal amino acid sequence that includes six repeats of the tripeptide Gly-X-Pro. This type of sequence is characteristic of animal collagens and collagen-like proteins which form triple helical structures. We have investigated the molecular organization of this bacterial pullulanase isolated from the cell surface of Escherichia coli cells that carry the cloned FG9 pulA (pullulanase encoding) gene. Non-denaturing polyacrylamide gel analysis shows that pullulanase exists as higher order, apparently homogeneous, structures. We have used highly purified bacterial collagenase to probe the role of the collagen-like region and we demonstrate that this feature is essential for non-covalent association of pullulanase homotrimers. In addition we show collagenase-specific release of cell-bound pullulanase.     

A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization.

A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chromatographies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), Sephadex G-100, single-stranded DNA cellulose and hydroxyapatite. The purified enzyme has an Mr of 34,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escherichia coli exonuclease III. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.     

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.     

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.     

The Escherichia coli pldC gene encoding lysophospholipase L(1) is identical to the apeA and tesA genes encoding protease I and thioesterase I, respectively.

We deduced the amino acid sequence of Escherichia coli lysophospholipase L(1) by determining the nucleotide sequence of the pldC gene encoding this enzyme. The translated protein was found to contain 208 amino acid residues with a hydrophobic leader sequence of 26 amino acid residues. The molecular weight of the purified enzyme (20,500) was in good agreement with the predicted size (20,399) of the processed protein. A search involving a data bank showed that the nucleotide sequence of the pldC gene was identical to those of the apeA and tesA genes encoding protease I and thioesterase I, respectively. Consistent with the identity of the pldC gene with these two genes, the enzyme purified from E. coli overexpressing the pldC gene showed both protease I and thioesterase I activities.