A Gene Encoding a Novel Multidomain β-1,4-Mannanase from Caldibacillus cellulovorans and Action of the Recombinant Enzyme on Kraft Pulp

Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function. The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a β-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85°C and pH 6.0 and was extremely thermostable at 70°C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto- and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable β-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.     

Characterization of Cyt2Bc Toxin from Bacillus thuringiensis subsp. medellin

We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin. Three IS240-like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus.     

A new NADH-dependent,zinc containing alcohol dehydrogenase from Bacillus thuringiensis serovar israelensis involved in oxidations of short to medium chain primary alcohols

The escalation in genome sequencing has presented a mass of potentially useful new alcohol dehydrogenases (ADHs) in the form of putative open reading frame (ORF). To take advantage of such available resources, PCR primers based on the genome sequence of Bacillus thuringiensis serovar israelensis were used to clone a gene encoding a hypothetical alcohol dehydrogenase (named as BtADH). Activity studies of the translation product revealed that the alcohol dehydrogenases catalyse the inter-conversion of aliphatic aldehydes and corresponding primary alcohol with chain length of two to ten carbons. The required co-factor for such inter-conversion was found to be NAD(H). The ADH gene was engineered for heterologous expression in Escherichia coli, and the enzyme was produced in a soluble form. The recombinant enzyme was purified to homogeneity and physical, spectral and catalytical properties were determined.The findings lead us to propose that BtADH represents a novel primary–secondary alcohol dehydrogenase that acts on primary alcohols of medium chain lengths and simple ketones. Besides, BtADH shares high sequence similarity with well known ADHs from thermophilic origins. Such biochemical characterisation of BtADH provides valuable information for the study of sequence–function relationship including source of thermal stability, cofactor and substrate preferences.     

Characterization of a new rhamnogalacturonan acetyl esterase from Bacillus halodurans C-125 with a new putative carbohydrate binding domain

BH1115 is a gene from Bacillus halodurans strain C-125 that hypothetically encodes a rhamnogalacturonan acetyl esterase (RGAE) of the CE-12 family. As confirmation, this gene was cloned, and the product was expressed in Escherichia coli strain Rosetta (DE3) cells and purified. The enzyme obtained was monomeric, with a molecular mass of 45 kDa, and exhibited alkaliphilic properties. A study of the inhibition of the activity by some modulators confirmed that the catalytic triad for the esterase activity was Ser-His-Asp. This enzyme also presents broad substrate specificity and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, β-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, RGAE from B. halodurans achieves a synergistic effect with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common α/β hydrolase fold. The homology between the folds of RGAE from Aspergillus aculeatus and the hypothetical YxiM precursor from Bacillus subtilis, which both belong to the SGNH family, illustrates the divergence of such proteins from a common ancestor. Furthermore, the enzyme possesses a putative substrate binding region at the N terminus of the protein which has never been described to date for any RGAE.     

A double mutant of highly purified Geobacillus stearothermophilus lactate dehydrogenase recognises l-mandelic acid as a substrate

Lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) (bsLDH) has a crucial role in producing chirally pure hydroxyl compounds. α-Hydroxy acids are used in many industrial situations, ranging from pharmaceutical to cosmetic dermatology products. One drawback of this enzyme is its limited substrate specificity. For instance, l-lactate dehydrogenase exhibits no detectable activity towards the large side chain of 2-hydroxy acid l-mandelic acid, an α-hydroxy acid with anti-bacterial activity. Despite many attempts to engineer bsLDH to accept α-hydroxy acid substrates, there have been no attempts to introduce the industrially important l-mandelic acid to bsLDH. Herein, we describe attempts to change the reactivity of bsLDH towards l-mandelic acid. Using the Insight II molecular modelling programme (except ‘program’ in computers) and protein engineering techniques, we have successfully introduced substantial mandelate dehydrogenase activity to the enzyme. Energy minimisation modelling studies suggested that two mutations, T246G and I240A, would allow the enzyme to utilise l-mandelic acid as a substrate. Genes encoding for the wild-type and mutant enzymes were constructed, and the resulting bsLDH proteins were overexpressed in Escherichia coli and purified using the TAGZyme system. Enzyme assays showed that insertion of this double mutation into highly purified bsLDH switched the substrate specificity from lactate to l-mandelic acid.     

Molecular characterization of an endochitinase from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. konkukian

A chitinase gene from Bacillus thuringiensis serovar konkukian S4 was cloned, sequenced, and heterologously expressed in Escherichia coli M15. Recombinant enzyme (Chi74) was purified by Ni-NTA affinity column chromatography. The chi74 gene contains an open reading frame (ORF), with a capacity to encode an endochitinase with a deduced molecular weight 74 kDa and predicted isoelectric point of 5.67. Comparison of Chi74 with other chitinases has shown that it contains a modular structure with an N-terminal family 18 catalytic-domain, a Fibronectin-III like domain and a C-terminal carbohydrate binding module (CBM-II). Turn over rate (K _cat ) of the enzyme was determined using colloidal chitin (28.3 ± 0.70 S⁻¹) as substrate. The Purified enzyme was active at a broad range of pH (pH 3.5–7.5) and temperature (20–70°C) with a peak activity at pH 5.5 and 55°C. However, the enzyme was found to be stable up to 30°C for longer incubation periods. Moreover, the purified enzyme was shown to inhibit fungal spore germination and hyphal growth in the pathogenic fungi Fusarium oxysporum and Aspergillus niger. These studies will lead us to develop broad spectrum resistance in the crop plants via co-expression of the chitinases and the insecticidal proteins.     

A novel carbohydrate:acceptor oxidoreductase from Microdochium nivale.

A Microdochium nivale carbohydrate:acceptor oxidoreductase was purified, cloned, heterologously expressed, and characterized. The gene encoding the protein showed one intron, and the ORF showed a sequence with low homology (< or = 25% identity or 65% similarity) to other known flavin-containing carbohydrate oxidases. The maturation of the protein required the cleavage of a tetrameric propeptide in addition to an 18 amino-acid signal peptide. The enzyme was found to have a relative molecular mass of 55 000 Da, an isoelectric point of 9, and one FAD per protein. It could oxidize mono-, oligo-, or polymeric saccharides, and transfer their electrons to O2 or other acceptors. When D-glucose served as electron-donating substrate, an activity of 2 s(-1) was observed at pH 5.5 and 23 degrees C. Among various oligosaccharides, the enzyme preferred tetrameric dextrins, indicating a favorable interaction of four linked glucose units with the substrate pocket. The unique structure and ability of oxidizing oligo/polymeric saccharides suggest a promising prospect of this enzyme for various industrial/medicinal applications.     

A new d-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression

A gene encoding a new d-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple amino acid sequence alignments of the D-2-hydroxyacid dehydrogenase from H. mediterranei showed high homology with D-2-hydroxyacid dehydrogenases from different organisms and other enzymes of this family. Analysis of the amino acid sequence showed catalytic residues conserved in hydroxyacid dehydrogenases with d-stereospecificity. In the reductive reaction, the enzyme showed broad substrate specificity, although α-ketoisoleucine was the most favourable of all α-ketocarboxylic acids tested. Kinetic data revealed that this new D-2-hydroxyacid dehydrogenase from H. mediterranei exhibits dual coenzyme-specificity, using both NADPH and NADH as coenzymes. To date, all D-2-hydroxyacid dehydrogenases have been found to be NADH-dependent. Here, we report the first example of a D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity.     

Nucleotide sequence of the gene encoding novel delta-endotoxin from Bacillus thuringiensis serovar japonensis strain Buibui specific to scarabaeid beetles

A new isolate of Bacillus thuringiensis serovar japonensis strain Buibui, which was specific to scarab beetles (M. Ohba et al., Lett. Appl. Microbiol. 14:54, 1992), was shown to have a 130-kDa insecticidal crystal protein (ICP) (H. Hori et al., J. Appl. Bacteriol. 76:307, 1994). CalI restriction enzyme fragments of total cell DNA of the isolate were cloned into E. coli (Sato et al., Curr. Microbiol. 28:15, 1994). Whole 3480-bp nucleotide sequence of the gene encoding 130-kDa ICP was determined, and the molecular weight of the ICP was estimated to be 130,424. The strongly conserved five blocks that occur in almost all ICP genes of B. thuringiensis were detected in the ORF with the same order and almost the same intervals as elsewhere. The amino acid sequence homologies of the whole ICP or N-terminus half portion to that of the CryIIIA, B, C, D, and CryV were about 35%.     

A unique enzyme of acetic acid bacteria, PQQ-dependent alcohol dehydrogenase, is also present in Frateuria aurantia

A membrane-bound, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) was purified from Frateuria aurantia LMG 1558^T. Although F. aurantia belongs to a group of γ-Proteobacteria, the characteristics of its PQQ-ADH were similar to the enzyme characteristics of the typical high-acetic acid-resistant bacterium Gluconacetobacter europaeus from the group of α-Proteobacteria. The PQQ-dependent ADH was solubilized from the membranes and purified after anionic, cationic, and affinity chromatography with specific activity of 117 U/mg. The purified enzyme was estimated to be composed of two subunits of ca. 72 and 45 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had maximum activity at pH 4.5 and showed the highest substrate specificity to ethanol, isoamyl alcohol, 1-butanol, and 1-propanol. The deduced sequences of cloned genes adhA and adhB encoding subunits I and II of PQQ-ADH showed 80 % amino acid (AA) identity to AdhA and 68 % AA identity to AdhB of Ga. europaeus V3 (LMG 18494). Because of the high similarity between genes encoding subunits I and II of PQQ-ADH and its homologous genes found in a distantly related taxonomic group of acetic acid bacteria, the results suggest the possibility of horizontal gene transfer between these two groups of genera.     

Cloning,expression, and characterization of an (R)-specific alcohol dehydrogenase from Lactobacillus kefir

Lactobacillus kefir DSM 20587 produces an (R)-specific NADP-dependent alcohol dehydrogenase (ADH) with a broad substrate specificity. The gene of this ADH was isolated and the complete nucleotide sequence determined. The adh gene comprises 759?bp and encodes a protein of 252 amino acids with a calculated molecular weight of 26 781?Da. The deduced amino acid sequence indicated a high degree of similarity to short-chain dehydrogenases. After cloning and expression in Escherichia coli the enzyme was purified and characterized. For the reduction of acetophenone the specific activity of the homogeneous recombinant ADH was 558?U?mg^?1. The enzyme shows its maximum activity at 50°C while the pH optimum was at pH?7.0. In order to demonstrate its preparative application, purified ADH was used for the stereoselective reduction of several aliphatic and aromatic ketones as well as β-keto esters. Glucose dehydrogenase was added for the regeneration of NADPH. All prochiral ketones were stereoselectively reduced to the corresponding alcohols with >99% ee and in the case of diketones >99% de.     

Iteron-binding ORF157 and FtsZ-like ORF156 proteins encoded by pBtoxis play a role in its replication in Bacillus thuringiensis subsp. israelensis

We recently identified a minireplicon of pBtoxis from Bacillus thuringiensis subsp. israelensis that contained an operon encoding two novel proteins (ORF156 and ORF157), both of which are required for replication. ORF157 contains a helix-turn-helix motif and shares no homology with known plasmid replication proteins (Rep), and ORF156 contains the signature motif present in FtsZ/tubulin proteins, the latter of which are known to function in cell division and chromosome segregation. Here we show that the minimal sequence composed of four 12-bp imperfect direct repeats (iterons) in the pBtoxis minireplicon was sufficient to replicate a reporter plasmid in B. thuringiensis subsp. israelensis when ORF156 and ORF157 functions were provided in trans. To further investigate the roles of ORF156 and ORF157 in pBtoxis replication, six-histidine-tagged recombinant rORF156 and rORF157 proteins were purified from Escherichia coli and used in electrophoretic mobility shift assays. Our results demonstrated that rORF157, but not rORF156, binds specifically to the pBtoxis iterons, suggesting that ORF157 functions as a Rep protein. Although rORF156 did not bind to the iteron sequence, we showed that it bound to rORF157-DNA complexes. In addition, we showed that rORF156 has GTPase activity characteristic of the FtsZ/tubulin superfamily of proteins. Taken together, these results suggest that the iterons compose the minimal replication origin (ori) of pBtoxis and that ORF157 and ORF156 are involved in the initiation of pBtoxis replication and possibly in the segregation and partitioning of this plasmid to daughter cells.     

Characterization of the pcp gene of Pseudomonas fluorescens and of its product, pyrrolidone carboxyl peptidase (Pcp).

The gene pcp, encoding pyrrolidone carboxyl peptidase (Pcp), from Pseudomonas fluorescens MFO was cloned and its nucleotide sequence was determined. This sequence contains a unique open reading frame (pcp) coding for a polypeptide of 213 amino acids (M(r) 22,441) which has significant homology to the Pcps from Streptococcus pyogenes, Bacillus subtilis, and Bacillus amyloliquefaciens. Comparison of the four Pcp sequences revealed two highly conserved motifs which may be involved in the active site of these enzymes. The cloned Pcp from P. fluorescens was purified to homogeneity and appears to exist as a dimer. This enzyme displays a Michaelis constant of 0.21 mM with L-pyroglutamyl-beta-naphthylamide as the substrate and an absolute substrate specificity towards N-terminal pyroglutamyl residues. Studies of inhibition by chemical compounds revealed that the cysteine and histidine residues are essential for enzyme activity. From their conservation in the four enzyme sequences, the Cys-144 and His-166 amino acids are proposed to form a part of the active site of these enzymes.     

Sequence analysis,cloning and extracellular expression of cyclodextrin glucanotransferase gene from the alkaliphilic Bacillus pseudalcaliphilus 8SB in Escherichia coli

The cgt-gene from the alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB was isolated and sequenced. An open reading frame (ORF) of 2112 bp encoding a polypeptide of 704 amino acids, composed of a 29-amino acid signal sequence and a 675-amino acid mature enzyme was found. The established low level of homology with nucleotide sequences of other Bacillus CGTases (less than 82%) suggested that the cgt-gene from Bacillus pseudalcaliphilus 8SB encodes a new enzyme. The cgt-gene was cloned as a PCR amplicon and thereby the construction of genome library was avoided. This is the first evidence for the use of pJET vector as an expression vector. The opportunity to apply its T7 promoter for efficient extracellular production of heterologous proteins in Escherichia coli BL21 (DE3) was demonstrated. The expression of extracellular recombinant CGTase improved 23-fold, concerning β-CGTase activity and 4.5-fold concerning γ-CGTase activity after IPTG induction and glycine supplementation was achieved.     

A thermostable leucine aminopeptidase from<Emphasis Type="Italic"> Bacillus kaustophilus</Emphasis> CCRC 11223

Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His₆-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65°C, respectively, and 50% of its activity remained after incubation at 60°C for 32 min. The enzyme preferentially hydrolyzed l-leucine-p-nitroanilide (l-Leu-p-NA) followed by Cys derivative.Communicated by G. Antranikian     

Molecular cloning and expression of a glycosyltransferase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> for modification of morin and related polyphenols

Objectives

To characterize glycosyltransferases from Bacillus subtilis ATCC 6633 and investigate their substrate specificity towards plant polyphenols.

Results

Among the cloned and expressed six UDP-glycosyltransferases (BsGT1-6), BsGT-1 showed activity with a wide range of polyphenols: morin, quercetin, alizarin, rehin, curcumin and aloe emodin. The gene of BsGT-1 has an ORF of 1206 bp encoding 402 amino acids. The recombinant enzyme was purified to homogeneity by Ni–NTA affinity chromatograph, and its biochemical characteristics were identified by HPLC–UV/MS, ¹H-NMR and ¹³C-NMR. BsGT-1 has an MW of approx. 46 kDa as indicated by SDS-PAGE; its activity was optimal at 40 °C and pH 8.5. The Km value of BsGT-1 towards morin was 110 μM.

Conclusions

BsGT-1 from B. subtilis was cloned. It had high catalytic capabilities towards polyphenols which would make it feasible for the structural modification of polyphenols.

     

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

Molecular cloning,sequence analysis,and characterization of a new cell wall hydrolase,CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

Simultaneous cloning and expression of two cellulase genes from Bacillus subtilis newly isolated from Golden Takin (Budorcas taxicolor Bedfordi)

A bacterial strain with high cellulase activity was isolated of feces sample of Golden Takin (Budorcas taxicolor Bedfordi). The bacterium was classified and designated Bacillus subtilis LN by morphological and 16SrDNA gene sequence analysis. Two putative cellulase genes, CelL15 and CelL73, were simultaneously cloned from the isolated strain by PCR. The putative gene CelL15 consisted of an open reading frame (ORF) of 1470 nucleotides and encoded a protein of 490 amino acids with a molecular weight of 54 kDa. The CelL73 gene consisted of an open reading frame (ORF) of 741 nucleotides and encoded a protein of 247 amino acids with a molecular weight of 27 kDa. Both genes were purified and cloned into pET-28a for expression in Escherichia coli BL21 (DE3). The ability of E. coli to degrade cellulose was enhanced when the two recombinants were cultured together.     

Molecular characterization and sequence of phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis

The gene encoding monophosphatidylinositol inositol phosphohydrolase (PI-specific phospholipase C, PI-PLC) of Bacillus thuringiensis was cloned in Staphylococcus carnosus TM300. The complete coding region comprises 987 base pairs corresponding to a precursor protein of 329 amino acids (molecular weight, 38095). The NH₂-terminal sequence of the purified enzyme from Escherichia coli indicated that the mature PI-PLC consists of 299 amino acid residues with a molecular weight of 34586. Polyacrylamide gel electrophoresis revealed the same molecular weight for the purified enzyme isolated from the DNA-donor strain of B. thuringiensis and from the E. coli clone. By computer analysis, the secondary structure was predicted. The enzyme from the E. coli recombinant shows no activity on other phospholipids and sphingo-myelin. The cleaving specifity of PI-PLC was examined by thin layer chromatography.