The aman6 Gene Encoding a Yeast Mannan Backbone Degrading 1,6-α-D-Mannanase in Bacillus circulans: Cloning,Sequence Analysis,and Expression

A gene (aman6) encoding endo-1,6-α-D-mannanase, a yeast mannan backbone degrading enzyme from Bacillus circulans was cloned. The putative aman6 was 1767 base pairs long and encoded a mature 1,6-α-D-mannanase protein of 589 amino acids and a signal peptide of 36 amino acids. The purified mature 1,6-α-D-mannanase from the Escherichia coli transformant showed 61-kDa protein, and N-terminal amino acid sequence and other general properties of the recombinant enzyme were identical to those of 1,6-α-D-mannanase from Bacillus circulans TN-31.     

Gene cloning, expression, and characterization of a novel beta-mannanase from Bacillus circulans CGMCC 1416

A DNA fragment containing 2,079 base pairs from Bacillus circulans CGMCC 1416 was cloned using degenerate PCR and inverse PCR. An open reading frame containing 981 bp was identified that encoded 326 amino acids residues, including a putative signal peptide of 31 residues. The deduced amino acid sequence showed the highest identity (68.1%) with endo-beta-1,4-D-mannanase from Bacillus circulans strain K-1 of the glycoside hydrolase family 5 (GH5). The sequence encoding the mature protein was cloned into the pET-22b(+) vector and expressed in Escherichia coli as a recombinant fusion protein containing an N-terminal hexahistidine sequence. The fusion protein was purified by Ni2+ affinity chromatography and its hexahistidine tag cleaved to yield a 31-kDa beta-mannanase having a specific activity of 481.55 U/mg. The optimal activity of the purified protein, MANB48, was at 58 degrees C and pH 7.6. The hydrolysis product on substrate locust bean gum included a monosaccharide and mainly oligosaccharides. The recombinant MANB48 may be of potential use in the feed industry.     

Cloning,sequencing, and expression of the gene from bacillus circulans that codes for a heparinase that degrades both heparin and heparan sulfate

The gene, designated hep, coding for a heparinase that degrades both heparin and heparan sulfate, was cloned from Bacillus circulans HpT298. Nucleotide sequence analysis showed that the open reading frame of the hep gene consists of 3,150 bp, encoding a precursor protein of 1,050 amino acids with a molecular mass of 116.5 kDa. A homology search found that the deduced amino acid sequence has partial similarity with enzymes belonging to the family of acidic polysaccharide lyases that degrade chondroitin sulfate and hyaluronic acid. Recombinant mature heparinase (111.2 kDa) was produced by the addition of IPTG from Escherichia coli harboring pETHEP with an open reading frame of the mature hep gene and was purified to homogeneity by SDS-polyacrylamide gel electrophoresis. Analyses of substrate specificity and degraded disaccharides indicated that the recombinant enzyme acts on both heparin and HS, as does heparinase purified from the wild-type strain.     

Purification, characterization, gene cloning, and sequencing of a new beta-glucosidase from Bacillus circulans subsp. alkalophilus.

An intracellular beta-glucosidase was purified from cell extracts of Bacillus circulans subsp. alkalophilus by NAD affinity and high-performance anion-exchange chromatographies. The enzyme was active against a wide range of aryl-beta-glucosides and beta-linked disaccharides. The structural gene for beta-glucosidase was cloned in Escherichia coli. The beta-glucosidase gene consisted of an open reading frame of 1,350 bp encoding a protein of 450 amino acids with a calculated M(r) of 51,303. The enzyme exhibited from 45 to 66% identity with five bacterial beta-glucosidases.     

Cloning, sequencing, and expression of the genes encoding an isocyclomaltooligosaccharide glucanotransferase and an alpha-amylase from a Bacillus circulans strain

The gene for a novel glucanotransferase, isocyclomaltooligosaccharide glucanotransferase (IgtY), involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}] from starch, was cloned from the genome of B. circulans AM7. The IgtY gene, designated igtY, consisted of 2,985 bp encoding a signal peptide of 35 amino acids and a mature protein of 960 amino acids with a calculated molecular mass of 102,071 Da. The deduced amino-acid sequence showed similarities to 6-alpha-maltosyltransferase, alpha-amylase, and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in this enzyme, indicating that this enzyme should be assigned to this family. The DNA sequence of 8,325-bp analyzed in this study contained two open reading frames (ORFs) downstream of igtY. The first ORF, designated igtZ, formed a gene cluster, igtYZ. The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme was purified. The enzyme acted on maltooligosaccharides that have a degree of polymerization (DP) of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme (IgtZ), which has a novel amino-acid sequence, should be assigned to alpha-amylase. It is notable that both IgtY and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to a family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.     

Purification and characterization of a Bacillus cereus exochitinase

Five extracellular chitinases of Bacillus cereus 6E1 were detected by a novel in-gel chitinase assay using carboxymethyl-chitin-remazol brilliant violet 5R (CM-chitin-RBV) as a substrate. The major chitinase activity was associated with a 36-kDa (Chi36) gel band. Chi36 was purified by a one-step, native gel purification procedure derived from the new in-gel chitinase assay. The purified Chi36 has optimal activity at pH 5.8 and retains some enzymatic activity between pH 2.5-8. The temperature optimum for Chi36 was 35 degrees C, but the enzyme was active between 4-70 degrees C. Based on its ability to hydrolyze mainly p-nitrophenyl-(N-acetyl-beta-D-glucosaminide)(2), Chi36 is characterized as a chitobiosidase, a type of exochitinase. The N-terminal amino acid sequence of mature Chi36 was determined (25 amino acids). Alanine is the first N-terminal amino acid residue indicating the cleavage of a signal peptide from a Chi36 precursor to form the mature extracellular Chi36. The N-terminal sequence of Chi36 demonstrated highest similarity with Bacillus circulans WL-12 chitinase D and significant similarity with several other bacterial chitinases.     

Purification, characterization, and gene analysis of cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both beta-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the beta-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to beta-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin

A chitinase gene of Bacillus circulans WL-12 was cloned into Escherichia coli by transforming HB101 cells with a recombinant plasmid composed of chromosomal DNA fragments prepared from B. circulans WL-12 and the plasmid vector pKK223-3. DNA sequencing analysis revealed that the region necessary for the normal expression of chitinase activity contained one open reading frame of 2097 base pairs which codes for the precursor of chitinase A1. The precursor of chitinase A1 contained a long signal sequence of 41 amino acids with an extremely long N-terminal hydrophilic segment of 15 amino acids. Cloned chitinase produced in E. coli had at its N terminus an additional 8 amino acids that were not found in B. circulans mature chitinase A1. The N-terminal two-thirds of the deduced amino acid sequence of chitinase A1 showed a 33% amino acid match to chitinase A of Serratia marcescens. This region of chitinase A1 is immediately followed by tandemly repeating 95-amino acid segments that are 70% homologous to each other. Statistical analysis revealed that these repeating segments are homologous to the type III homology units of fibronectin, a multifunctional extracellular matrix and plasma protein of higher eukaryotes. This observation indicates that type III homology units originated prior to the emergence of eukaryotes and may be distributed in a wide range of organisms.     

Enzymatic properties,crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans

A high-isoelectric-point (pI), alkaline endo-1,4-beta-glucanase (Egl-257) of Bacillus circulans KSM-N257 was purified to homogeneity and crystallized. The purified enzyme hydrolyzed carboxymethyl cellulose (CMC) with optima of pH 8.5 and 55 degrees C. The molecular mass was 43 kDa, and the pI was pH 9.3. The structural gene contained a single open reading frame of 1221 bp, corresponding to 407 amino acids (aa), including a 30-aa signal peptide (377 aa and 41,680 Da for the mature enzyme). Egl-257 hydrolyzed lichenan and showed 76.3% aa identity to a lichenase from B. circulans WL-12 belonging to glycosyl hydrolase family 8 but did not hydrolyze laminarin, curdran, and xylan at all. This indicates that Egl-257 is a true endo-1,4-beta-glucanase. However, this enzyme was not active on p-nitrophenyl beta-D-cellotrioside and p-nitrophenyl beta-D-cellotetraoside. It was crystallized by the hanging-drop vapor-diffusion method with phosphate plus CdCl(2) as precipitant. Pyramid-like crystals were formed, and they diffracted X-rays beyond 2.2 A resolution. It belongs to the space group P2(1)2(1)2(1) with unit cell parameters of a=62.5 A, b=71.7 A, and c=88.6 A.     

Gene cloning and biochemical analysis of thermostable chitosanase (TCH-2) from Bacillus coagulans CK108

The DNA sequence of the thermostable chitosanase TCH-2 gene from Bacillus coagulans CK108 showed a 843-bp open reading frame that encodes a protein of 280 amino acids with a signal peptide corresponding to 32 kDa in size. The deduced amino acid sequence of the chitosanase from Bacillus coagulans CK108 has 61.6%, 48.0%, and 12.6% identities to those from Bacillus ehemensis, Bacillus circulans, and Bacillus subtilis, respectively. C-Terminal homology analysis shows that the enzyme belongs to the Cluster I group. The size of the gene was similar to those from mesophiles of the Cluster I group with regard to higher preference for codons ending in G or C. The recombinant chitosanase was electrophoretically purified to homogeneity by only two steps with column chromatography. The half-life of the enzyme was 40 min at 90 degrees C. The purified protein was also highly stable, retaining above 50% residual activities during treatment with denaturants such as urea (8 M) and guanidine x HCl (4 M) at 37 degrees C for 30 min. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, producing the tetramer as a major product.     

Synthesis and secretion of a Bacillus circulans WL-12 1,3-1,4-beta-D-glucanase in Escherichia coli.

The synthesis and secretion of a 1,3-1,4-beta-D-glucanase were studied in different strains of Escherichia coli transformed with plasmids carrying the Bacillus circulans WL-12 1,3-1,4-beta-D-glucanase structural gene. This gene (named BGC) is contained within a 1.9-kilobase BamHI-HindIII fragment and directs the synthesis in E. coli of an enzyme that specifically degrades lichenan. Only one active form of the enzyme was found when the gene was expressed in different E. coli strains. The electrophoretic pattern of this protein showed a molecular weight that was approximately the same as that of the mature beta-glucanase secreted from B. circulans WL-12, suggesting that the processing of this protein may be similar in both species. As deduced from maxicell experiments, the Bacillus parental promoter directs the synthesis in E. coli. Pulse-chase experiments showed that the protein may be cotranslationally processed.     

Cloning and expression of an alpha-1,3-glucanase gene from Bacillus circulans KA-304: the enzyme participates in protoplast formation of Schizophyllum commune

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of alpha-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of alpha-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of alpha-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (alpha-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases.The recombinant alpha-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant alpha-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-beta-D-thiogalactopyranoside. The recombinant alpha-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.     

Molecular cloning and characterization of the beta-amylase gene from Bacillus circulans

A gene encoding the beta-amylase of Bacillus circulans was isolated from a lambda library and sequenced. The structural gene consists of a 1725 bp open reading frame encoding a polypeptide with a predicted molecular wt of 62830 Daltons. Two active forms of the enzyme were found when the gene was expressed in E. coli. The larger 60 kD form was approximately 3 kD larger than the mature beta-amylase secreted from B. circulans, suggesting that processing of this protein is different between the two species. The smaller 49 kD form is also present at a low level in B. circulans and may result from proteolytic cleavage. The enzyme has a temperature optimum of 50 degrees C. Two other genes, one encoding an alpha-amylase and one a pullulanase, were also isolated from the lambda library.     

Cloning and expression of a Bacillus circulans KA-304 gene encoding chitinase I, which participates in protoplast formation of Schizophyllum commune

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, isolated from KA-prep, brings about the protoplast-forming activity. The gene of chitinase I was cloned from B. circulans KA-304 into pGEM-T Easy vector. The gene consists of 1,239 nucleotides, which encodes 413 amino acids including a putative signal peptide (24 amino acid residues). The molecular weight of 40,510, calculated depending on the open reading frame without the putative signal peptide, coincided with the apparent molecular weight of 41,000 of purified chitinase I estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal domain of the deduced amino acid sequence showed high similarity to that of family 19 chitinases of actinomycetes and other organisms, indicating that chitinase I is the first example of family 19 chitinase in Bacillus species. Recombinant chitinase I without the putative signal peptide was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the purified recombinant enzyme were almost the same as those of chitinase I purified from KA-prep, and showed the protoplast-forming activity when it was combined with alpha-1,3-glucanase from KA-prep. Recombinant chitinase I as well as the native enzyme inhibited hyphal extension of Trichoderma reesei.     

Thermostable chitosanase from Bacillus sp. Strain CK4: cloning and expression of the gene and characterization of the enzyme

A thermostable chitosanase gene from the environmental isolate Bacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. strain CK4 belongs to cluster III with B. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80 degrees C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.     

Identification of catalytic amino acids of cyclodextran glucanotransferase from Bacillus circulans T-3040

In glycoside hydrolase family 66 (see http://afmb.cnrs-mrs.fr/CAZY/), cyclodextran glucanotransferase (CITase) is the only transglycosylation enzyme, all the other family 66 enzymes being dextranases. To analyze the catalytic amino acids of CITase, we modified CITase chemically from the T-3040 strain of Bacillus circulans with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). EDC inactivated the enzyme by following pseudo-first order kinetics. In addition, the substrates of an isomaltooligosaccharide and a cyclodextran inhibited EDC-induced enzyme inactivation, implicating the carboxyl groups of CITase as the catalytic amino acids of the enzyme. When two conserved aspartic acid residues, Asp145 and Asp270, were replaced with Asn in T-3040 mature CITase, CIT-D270N was completely inactive, and CIT-D145N had reduced activity. The V(max) of CIT-D145N was 1% of that of wild-type CITase, whereas the K(m) of CIT-D145N was about the same as that of the wild-type enzyme. These findings indicate that Asp145 and Asp270 play an important role in the enzymatic reaction of T-3040 CITase.     

Cloning, expression, purification and characterization of fructose-1,6-bisphosphate aldolase from Anoxybacillus gonensis G2

The fructose-1,6-bisphosphate aldolase gene from the thermophilic bacterium, Anoxybacillus gonensis G2, was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame coding for a 30.9 kDa protein of 286 amino acids. The amino acid sequence shared approximately 80-90% similarity to the Bacillus sp. class II aldolases. The motifs that are responsible for the binding of a divalent metal ion and catalytic activity completely conserved. The gene encoding aldolase was overexpressed under T7 promoter control in Escherichia coli and the recombinant protein purified by nickel affinity chromatography. Kinetic characterization of the enzyme was performed at 60 degrees C, and K(m) and V(max) were found to be 576 microM and 2.4 microM min(-1) mg protein(-1), respectively. Enzyme exhibits maximal activity at pH 8.5. The activity of enzyme was completely inhibited by EDTA.     

Cloning and expression of an endo-1,6-beta-D-glucanase gene (neg1) from Neurospora crassa

A gene (neg1) encoding an endo-1,6-beta-D-glucanase from Neurospora crassa was cloned. The putative neg1 was 1443-bp long and encoded a mature endo-1,6-beta-D-glucanase protein of 463 amino acids and signal peptide of 17 amino acids. The purified recombinant protein (Neg1) obtained from Escherichia coli showed 1,6-beta-D-glucanase activity. No genes similar in sequence were found in yeasts and fungi.     

Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3

In the present study, we report the identification of a new gene from the Bacillus subtilis B3 strain (aatB3), which comprises 1308 bp encoding a 436 amino acid protein with a monomer molecular weight of 49.1 kDa. Phylogenetic analyses suggested that this enzyme is a member of the Ib subgroup of aspartate aminotransferases (AATs; EC 2.6.1.1), although it also has conserved active residues and thermostability characteristic of Ia-type AATs. The Asp232, Lys270 and Arg403 residues of AATB3 play a key role in transamination. The enzyme showed maximal activity at pH 8.0 and 45 °C, had relatively high activity over an alkaline pH range (pH 7.0-9.0) and was stable up to 50 °C. AATB3 catalyzed the transamination of five amino acids, with L-aspartate being the optimal substrate. The K(m) values were determined to be 6.7 mM for L-aspartate, 0.3 mM for α-ketoglutarate, 8.0 mM for L-glutamate and 0.6 mM for oxaloacetate. A 32-residue N-terminal amino acid sequence of this enzyme has 53% identity with that of Bacillus circulans AAT, although it is absent in all other AATs from different organisms. Further studies on AATB3 may confirm that it is potentially beneficial in basic research as well as various industrial applications.     

Deduced amino acid sequence and possible catalytic residues of a thermostable, alkaline cellulase from an Alkaliphilic bacillus strain

Alkaliphilic Bacillus sp. strain KSM-S237 (a relative of Bacillus pseudofirmus) produces a thermostable, alkaline endo-1,4-beta-glucanase (Egl). The entire gene for the enzyme harbored a 2,472-bp open reading frame (ORF) encoding 824 amino acids, including a 30-aminoacid signal peptide. The deduced amino acid sequence of the mature enzyme (794 amino acids, 88,284 Da) showed very high similarity to those of family 5 mesophilic, alkaline Egls from some alkaliphilic bacilli. The enzyme had a region similar to a novel cellulose binding domain proposed for an Egl (EngF) from Clostridium cellulovorans. Expression of the Bacillus Egl gene in Bacillus subtilis resulted in high carboxymethy cellulase activity (2.0 g/l) in the culture broth, concomitant with the appearance of a protein band on an SDS gel at 86 kDa. Site-directed mutagenesis delineated the importance of Arg111, His151, Glu190, His262, Tyr264, and Glu305 in catalysis and/or substrate binding of the enzyme.