Molecular cloning of isomaltotrio-dextranase gene from Brevibacterium fuscum var. dextranlyticum strain 0407 and its expression in Escherichia coli.

The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum strain 0407, and expressed in Escherichia coli. A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (M(r), 68,300) was found. The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG. The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.     

Cloning, sequencing, and expression of the meso-diaminopimelate dehydrogenase gene from Bacillus sphaericus

The gene encoding the meso-diaminopimelate dehydrogenase of Bacillus sphaericus was cloned into E. coli cells and its complete DNA sequence was determined. The meso-diaminopimelate dehydrogenase gene consisted of 978 nucleotides and encoded 326 amino acid residues corresponding to the subunit of the dimeric enzyme. The amino acid sequence deduced from the nucleotide sequence of the enzyme gene of B. sphaericus showed 50% identity with those of the enzymes from Corynebacterium glutamicum and Brevibacterium flavum. The enzyme gene from B. sphaericus was highly expressed in E. coli cells. We purified the enzyme to homogeneity from a transformant with 76% recovery. The N-terminal amino acid of both the enzyme from B. sphaericus and the transformant were serine, indicating that the N-terminal methionine is removed by post-translational modification in B. sphaericus and E. coli cells.     

Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis

A bacterial strain, which assimilated dextran and water-insoluble glucan produced by Streptococcus mutans, was isolated from soil. The bacterium produced and secreted potent dextranase activity, which was identified as Arthrobacter sp. and named CB-8. The dextranase was purified and some enzymatic properties were characterized. The enzyme efficiently decomposed the water-insoluble glucan as well as dextran. A gene library from the bacteria was constructed with Escherichia coli, using plasmid pUC19, and clones producing dextranase activity were selected. Based on the result of nucleotide sequencing analysis, it was deduced that the dextranase was synthesized in CB-8 cells as a polypeptide precursor consisting of 640 amino acid residues, including 49 N-terminal amino acid residues which could be regarded as a signal peptide. In the E. coli transformant, the dextranase activity was detected mostly in the periplasmic space. The gene for the dextranase was introduced into Streptococcus sanguis, using an E. coli-S. sanguis shuttle vector that contained the promoter sequence of a gene for glucosyltransferase derived from a strain of S. mutans. The active dextranase was also expressed and accumulated in S. sanguis cells.     

Molecular Cloning and Expression of Proteinaceous α-Amylase Inhibitor Gene from Streptomyces nitrosporeus in Escherichia coli

The proteinaceous α-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor. The nucleotide sequence of the insert of a positive clone had an open reading frame of 330 bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11,306 daltons. The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor. Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space. The amino-terminal sequence of the inhibitor produced by an E. coli transformant was identical to that of the T-76 inhibitor secreted by S. nitrosporeus. The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous α-amylase inhibitors.     

Cloning, expression, and characterization of Bacillus sp. snu-7 inulin fructotransferase

A gene encoding inulin fructotransferase (di-D-fructofuranose 1,2': 2,3' dianhydride [DFA III]-producing IFTase, EC 4.2.2.18) from Bacillus sp. snu-7 was cloned. This gene was composed of a single, 1,353-bp open reading frame encoding a protein composed of a 40-amino acid signal peptide and a 410-amino acid mature protein. The deduced amino acid sequence was 98% identical to Arthrobacter globiformis C11-1 IFTase (DFA III-producing). The enzyme was successfully expressed in E. coli as a functionally active, His-tagged protein, and it was purified in a single step using immobilized metal affinity chromatography. The purified enzyme showed much higher specific activity (1,276units/mg protein) than other DFA III-producing IFTases. The recombinant and native enzymes were optimally active in very similar pH and temperature conditions. With a 103-min half-life at 60 degrees C, the recombinant enzyme was as stable as the native enzyme. Acidic residues and cysteines potentially involved in the catalytic mechanism are proposed based on an alignment with other IFTases and a DFA IIIase.     

Purification, characterization, and gene cloning of a novel maltosyltransferase from an Arthrobacter globiformis strain that produces an alternating alpha-1,4- and alpha-1,6-cyclic tetrasaccharide from starch

A glycosyltransferase, involved in the synthesis of cyclic maltosylmaltose [CMM; cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}] from starch, was purified to homogeneity from the culture supernatant of Arthrobacter globiformis M6. The CMM-forming enzyme had a molecular mass of 71.7 kDa and a pI of 3.6. The enzyme was most active at pH 6.0 and 50 degrees C and was stable from pH 5.0 to 9.0 and up to 30 degrees C. The addition of 1 mM Ca2+ enhanced the thermal stability of the enzyme up to 45 degrees C. The enzyme acted on maltooligosaccharides that have degrees of polymerization of > or =3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan, and dextran. The mechanism for the synthesis of CMM from maltotetraose was determined as follows: (i) maltotetraose + maltotetraose --> 6(4)-O-alpha-maltosyl-maltotetraose + maltose and (ii) 6(4)-O-alpha-maltosyl-maltotetraose --> CMM + maltose. Thus, the CMM-forming enzyme was found to be a novel maltosyltransferase (6MT) catalyzing both intermolecular and intramolecular alpha-1,6-maltosyl transfer reactions. The gene for 6MT, designated cmmA, was isolated from a genomic library of A. globiformis M6. The cmmA gene consisted of 1,872 bp encoding a signal peptide of 40 amino acids and a mature protein of 583 amino acids with a calculated molecular mass of 64,637. The deduced amino acid sequence showed similarities to alpha-amylase and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in 6MT, indicating that 6MT should be assigned to this family.     

Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene.

The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci.     

Site-directed mutagenesis establishes aspartic acids-227 and -342 as essential for enzyme activity in an isomalto-dextranase from Arthrobacter globiformis

Isomalto-dextranase, from Arthrobacter globiformis T6, is a member of the glycoside hydrolase family 27. However, the alignments of the whole amino acid sequence are distinct from other members of this family. The enzymes cleave the glycosidic bond of the substrate in two different manners: either retaining or inverting the anomeric configuration. We believe that a retaining enzyme is involved in a two-step, double-displacement mechanism utilizing active site carboxylic acids as the nucleophile and general acid/base catalysts in the hydrolytic reaction. The critical amino acid residues at the isomalto-dextranase active site that catalyzes the hydrolysis reaction of dextran have been identified and the roles of nine amino acid residues (D107, D163, D227, D295, D340, D342, D373, D396, and E420) in the isomalto-dextranase from A. globiformis analyzed by site-directed mutagenesis. Of 15 mutant enzymes that were prepared, eight had reduced activities for dextran hydrolysis. Aspartic acids-227 and -342, which are part of the apparent catalytic dyad, were essential for hydrolase activity toward dextran.     

Diverse dextranase genes from <Emphasis Type="Italic">Paenibacillus</Emphasis> species

Genes encoding dextranolytic enzymes were isolated from Paenibacillus strains Dex40-8 and Dex50-2. Single, similar but non-identical dex1 genes were isolated from each strain, and a more divergent dex2 gene was isolated from strain Dex50-2. The protein deduced from the Dex40-8 dex1 gene sequence had 716 amino acids, with a predicted M_r of 80.8 kDa. The proteins deduced from the Dex50-2 dex1 and dex2 gene sequences had 905 and 596 amino acids, with predicted M_r of 100.1 kDa and 68.3 kDa, respectively. The deduced amino acid sequences of all three dextranolytic proteins had similarity to family 66 glycosyl hydrolases and were predicted to possess cleavable N-terminal signal peptides. Homology searches suggest that the Dex40-8 and Dex50-2 Dex1 proteins have one and two copies, respectively, of a carbohydrate-binding module similar to CBM_4_9 (pfam02018.11). The Dex50-2 Dex2 deduced amino acid sequence had highest sequence similarity to thermotolerant dextranases from thermophilic Paenibacillus strains, while the Dex40-8 and Dex50-2 Dex1 deduced protein sequences formed a distinct sequence clade among the family 66 proteins. Examination of seven Paenibacillus strains, using a polymerase chain reaction-based assay, indicated that multiple family 66 genes are common within this genus. The three recombinant proteins expressed in Escherichia coli possessed dextranolytic activity and were able to convert ethanol-insoluble blue dextran into an ethanol-soluble product, indicating they are endodextranases (EC 3.2.1.11). The reaction catalysed by each enzyme had a distinct temperature and pH dependence.     

The aman6 gene encoding a yeast mannan backbone degrading 1,6-alpha-D-mannanase in Bacillus circulans: cloning, sequence analysis, and expression

A gene (aman6) encoding endo-1,6-alpha-D-mannanase, a yeast mannan backbone degrading enzyme from Bacillus circulans was cloned. The putative aman6 was 1,767 base pairs long and encoded a mature 1,6-alpha-D-mannanase protein of 589 amino acids and a signal peptide of 36 amino acids. The purified mature 1,6-alpha-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-alpha-D-mannanase from Bacillus circulans TN-31.     

Molecular cloning and nucleotide sequence of the gene encoding a H2O2-forming NADH oxidase from the extreme thermophilic Thermus thermophilus HB8 and its expression in Escherichia coli.

The sequence of the 32 N-terminal amino acids of the NADH oxidase from the extreme thermophile, Thermus thermophilus HB8, was used to synthesize oligonucleotides to probe for the respective gene in a genomic library of T. thermophilus HB8. The gene encoding the NADH oxidase, designated nox, was cloned, its nucleotide sequence was determined and found to be colinear with the N-terminal sequence of the enzyme. The molecular mass of 26835 Da, as deduced from the nox gene, agrees with that of the purified NADH oxidase from T. thermophilus HB8 (25,000 Da), as estimated by polyacrylamide gel electrophoresis under denaturing conditions. The nox gene was overexpressed in Escherichia coli and a protocol for the rapid purification of the enzyme was developed. The E. coli-borne T. thermophilus HB8 NADH oxidase has properties identical to those of the authentic T. thermophilus HB8 enzyme and possesses a high thermal stability.     

A manganese-dependent dioxygenase from Arthrobacter globiformis CM-2 belongs to the major extradiol dioxygenase family.

Almost all bacterial ring cleavage dioxygenases contain iron as the catalytic metal center. We report here the first available sequence for a manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxygenase and its further characterization. This manganese-dependent extradiol dioxygenase from Arthrobacter globiformis CM-2, unlike iron-dependent extradiol dioxygenases, is not inactivated by hydrogen peroxide. Also, ferrous ions, which activate iron extradiol dioxygenases, inhibit 3,4-DHPA 2,3-dioxygenase. The gene encoding 3,4-DHPA 2,3-dioxygenase, mndD, was identified from an A. globiformis CM-2 cosmid library. mndD was subcloned as a 2.0-kb SmaI fragment in pUC18, from which manganese-dependent extradiol dioxygenase activity was expressed at high levels in Escherichia coli. The mndD open reading frame was identified by comparison with the known N-terminal amino acid sequence of purified manganese-dependent 3,4-DHPA 2,3-dioxygenase. Fourteen of 18 amino acids conserved in members of the iron-dependent extradiol dioxygenase family are also conserved in the manganese-dependent 3,4-DHPA 2,3-dioxygenase (MndD). Thus, MndD belongs to the extradiol family of dioxygenases and may share a common ancestry with the iron-dependent extradiol dioxygenases. We propose the revised consensus primary sequence (G,T,N,R)X(H,A)XXXXXXX(L,I,V,M,F)YXX(D,E,T,N,A)PX(G,P) X(2,3)E for this family. (Numbers in brackets indicate a gap of two or three residues at this point in the sequence.) The suggested common ancestry is also supported by sequence obtained from genes flanking mndD, which share significant sequence identity with xylJ and xylG from Pseudomonas putida.     

Organization of the genes involved in dimethylglycine and sarcosine degradation in Arthrobacter spp.: implications for glycine betaine catabolism.

The nucleotide sequences of two cloned DNA fragments containing the structural genes of heterotetrameric sarcosine oxidase (soxBDAG) and dimethylglycine dehydrogenase (dmg) from Arthrobater spp. 1-IN and Arthrobacter globiformis, respectively, have been determined. Open reading frames were identified in the soxBDAG operon corresponding to the four subunits of heterotetrameric sarcosine oxidase by comparison with the N-terminal amino-acid sequences and the subunit relative molecular masses of the purified enzyme. Alignment of the deduced sarcosine oxidase amino-acid sequence with amino-acid sequences of functionally related proteins indicated that the arthrobacterial enzyme is highly homologous to sarcosine oxidase from Corynebacterium P-1. Deletion and expression analysis, and alignment of the deduced amino-acid sequence of the dmg gene, showed that dmg encodes a novel dimethylglycine oxidase, which is related to eukaryotic dimethylglycine dehydrogenase, and contains nucleotide-binding, flavinylation and folate-binding motifs. The recombinant dimethylglycine oxidase was purified to homogeneity and characterized. The DNA located upstream and downstream of both the soxBDAG and dmg genes is predicted to encode enzymes involved in the tetrahydrofolate-dependent assimilation of methyl groups. Based on the sequence analysis reported herein, pathways are proposed for glycine betaine catabolism in Arthrobacter species, which involve the identified folate-dependent enzymes.     

Molecular cloning and nucleotide sequence of the beta-lytic protease gene from Achromobacter lyticus.

Two bacteriolytic enzymes secreted by Achromobacter lyticus M497-1 were purified and identified as being very similar (considering their amino acid composition and N-terminal sequence) to alpha- and beta-lytic proteases from Lysobacter enzymogenes. A 1.8-kb EcoRI fragment containing the structural gene for beta-lytic protease was cloned from A. lyticus chromosomal DNA. The protein sequence deduced from the nucleotide sequence was identical to the known sequence of beta-lytic protease, except for six residues. The nucleotide sequence revealed that the mature enzyme is composed of 179 amino acid residues with an additional 195 amino acids at the amino-terminal end of the enzyme, which includes the signal peptide, thus indicating that the enzyme is synthesized as a precursor protein.     

Novel prolyl tri/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.     

Cloning and overexpression of ketopantoic acid reductase gene from Stenotrophomonas maltophilia and its application to stereospecific production of D-pantoic acid

Ketopantoic acid (KPA) reductase catalyzes the stereospecific reduction of ketopantoic acid to d-pantoic acid. Based on the N-terminal amino acid sequence of KPA reductase from Stenotrophomonas maltophilia 845, the KPA reductase gene was cloned from S. maltophilia NBRC14161 and sequenced. This gene contains an open reading frame of 777 bp encoding 258 amino acid residues, and the deduced amino acid sequence showed high similarity to the SDR superfamily proteins. An expression vector, pETSmKPR, containing the full KPA reductase gene was constructed and introduced into Escherichia coli BL21 (DE3) to overexpress the enzyme. Bioreduction of KPA using E. coli transformant cells coexpressing KPA reductase together with cofactor regeneration enzyme gene was also performed. The conversion yield of KPA to d-pantoic acid reached over 88% with a substrate concentration up to 1.17 M.     

节杆菌A．pascens DMDC12中SOD基因的克隆和序列分析

根据基因库中细菌SOD的基因保守序列和Arthrobacter pascens DMDC12的N-末端氨基酸序列设计引物,采用PCR技术,以A.pascens DMDC12基因组为模板,克隆了A.pascens DMDC12中Mn-SOD的567 bp的基因片段;再结合该基因片段和A.pascens DMDC12中SOD的N-末端氨基酸序列的有关信息,设计包含该sod完整ORF区域的引物,成功扩增了A.pascens DMDC12中Mn-SOD的基因序列,新sod序列(sodAP)已提交Gen-Bank(收录号DQ779150)。利用生物学软件对该序列进行分析表明,该sodAP序列的ORF区域全长651 bp,编码216个氨基酸;该序列和Arthrobacter sp.FB24的SOD基因序列同源性为86%。