The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral

The quaternary structure of the cyanide dihydratase from Pseudomonas stutzeri AK61 was determined by negative stain electron microscopy and three-dimensional reconstruction using the single particle technique. The structure is a spiral comprising 14 subunits with 2-fold symmetry. Interactions across the groove cause a decrease in the radius of the spiral at the ends and the resulting steric hindrance prevents the addition of further subunits. Similarity to two members of the nitrilase superfamily, the Nit domain of NitFhit and N-carbamyl-D-amino acid amidohydrolase, enabled the construction of a partial atomic model that could be unambiguously fitted to the stain envelope. The model suggests that interactions involving two significant insertions in the sequence relative to these structures leads to the left-handed spiral assembly.     

耐碱性木聚糖酶基因在短小芽孢杆菌中高效分泌表达的研究

从木聚糖酶高产短小芽孢杆菌 (Bacilluspumilus)BP5 1中克隆得到木聚糖酶基因xynA ,将其构建在芽孢杆菌表达载体pWH1 5 2 0中得到重组质粒pWSX1 1。xynA由木糖诱导xylA启动子调控xynA表达。采用同源高效表达策略 ,以原生质体转化方法将pWSX1 1转回原始菌株BP5 1中 ,获得重组菌株BPX1 1。通过木糖诱导重组菌株中的xy nA基因高效分泌表达 ,使木聚糖酶产酶活力比原菌株BP5 1提高了 87% ,同时对重组表达的木聚糖酶的酶学性质进行了初步研究     

Chloramphenicol acetyltransferase specified by cat-86: relationship between the gene and the protein

Gene cat-86 is chloramphenicol (Cm)-inducible and specifies Cm acetyltransferase, CAT-86. The gene was previously cloned from the DNA of a strain of Bacillus pumilus. In the present study we report the construction of a constitutively expressed version of cat-86 that permits high-level expression of the gene on a plasmid in B. subtilis. A method is described that allows very rapid purification of CAT-86 protein to homogeneity. The sequence of 13 N-terminal amino acids of purified CAT-86, as well as the 26.6-kDa size of the subunit protein, agree with predictions made based on the nucleotide sequence of the gene. The Mr of the native enzyme suggests that CAT-86 is a trimer consisting of three identical protein subunits. Our studies demonstrate that cat-86 provides a convenient system for analyzing relationships between a gene and a multimeric enzyme in the B. subtilis background.     

Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency

We have described previously the potential use of an alkaline protease from Bacillus pumilus CBS as an effective additive in laundry detergent formulations [B. Jaouadi, S. Ellouz-Chaabouni, M. Ben Ali, E. Ben Messaoud, B. Naili, A. Dhouib, S. Bejar, A novel alkaline protease from Bacillus pumilus CBS having a high compatibility with laundry detergent and a high feather-degrading activity, Process Biochem, submitted for publication]. Here, we purified this enzyme (named SAPB) and we cloned, sequenced and over-expressed the corresponding gene. The enzyme was purified to homogeneity using salt precipitation and gel filtration HPLC. The pure protease was found to be monomeric protein with a molecular mass of 34598.19Da as determined by MALDI-TOF mass spectrometry. The NH(2)-terminal sequence of first 21 amino acids (aa) of the purified SAPB was AQTVPYGIPQIKAPAVHAQGY and was completely identical to proteases from other Bacillus pumilus species. This protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine proteases superfamily. Interestingly, the optimum pH is 10.6 while the optimum temperature was determined to be 65 degrees C. The enzyme was completely stable within a wide range of pH (7.0-10.6) and temperature (30-55 degrees C). One of the distinguishing properties is its catalytic efficiency (k(cat)/K(m)) calculated to be 45,265min(-1)mM(-1) and 147,000min(-1)mM(-1) using casein and AAPF as substrates, respectively, which is higher than that of Subtilisin Carlsberg, Subtilisin BPN' and Subtilisin 309 determined under the same conditions. In addition, SAPB showed remarkable stability, for 24h at 40 degrees C, in the presence of 5% Tween-80, 1% SDS, 15% urea and 10% H(2)O(2), which comprise the common bleach-based detergent formulation. The sapB gene encoding SAPB was cloned, sequenced and over-expressed in Escherichia coli. The purified recombinant enzyme (rSAPB) has the same physicochemical and kinetic properties as the native one. SapB gene had an ORF of 1149bp encoding a protein of 383 aa organized into a signal peptide (29 aa), a pro-protein (79 aa) and a mature enzyme (275 aa). The deduced amino acid sequence inspection displays an important homology with other bacterial proteases. The highest homology of 98.1% was found with BPP-A protease from Bacillus pumilus MS-1, with only 8 aa of difference.     

Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1.

A cyanide-degrading enzyme from Bacillus pumilus C1 has been purified and characterized. This enzyme consisted of three polypeptides of 45.6, 44.6, and 41.2 kDa; the molecular mass by gel filtration was 417 kDa. Electron microscopy revealed a multimeric, rod-shaped protein approximately 9 by 50 nm. Cyanide was rapidly degraded to formate and ammonia. Enzyme activity was optimal at 37 degrees C and pH 7.8 to 8.0. Activity was enhanced by Sc3+, Cr3+, Fe3+, and Tb3+; enhancement was independent of metal ion concentration at concentrations above 5 microM. Reversible enhancement of enzymatic activity by azide was maximal at 4.5 mM azide and increased with time. No activity was recorded with the cyanide substrate analogs CNO-, SCN-, CH3CN, and N3- and the possible degradation intermediate HCONH2. Kinetic studies indicated a Km of 2.56 +/- 0.48 mM for cyanide and a Vmax of 88.03 +/- 4.67 mmol of cyanide per min/mg/liter. The Km increased approximately twofold in the presence of 10 microM Cr3+ to 5.28 +/- 0.38 mM for cyanide, and the Vmax increased to 197.11 +/- 8.51 mmol of cyanide per min/mg/liter. We propose naming this enzyme cyanide dihydratase.     

Molecular characterisation of the gene encoding an esterase from Bacillus licheniformis sharing significant similarities with lipases

An esterase gene from the moderate thermophilic strain Bacillus licheniformis LCB40 was cloned and expressed in Escherichia coli. Comparison of the amino acid sequence of the esterase with those of known lipases and esterases showed the presence of the well-conserved Gly-X-Ser-X-Gly pentapeptide, with an alanine replacing the first glycine. This substitution has never been reported for an esterase but it is present in the lipases from Bacillus subtilis, Bacillus pumilus and Galactomyces candidum. The amino acid sequence showed similarities with lipases and with mammalian lecithin-cholesterol acyltranferases and no similarities with esterases. The enzyme activity of a crude extract from a recombinant Escherichia coli strain showed hydrolysis of p-nitrophenyl caprylate (pNPC8) as for esterases, but not of p-nitrophenyl palmitate (pNPC16) or olive oil such as for lipases. Thus, the enzyme displays the original property of associating the activity of an esterase with a primary sequence showing high similarity with lipases.     

First report of a bifunctional chitinase/lysozyme produced by Bacillus pumilus SG2

Bacillus pumilus SG2 isolated from high salinity ecosystem in Iran produces two chitinases (ChiS and ChiL) and secretes them into the medium. In this study, chiS and chiL genes were cloned in pQE-30 expression vector and were expressed in the cytoplasm of Escherichia coli strain M15. The recombinant proteins were purified using Ni-NTA column. The optimum pH and optimum temperature for enzyme activity of ChiS were pH 6, 50°C; those of ChiL were pH 6.5, 40°C. The purified chitinases showed antifungal activity against Fusarium graminearum, Rhizoctonia solani, Magnaporthe grisea, Sclerotinia sclerotiorum, Trichoderma reesei, Botrytis cinerea and Bipolaris sp. Moreover, purified ChiS was identified as chitinase/lysozyme, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of cell walls with many kinds of bacteria (Xanthomonas translucens pv. hordei, Xanthomonas axonopodis pv. citri, Bacillus licheniformis, E. coli C600, E. coli TOP10, Pseudomonas aeruginosa and Pseudomonas putida). Strong homology was found between the three-dimensional structures of ChiS and a chitinase/lysozyme from Bacillus circulans WL-12. This is the first report of a bifunctional chitinase/lysozyme from B. pumilus.     

Cloning of the gene encoding a novel thermostable alpha-galactosidase from Thermus brockianus ITI360.

An alpha-galactosidase gene from Thermus brockianus ITI360 was cloned, sequenced, and expressed in Escherichia coli, and the recombinant protein was purified. The gene, designated agaT, codes for a 476-residue polypeptide with a calculated molecular mass of 53, 810 Da. The native structure of the recombinant enzyme (AgaT) was estimated to be a tetramer. AgaT displays amino acid sequence similarity to the alpha-galactosidases of Thermotoga neapolitana and Thermotoga maritima and a low-level sequence similarity to alpha-galactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme is thermostable, with a temperature optimum of activity at 93 degrees C with para-nitrophenyl-alpha-galactopyranoside as a substrate. Half-lives of inactivation at 92 and 80 degrees C are 100 min and 17 h, respectively. The pH optimum is between 5.5 and 6.5. The enzyme displayed high affinity for oligomeric substrates. The K(m)s for melibiose and raffinose at 80 degrees C were determined as 4.1 and 11.0 mM, respectively. The alpha-galactosidase gene in T. brockianus ITI360 was inactivated by integrational mutagenesis. Consequently, no alpha-galactosidase activity was detectable in crude extracts of the mutant strain, and it was unable to use melibiose or raffinose as a single carbohydrate source.     

Engineering pH-tolerant mutants of a cyanide dihydratase

Cyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of Bacillus pumilus C1 cyanide dihydratase (CynD_pum) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynD_pum mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance.     

短小芽孢杆菌HZbp脂肪酶基因的克隆表达

以短小芽孢杆菌HZbp总DNA为模板以PCR的方式获得512 bp的脂肪酶基因,并在该基因的两端引入了EcoR1和Sal1的酶切位点,将该基因与大肠杆菌表达质粒pSE380连接,获得重组质粒pSE380-BPL。重组质粒转入大肠杆菌表达细胞株BL21,获得工程菌株BL21-BPL。序列分析显示所克隆的基因具有脂肪酶的保守G-X-S-X-G序列,SDS-PAGE电泳显示该脂脂肪酶的分子质量约为20 kDa。在LB培养基中,IPTG诱导浓度为1.0 mmol/L,33℃诱导培养10 h后,发酵液酶活达到8 U/mL。     

Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures

An aberrant random amplified polymorphic DNA (RAPD) marker in genomic DNA of tissue culture plantlets was frequently observed during a comparison of DNA fingerprints derived from potato germplasm grown in tissue culture and the field. The RAPD marker was cloned, sequenced and determined to be of bacterial origin. A bacterial contaminant was isolated from the tissue culture plants and identified as a Bacillus pumilus. A set of sequence characterised amplified region (SCAR) primers were designed from the sequence of the cloned fragment and tested for the specific detection of B. pumilus. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) were also used to generate B. pumilus profiles specific to our isolate in order to test and confirm the sequence homology of amplified markers generated from a range of DNA samples isolated from tissue culture plants and pure isolates of B. pumilus-like bacteria.     

Characterization of the H(+)-pumping F1F0 ATPase of Vibrio alginolyticus.

The F1F0 ATPase of Vibrio alginolyticus was cloned from a chromosomal lambda library. The unc operon, which contains the structural genes for the ATPase, was sequenced and shown to have a gene organization of uncIBEFHAGDC. The sequence of each subunit was compared with those of other eubacterial ATPases. The V. alginolyticus unc genes exhibited greater similarity to the Escherichia coli unc genes than to any of the other bacterial unc genes for which the sequence is available. The ATPase was expressed in an E. coli unc deletion strain, and the ATP hydrolytic activity was characterized. It has a pH optimum of 7.6 and is stimulated by the addition of Triton X-100 or any of a variety of salts. The recombinant F1F0 was purified 30.4-fold and reconstituted into proteoliposomes. This enzyme catalyzed the pumping of protons coupled to ATP hydrolysis as measured in fluorescence quenching experiments but would not pump Na+ ions under similar conditions.     

短小芽孢杆菌碱性蛋白酶基因启动子的克隆、鉴定及其应用

利用TAIL-PCR(Thermal asymmetric interlaced PCR)从短小芽孢杆菌基因组中扩增到碱性蛋白酶基因编码区上游的启动子片段。对该片段的序列测定和分析表明, 此片段长797 bp, 但与基因表达有关的序列长约390 bp。对启动子片段进行不同长度的缺失突变, 以获得最小的基因启动子片段, 结果表明, 该基因起始密码子上游约160 bp的DNA片段就可以启动基因的表达。将含有该片段的碱性蛋白酶基因WApQ3插入大肠杆菌-芽孢杆菌穿梭质粒载体pSUGV4中, 构建了碱性蛋白酶基因表达质粒pSUBpWApQ3。将该质粒分别转入枯草芽孢杆菌和短小芽孢杆菌中表达, 可在胞外检测到碱性蛋白酶活性, 最高酶活分别为466.5 U/mL和3060 U/mL。     

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli.

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

A Maltooligosaccharide-Forming Amylase Gene from Brachybacterium sp. Strain LB25: Cloning and Expression in Escherichia coli

Brachybacterium sp. strain LB25 produces a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents. The enzyme hydrolyzed starch to produce maltotriose primarily. The structural gene encoding the amylase from strain LB25 was cloned and sequenced. The amino acid sequence of the product showed significant similarity (45 to 49%) to amylases from the genus Streptomyces. The amylase gene was expressed in Escherichia coli, but the specific activity of the recombinant amylase was lower than that of the amylase purified from strain LB25.     

Expression and characterization of Ca(2+)-independent lipase from Bacillus pumilus B26

A lipase-producing Bacillus pumilus strain (B26) was isolated from a soil sample collected in Korea. The cloned gene showed that the lipase B26 composed of a 34-amino-acid signal sequence and a 181-amino-acid mature part corresponding to a molecular mass (M(r)) of 19,225. Based on the M(r) and the protein sequence, the lipase B26 belongs to the lipase family I.4. The optimum temperature and pH of the purified enzyme were 35 degrees C and 8.5, respectively. The lipase B26 showed a 'Ca(2+)-independent thermostability and catalytic activity'. These are novel properties observed for the first time in lipase B26 among all bacterial lipases and correspond with the suggestion that this enzyme had no Ca(2+)-binding motif around the catalytic His156 residue. This enzyme seems to be a true lipase based on the experimental results that it could hydrolyze various long-chain triglycerides (C(14)-C(18)) and triolein (C(18:1)) and that it showed a typical interfacial activation mechanism toward both tripropionin and p-nitrophenyl butyrate.     

Controlled specific expression and purification of 6×His-tagged proteins in Pseudomonas

The Coxiella burnetii icd gene encoding an immunogenic dimeric NADP(+)-dependent isocitrate dehydrogenase (IDH) was cloned by screening a C. burnetii genomic library with a human positive serum and sequenced. The predicted gene product consists of 427 amino acids (M(r) = 46,600) and showed high identity to the IDHs of Escherichia coli (74%), Salmonella enterica (73%) and IDH-I of Vibrio sp. (71%). The cloned gene complemented an icd-defective E. coli mutant producing a recombinant IDH that had the same biochemical properties as the enzyme from purified C. burnetii. Unlike the homologs from other bacteria, the cloned enzyme was expressed to the highest level in low pH conditions. This distinct property of the cloned IDH suggests that C. burnetii icd gene may have a role in the adaptation of the organism to the harsh acidic environment of the eucaryotic phagolysosomes.     

Expression of chitinase A (chiA) gene from a local isolate of Serratia marcescens in Coleoptera-specific Bacillus thuringiensis

Aims:  The present study focused on cloning and expression of chiA gene from a highly chitinolytic local isolate of Serratia marcescens in an anti-Coleopteran Bacillus thuringiensis and comparison of the characteristics of the native and recombinant ChiAs.
Methods and Results:  chiA gene from Ser . marcescens was cloned, sequenced and compared with the previously cloned chiA genes. chiA gene was PCR cloned and expressed in anti-Coleopteran B. thuringiensis strain 3023 as verified by Western blot analysis. Specific ChiA activity of the recombinant B. thuringiensis (strain 3023-SCHI) reached its highest level at 21st hour of growth (16·93 U mg⁻¹), which was 5·2- and 1·3-fold higher than that of its parental strain and Ser . marcescens , respectively. Temperature and pH effects on native and recombinant ChiAs were next determined. The recombinant plasmid was quite stable over 240 generations.
Conclusions:  Serratia marcescens ChiA was heterologously expressed in an anti-Coleopteran B. thuringiensis at levels even higher than that produced by the source organism.
Significance and Impact of the Study:  Bacillus thuringiensis 3023-SCHI co-expressing anti-Coleopteran Cry3Aa protein and Ser . marcescens chitinase offers a viable alternative to the use of chitinolytic microbes/enzymes in combination with entamopathogenic bacteria for an increased potency because of synergistic interaction between them.