Characterization of maltotriose production by hydrolyzing of soluble starch with α-amylase from Microbulbifer thermotolerans DAU221.

A maltotriose-producing α-amylase, AmyA, from a newly isolated bacterial strain Microbulbifer thermotolerans DAU221 was purified and characterized in the heterologous host, Escherichia coli, using the pCold I vector. The amyA gene encoded a 761-residue protein composed of a 33 amino acid secretion signal peptide. The purified α-amylase with a molecular mass of 80 kDa, approximately, shared a sequence motif characteristic of the glycoside hydrolase family 13. The enzyme was optimally active, at 50 °C in sodium phosphate buffer (pH 6.0), by the traditional one factor-at-a-time method. But the optimal conditions of time, temperature, and pH for production of maltotriose from soluble starch were 1.76 h, 44.95 °C, and pH 6.35 by response surface methodology, respectively. Maltotriose, as the major enzyme reaction product, was analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The enzyme was found to be inhibited by the addition of 10 mM Cu(2+), Fe(3+), Hg(2+), Zn(2+), and EDTA, but exhibited extreme stability toward hexane. The K m and V max values for the hydrolysis of soluble starch were 1.08 mg/mL and 1.736 mmol maltotriose/mg protein/min, respectively.

Cloning and characterization of a new broadspecific ß-glucosidase from Lactococcus sp. FSJ4.

A ß-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure-function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this ß-glucosidase of great interest for further study on physiological and catalytic reaction processes.

ß-cyclodextrin production by the cyclodextrin glucanotransferase from Paenibacillus illinoisensis ZY-08: cloning, purification, and properties.

The gene encoding the cyclodextrin glucanotransferase (CGTase, EC2.4.1.19) of Paenibacillus illinoisensis was isolated, cloned, sequenced and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34-residues. The deduced amino acid sequence of the CGTase from P. illinoisensis ZY-08 exhibited highest identity (99 %) to the CGTase sequence from Bacillus licheniformis (P14014). The four consensus regions of carbohydrate converting domain and Ca(2+) binding domain could be identified in the sequence. The CGTase was purified by using cold expression vector, pCold I, and His-tag affinity chromatography. The molecular weight of the purified enzyme was about 74 kDa. The optimum temperature and pH of the enzyme were 40 °C and pH 7.4, respectively. The enzyme activity was increased by the addition of Ca(2+) and inhibited by Ba(2+), Cu(2+), and Hg(2+). The K m and V max values calculated were 0.48 mg/ml and 51.38 mg of ß-cyclodextrin/ml/min. The ZY-08 and recombinant readily converted soluble starch to ß-cyclodextrin but ZY-08 did not convert king oyster mushroom powder and enoki mushroom powder. However the recombinant CGTase converted king oyster mushroom powder and enoki mushroom powder to ß-cyclodextrin.

Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization.

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.

Characterization of an organic solvent-tolerant polysaccharide lyase from Microbulbifer thermotolerans DAU221.

Alginate and its derivatives are annually produced approximately 30,000 tons or more and are applied to various industries as they are natural polymers. The global market for alginate and its derivatives has been growing steadily. There is little research compared to other enzymes produced through biomass degradation or modification. An alginate lyase, MtAl138, from Microbulbifer thermotolerans DAU221 was cloned and identified in Escherichia coli BL21 (DE3). MtAl138 contains a highly conserved motif (R538TELR, Q607IH609, and YFKAGVY716NQ), which indicates that it belongs to the polysaccharide lyase family 7 (PL7). MtAl138, with a molecular weight of 77 kDa worked optimally at 45 °C and pH 7.4. MtAl138 showed twice as much activity as when there was no NaCl when there was between 100 and 600 mM NaCl. Moreover, its activity increased in organic solvents such as benzene, hexane, methanol, and toluene. Based on the thin layer chromatography analyses, MtAl38 is an endo-type enzyme that produces di-, tri-, or tetrasaccharides from polyG and polyM. This study provided that MtAl138 is an endoenzyme that showed outstanding enzymatic activity at concentrated salt solutions and organic solvents, which makes it a reasonably attractive enzyme for use in various industries.

Molecular cloning, purification and characterization of thermostable beta-1,3-1,4 glucanase from Bacillus subtilis A8-8.

A gene encoding a beta-1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant beta-1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60 degrees C, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60 degrees C and retained 30% of its original activity at 70 degrees C for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.

Gene cloning, purification, and characterization of a cold-adapted lipase produced by Acinetobacter baumannii BD5.

Acinetobacter baumannii BD5 was isolated from waters of Baek-du mountain, and the lipase gene was cloned using a PCR technique. The deduced amino acid sequence of the lipase and lipase chaperone were found to encode proteins of 325 aa and 344 aa with a molecular mass of 35 kDa and 37 kDa, respectively. The lipase gene was cloned and expressed in Escherichia coli BL21 (trxB) as an inclusion body, which was subsequently solubilized by urea, and then purified using Ni-affinity chromatography. After being purified, the lipase was refolded by incubation at 4oC in the presence of a 1:10 molar ratio of lipase:chaperone. The maximal activity of the refolded lipase was observed at a temperature of 35 degrees and pH 8.3 when p-NP caprate (C10) was used as a substrate; however, 28% of the activity observed at 35 degrees was still remaining at 0 degrees . The stability of the purified enzyme at low temperatures indicates that it is a cold-adapted enzyme. The refolded lipase was activated by Ca2+, Mg2+, and Mn2+, whereas Zn2+ and Cu2+ inhibited it. Additionally, 0.1% Tween 20 increased the lipase activity by 33%, but SDS and Triton X-100 inhibited the lipase activity by 40% and 70%, respectively.

Cloning, purification, and characterization of GH3 ß-glucosidase, MtBgl85, from Microbulbifer thermotolerans DAU221.

BACKGROUND: ß-Glucosidases have attracted considerable attention due to their important roles in various biotechnological processes such as cellulose degradation to make energy and hydrolysis of isoflavone. Microbulbifer thermotolerans (M. thermotolerans) is isolated from deep-sea sediment and has not been researched much yet. As a potential candidate for a variety of biotechnological industries, ß-glucosidases from the novel bacterial species should be researched extensively. METHODS: ß-Glucosidase, MtBgl85, from M. thermotolerans DAU221 was purified by His-tag affinity chromatography and confirmed by SDS-PAGE and zymogram. Its biochemical and physiological properties, such as effects of temperature, pH, metal ions, and organic solvents, substrate specificity, and isoflavone hydrolysis, were investigated. RESULTS: M. thermotolerans DAU221 showed ß-glucosidase activity in a marine broth plate containing 0.1% esculin and 0.25% ammonium iron (III) citrate. The ß-glucosidase gene, mtbgl85, was isolated from the whole genome sequence of M. thermotolerans DAU221. The ß-glucosidase gene was 2,319 bp and encoded 772 amino acids. The deduced amino acid sequence had a 43% identity with OaBGL84 from Olleya aquimaris and 35% and 32% identity with to CfBgl3A and CfBgl3C from Cellulomonas fimi among bacterial glycosyl hydrolase family 3, respectively. The optimal temperature of MtBgl85 was 50 °C and the optimum pH was 7.0. MtBgl85 activity was strongly reduced in the presence of Hg2+ and Cu2+ ions. As a result of measuring the activity at various concentrations of NaCl, it was confirmed that the activity was maintained up to the concentration of 1 M, but gradually decreased with increasing concentration. MtBgl85 showed higher enzyme stability at non-polar solvents (high Log Pow ) than polar solvents (low Log Pow ). The hydrolyzed products of isoflavone glycosides and arbutin were analyzed by HPLC.

Novel roles of Bacillus thuringiensis to control plant diseases.

Bacillus thuringiensis is well known as an effective bio-insecticidal bacterium. However, the roles of B. thuringiensis to control plant diseases are not paid great attention to. In recent years, many new functions in protecting plants from pathogen infection have been discovered. For example, acyl homoserine lactone lactonase produced by B. thuringiensis can open the lactone ring of N-acyl homoserine lactone, a signal molecule in the bacterial quorum-sensing system. This in turn, significantly silences bacterial virulence. This finding resulted in the development of a new strategy against plant bacterial diseases by quenching bacterial quorum sensing. Another new discovery about B. thuringiensis function is zwittermicin A, a linear aminopolyol antibiotic with high activity against the Oomycetes and their relatives, as well as some gram-negative bacteria. This paper summarized the relative progresses of B. thuringiensis in plant disease control and its favorable application prospects.

Characterization of new biosurfactant produced by Klebsiella sp. Y6-1 isolated from waste soybean oil.

To obtain predominant bacteria degrading crude oil, we isolated some bacteria from waste soybean oil. Isolated bacterial strain had a marked tributyrin (C4:0) degrading activity as developed clear zone around the colony after incubation for 24h at 37 degrees C. It was identified as Klebsiella sp. Y6-1 by analysis of 16S rRNA gene. Crude biosurfactant was extracted from the culture supernatant of Klebsiella sp. Y6-1 by organic solvent (methanol:chloroform:1-butanol) after vacuum freeze drying and the extracted biosurfactant was purified by silica gel column chromatography. When the purified biosurfactant dropped, it formed degrading zone on crude oil plate. When a constituent element of the purified biosurfactant was analyzed by TLC and SDS-PAGE, it was composed of peptides and lipid. The emulsification activity and stability of biosurfactant was measured by using hydrocarbons and crude oil. The emulsification activity and stability of the biosurfactant showed better than the chemically synthesized surfactant. It reduced the surface tension of water from 72 to 32 mN/m at a concentration of 40 mg/l.

Solubilization of insoluble inorganic phosphate by Burkholderia cepacia DA23 isolated from cultivated soil.

A mineral phosphate solubilizing bacterium, Burkholderia cepacia DA23 has been isolated from cultivated soils. Phosphate-solubilizing activities of the strain against three types of insoluble phosphate were quantitatively determined. When 3% of glucose concentration was used for carbon source, the strain had a marked mineral phosphate-solubilizing activity. Mineral phosphate solubilization was directly related to the pH drop by the strain. Analysis of the culture medium by high pressure liquid chromatography identified gluconic acid as the main organic acid released by Burkholderia cepacia DA23. Gluconic acid production was apparently the result of the glucose dehydrogenase activity and glucose dehydrogenase was affected by phosphate regulation.

Cloning, purification, and characterization of an organic solvent-tolerant chitinase, MtCh509, from Microbulbifer thermotolerans DAU221.

BACKGROUND: The ability to use organic solvents in enzyme reactions offers a number of industrially useful advantages. However, most enzymes are almost completely inactive in the presence of organic solvents. Thus, organic solvent-tolerant enzymes have potential applications in industrial processes. RESULTS: A chitinase gene from Microbulbifer thermotolerans DAU221 (mtch509) was cloned and expressed in Escherichia coli BL21 (DE3). The molecular weight of the expressed MtCh509 protein was approximately 60 kDa, and it was purified by His-tag affinity chromatography. Enzymatic assays showed that the optimum temperature for MtCh509 chitinase activity was 55 °C, and the enzyme was stable for 2 h at up to 50 °C. The optimum pH for MtCh509 activity was in the sub-acidic range, at pH 4.6 and 5.0. The activity of MtCh509 was maintained in presence of 1 M salt, gradually decreasing at higher concentrations, with residual activity (20%) detected after incubation in 5 M salt. Some organic solvents (benzene, DMSO, hexane, isoamyl alcohol, isopropyl alcohol, and toluene; 10-20%, v/v) increased the reactivity of MtCh509 relative to the aqueous system. When using NAG3, as a substrate, MtCh509 produced NAG2 as the major product, as well as NAG4, demonstrating that MtCh509 has transglycosylation activity. The K m and V max values for MtCh509 using colloidal chitin as a substrate were 9.275 mg/mL and 20.4 U/mg, respectively. Thus, MtCh509 could be used in extreme industrial conditions. CONCLUSION: The results of the hydrolysate analysis and the observed increase in enzyme activity in the presence of organic solvents show that MtCh509 has industrially attractive advantages. This is the first report on an organic solvent-tolerant and transglycosylating chitinase from Microbulbifer species.

Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101.

A chitinase encoding gene from Bacillus sp. DAU101 was cloned in Escherichia coli. The nucleotide sequencing revealed a single open reading frame containing 1781 bp and encoding 597 amino acids with 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram. The chitinase was composed of three domains: a catalytic domain, a fibronectin III domain, and a chitin binding domain. The chitinase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 7.5 and 60 degrees C, respectively. The metal ions, Zn(2+), Cu(2+), and Hg(2+), were strongly inhibited chitinase activity. However, chitinase activity was increased 1.4-fold by Co(2+). Chisb could hydrolyze GlcNAc(2) to N-acetylglucosamine and was produced GlcNAc(2), when chitin derivatives were used as the substrate. This indicated that Chisb was a bifunctional enzyme, N-acetylglucosaminase and chitobiosidase. The enzyme could not hydrolyze glycol chitin, glycol chitosan, or CMC, but hydrolyzed colloidal chitin and soluble chitosan.

Complete genome sequence and analysis of three kinds of ß-agarase of Cellulophaga lytica DAU203 isolated from marine sediment.

Cellulophaga lytica DAU203 (KACC 19187), isolated from the marine sediment in Korea, has a strong ability to degrade agar. The genome of C. lytica DAU203 contains a single chromosome that is 3,952,957bp in length, with 32.02% G+C contents. The genomic information predicted that the DAU203 has the potential to be utilized in various enzymatic industries.

Complete genome sequence of cold-adapted enzyme producing Microbulbifer thermotolerans DAU221.

Microbulbifer thermotolerans DAU221 was preliminary isolated from the marine sediment samples in the Republic of Korea. Here, we present the complete genome sequence of M. thermotolerans DAU221, which consisted of 3,938,396 base pairs with a GC content of 56.57%. This genomic information should help us find the industrially useful enzymes.

Overexpression and characterization of a novel chitinase gene from a marine bacterium Pseudomonas sp. BK1.

The chitinase A (ChiA)-coding gene of Pseudomonas sp. BK1, which was isolated from a marine red alga Porphyra dentata, was cloned and expressed in Escherichia coli. The structural gene consists of 1602 bp encoding a protein of 534 amino acids, with a predicted molecular weight of 55,370 Da. The deduced amino acid sequence of ChiA showed low identity (less than 32%) with other bacterial chitinases. The ChiA was composed of multiple domains, unlike the arrangement of domains in other bacterial chitinases. Recombinant ChiA overproduced as inclusion bodies was solubilized in the presence of 8 M urea, purified in a urea-denatured form and re-folded by removing urea. The purified enzyme showed maximum activity at pH 5.0 and 40 degrees C. It exhibited high activity towards glycol chitosan and glycol chitin, and lower activity towards colloidal chitin. The enzyme hydrolyzed the oligosaccharides from (GlcNAc)4 to (GlcNAc)6, but not GlcNAc to (GlcNAc)3. The results suggest that the ChiA is a novel enzyme, with different domain structure and action mode from bacterial family 18 chitinases.

Molecular cloning and characterization of CMCase gene (celC) from Salmonella typhimurium UR.

The sequence coding for carboxymethylcellulase (CMCase, CelC) was isolated from the DNA of Salmonella typhimurium UR1. Comparison between the deduced amino acid sequence of CelC (368 amino acid residues, Molecular mass 41 kDa) and that of the previously published CMCase revealed that this enzyme belongs to the cellulase family 8 and D. The protein was overproduced in Escherichia coli using T7 expression system, and its activity was confirmed by CMC-SDS-PAGE. When the overexpressed CelC protein was tested on cellulose-type substrates, the recombinant protein is able to degrade cellulose-type substrates, such as CM-cellulose, xylan, avicel, lichenan, and laminarin. Optimal temperature and pH for enzyme activity were found to be 50 degrees C and pH 6.5, respectively.

Isolation of Citrobacter sp. mutants defective in decolorization of brilliant green by transposon mutagenesis.

To identify genes involved in the decolorization of brilliant green, we isolated random mutants generated by transposon insertion in brilliant green-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 19 mutants with a complete defect in terms of the brilliant green color removing ability. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants and these mutants appeared to have insertions at different sites of the chromosome. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. By comparing these with a sequence database, putative protein products encoded by bg genes were identified as follows: bg 3 as a LysR-type regulatory protein; bg 11 as a MalG protein in the maltose transport system; bg 14 as an oxidoreductase; and bg 17 as an ABC transporter. The sequences deduced from the three bg genes, bg 2, bg 7 and bg 16, showed no significant similarity to any protein with a known function, suggesting that these three bg genes may encode unidentified proteins responsible for the decolorization of brilliant green.

Cloning, high-level expression and enzymatic properties of an intracellular serine protease from Bacillus sp. WRD-2.

A gene, isp-B, encoding an intracellular serine protease from a newly isolated Bacillus sp. WRD-2 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 960 bp encoding a polypeptide comprised of 319 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other intracellular serine proteases, including active sites, Ser, His and Asp, as well as no signal sequence. The predicted amino acid sequence showed more than 60% homology with the intracellular serine proteases from Bacillus species. When expressed in E. coli, the recombinant enzyme (rISP-B) was overproduced in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, EDTA and antipain. The enzyme showed maximum activity at pH 8.0 and 45 degrees C. It was stable atpH from 7.5 to 11.0 and below 50 degrees C.

A cold-adapted carbohydrate esterase from the oil-degrading marine Bacterium Microbulbifer thermotolerans DAU221: gene cloning, purification, and characterization.

A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6, was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids with the first 22 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG, QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST belongs to the α/ß-class of protein consisted of a central six-stranded ß-sheet flanked by eight α-helices. The recombinant CEST was purified by His-tag affinity chromatography and the characterization showed its optimal temperature and pH were 15°C and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at 50°C or 60°C for 6 h, and 89% of its enzyme activity when preincubated at 70°C for 1h . The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by Na(+) and Mg(2+) ions but was strongly inhibited by Cu(+) and Hg(2+) ions, at all ion concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a Km of 0.278 mM and a kcat of 1.9 s(-1). Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192, and His281) and Asp278 were essential for the enzyme activity.