Identification of crucial amino acid residues involved in large ring cyclodextrin synthesis by amylomaltase from Corynebacterium glutamicum.

Amylomaltase can be used to synthesize large ring cyclodextrins (LR-CDs), applied as drug solubilizer, gene delivery vehicle and protein aggregation suppressor. This study aims to determine the functional amino acid positions of Corynebacterium glutamicum amylomaltase (CgAM) involved in LR-CD synthesis by site-directed mutagenesis approach and molecular dynamic simulation. Mutants named Δ167, Y23A, P228Y, E231Y, A413F and G417F were constructed, purified, and characterized. The truncated CgAM, Δ167 exhibited no starch transglycosylation activity, indicating that the N-terminal domain of CgAM is necessary for enzyme activity. The P228Y, A413F and G417F produced larger LR-CDs from CD36-CD40 as compared to CD29 by WT. A413F and G417F mutants produced significantly low LR-CD yield compared to the WT. The A413F mutation affected all tested enzyme activities (starch tranglycosylation, disproportionation and cyclization), while the G417F mutation hindered the cyclization activity. P228Y mutation significantly lowered the k cat of disproportionation activity, while E231Y mutant exhibited much higher k cat and K m values for starch transglycosylation, compared to that of the WT. In addition, Y23A mutation affected the kinetic parameters of starch transglycosylation and cyclization. Molecular dynamic simulation further confirmed these mutations' impacts on the CgAM and LR-CD interactions. Identified functional amino acids for LR-CD synthesis may serve as a model for future modification to improve the properties and yield of LR-CDs.

Localization of functional ß-xylosidases, encoded by the same single gene, xlsIV (xlnD), from Aspergillus niger E-1.

Cell wall-associated ß-xylosidase was isolated from Aspergillus niger E-1 and identified as XlsIV, corresponding to the extracellular enzyme XlnD reported previously. xlsIV was transcribed only in the early cultivation period. Cell wall-associated enzyme activity gradually decreased, but extracellular activity increased as the strain grew. These results indicate that XlsIV (XlnD) was secreted into culture after localizing at cell wall.

Analysis of functional xylanases in xylan degradation by Aspergillus niger E-1 and characterization of the GH family 10 xylanase XynVII.

Xylanases produced by Aspergillus niger are industrially important and many types of xylanases have been reported. Individual xylanases have been well studied for their enzymatic properties, gene cloning, and heterologous expression. However, less attention has been paid to the relationship between xylanase genes carried on the A. niger genome and xylanases produced by A. niger strains. Therefore, we examined xylanase genes encoded on the genome of A. niger E-1 and xylanases produced in culture. Seven putative xylanase genes, xynI-VII (named in ascending order of the molecular masses of the deduced amino acid sequences), were amplified from the strain E-1 genome using primers designed from the genome sequence of A. niger CBS 513.88 by PCR and phylogenetically classified into three clusters. Additionally, culture supernatant analysis by DE52 anion-exchange column chromatography revealed that this strain produced three xylanases, XynII, XynIII, and XynVII, which were identified by N-terminal amino acid sequencing and MALDI-TOF-MS analyses, in culture when gown in 0.5% xylan medium supplemented with 50 mM succinate. Furthermore, XynVII, the only GH family 10 xylanase in A. niger E-1, was purified and characterized. The purified enzyme showed a single band with a molecular mass of 35 kDa by SDS-PAGE. The highest activity of purified XynVII was observed at 55°C and pH 5.5. The enzyme was stable in the broad pH range of 3-10 and up to 60°C and was resistant to most metal ions and modifying regents. XynVII showed high specificity against beechwood xylan with K m and V max values of 2.8 mg mL(-1) and 127 µmol min(-1)mg(-1), respectively. TLC and MALDI-TOF-MS analyses showed that the final hydrolyzed products of the enzyme from beechwood xylan were xylose, xylobiose, and xylotriose substituted with a 4-o-metylglucuronic acid residue.

Crystallization and preliminary X-ray crystallographic analysis of the amylomaltase from Corynebacterium glutamicum.

Amylomaltase (AM; EC 2.4.1.25) belongs to the 4-α-glucanotransferase group of the α-amylase family. The enzyme can produce cycloamylose or large-ring cyclodextrin through intramolecular transglycosylation or cyclization reactions of α-1,4-glucan. Amylomaltase from the mesophilic bacterium Corynebacterium glutamicum (CgAM) contains extra residues at the N-terminus for which the three-dimensional structure is not yet known. In this study, CgAM was overexpressed and purified to homogeneity using DEAE FF and Phenyl FF columns. The purified CgAM was crystallized by the vapour-diffusion method. Preliminary X-ray data showed that the CgAM crystal diffracted to 1.7 Šresolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 73.28, b = 82.61, c = 118.64 Å. To obtain the initial phases, crystals of selenomethionyl-substituted amylomaltase were produced, and multiple-wavelength anomalous dispersion phasing and structure refinement are now in progress.

Molecular cloning and sequence analysis of two distinct halotolerant extracellular proteases from Bacillus subtilis FP-133.

We cloned the genes encoding the two distinct extracellular halotolerant proteases of Bacillus subtilis FP-133 Expro-I and Expro-II, which were classified as alkaline serine and neutral proteases respectively. Three-dimensional modeling suggested that acidic and polar amino acid residues located on the surface stabilize protein structure in the presence of relatively high NaCl concentrations.

Fe-superoxide dismutase and 2-hydroxy-1,4-benzoquinone reductase preclude the auto-oxidation step in 4-aminophenol metabolism by Burkholderia sp. strain AK-5.

Burkholderia sp. strain AK-5 converts 4-aminophenol to maleylacetic acid via 1,2,4-trihydroxybenzene, which is unstable in vitro and non-enzymatically auto-oxidized to 2-hydroxy-1,4-benzoquinone. Crude extract of strain AK-5 retarded the auto-oxidation and reduced the substrate analogue, 2,6-dimethoxy-1,4-benzoquinone, in the presence of NADH. The two enzymes responsible were purified to homogeneity. The deduced amino acid sequence of the enzyme that inhibited the auto-oxidation showed a high level of identity to sequences of iron-containing superoxide dismutases (Fe-SODs) and contained a conserved metal-ion-binding site; the purified enzyme showed superoxide dismutase activity and contained 1 mol of Fe per mol of enzyme, identifying it as Fe-SOD. Among three type SODs tested, Fe-SOD purified here inhibited the auto-oxidation most efficiently. The other purified enzyme showed a broad substrate specificity toward benzoquinones, including 2-hydroxy-1,4-benzoquinone, converting them to the corresponding 1,4-benzenediols; the enzyme was identified as 2-hydroxy-1,4-benzoquinone reductase. The deduced amino acid sequence did not show a high level of identity to that of benzoquinone reductases from bacteria and fungi that degrade chlorinated phenols or nitrophenols. The indirect role of Fe-SOD in 1,2,4-trihydroxybenzene metabolism is probably to scavenge and detoxify reactive species that promote the auto-oxidation of 1,2,4-trihydroxybenzene in vivo. The direct role of benzoquinone reductase would be to convert the auto-oxidation product back to 1,2,4-trihydroxybenzene. These two enzymes together with 1,2,4-trihydroxybenzene 1,2-dioxygenase convert 1,2,4-trihydroxybenzene to maleylacetic acid.

Expression and characterization of a fusion protein-containing cyclodextrin glycosyltransferase from Paenibacillus sp. A11.

A recombinant cyclodextrin glycosyltransferase (CGTase) gene fused with thioredoxin (Trx), hexa-histidine (His(6)) and S-protein (S) at the N terminus and a proline-rich peptide (PRP) at the C terminus, was constructed using the wild-type gene from Paenibacillus sp. A11, the pET-32a vector and Escherichia coli BL21(DE3) as the host cell. The expression levels and enzyme characteristics of the Trx-His(6)-CGTase-PRP fusion protein, the recombinant CGTase without fusion peptides, and the wild-type CGTase were compared. The maximum specific activity for the Trx-His(6)-CGTase-PRP fusion enzyme was 2.7 fold higher than that of the non-fusion form at the optimal IPTG concentration. The Trx-His(6)-CGTase-PRP fusion protein was purified to homogeneity by starch adsorption and Ni-NTA affinity chromatography, with a specific activity of 2,268 units/mg protein at a 61% yield. The ease of purification and the higher enzyme yield were obtained with the fusion form when compared to the non-fusion and wild-type enzymes. The fusion enzyme was superior than its wild-type counterpart in terms of stability against high temperature and organic solvents. Moreover, the fusion enzyme could catalyze the synthesis of cyclodextrins in 20% (v/v) dimethylformamide with a higher product yield of CD(7) and CD(8) compared to that of the wild-type enzyme in the same buffer-solvent system.

Gene cloning and characterization of a deaminase from the 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d.

The 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d produces a deaminase that catalyzes the deamination of 2-amino-5-carboxymuconic 6-semialdehyde. A gene encoding the deaminase, ahdB, was cloned and expressed in Escherichia coli; ahdB is located downstream from the previously reported genes encoding 4-amino-3-hydroxybenzoate 2,3-dioxygenase (ahdA) and a LysR-type regulator. The deduced amino acid sequence of ahdB shows 30-33% identity to those of previously reported 2-aminomuconate deaminases. We identified a region (RAGDFLXVSG) conserved in AhdB and three other deaminases. The recombinant enzyme AhdB was purified to homogeneity. After a coupled enzyme assay with purified AhdA, AhdB, and the substrate 4-amino-3-hydroxybenzoate, the final product, formed by the action of AhdA, AhdB, and by nonenzymatic decarboxylation, was identified by HPLC, MS, and (1)H-nuclear magnetic resonance analyses as 2-hydroxymuconic 6-semialdehyde.

Purification and characterization of an eggshell membrane decomposing protease from Pseudomonas aeruginosa strain ME-4.

A bacterial strain, ME-4, isolated from farm soil and identified as Pseudomonas aeruginosa, grew well on a medium containing eggshell membrane (ESM). P. aeruginosa strain ME-4 decomposed the ESM by producing an extracellular protease able to solubilize it. The protease was purified to homogeneity from culture supernatant by fractionation with (NH(4))(2)SO(4), as well as CM52 cellulose and DE52 cellulose column chromatography, with a final yield of 47%. The molecular mass of the enzyme was 33 kDa. The isolated enzyme was a metalloprotease and was strongly inhibited by EDTA, o-phenanthroline, and phosphoramidon. The enzyme inhibited by these reagents was reactivated in the presence of several metal ions. The enzyme acted on various proteins and showed higher activity with collagen than collagenase from Clostridium histolyticum. Results of assays with the FRETS combinatorial libraries revealed that the enzyme preferred Ser at the P1 position and Lys at the P2 position. It also preferred hydrophobic amino acid residues at the P1' and P2' positions. The enzyme showed a much higher solubilization activity with the ESM substrate than commercially obtained enzymes. The enzyme decomposed ESM to produce water-soluble peptides, Val-Leu-Pro-Pro and (X)-Val-Pro-Pro, and a free amino acid, tryptophan.

Purification and characterization of an extracellular laccase from Phlebia radiata strain BP-11-2 that decolorizes fungal melanin.

Phlebia radiata strain BP-12-2, isolated from forest soil, decolorized fungal melanin and produced an extracellular laccase in culture. The enzyme was purified to homogeneity and characterized. It showed no absorption bands in the ultraviolet above 300 nm or visible regions. It differed from the reported laccases in substrate specificity, kinetic parameters, and NH2-terminal amino acid sequences.

Gene cloning and characterization of arylamine N-acetyltransferase from Bacillus cereus strain 10-L-2.

Bacillus cereus strain 10-L-2 synthesizes two arylamine N-acetyltransferases (Nat-a and Nat-b) with broad substrate specificities toward aniline and its derivatives. In southern blot analysis using probes encoding the NH2-terminus of Nat-b and a conserved region of N-acetyltransferases, digested total DNA of strain 10-L-2 showed one positive band. We cloned and sequenced the gene encoding Nat-b. The NH2-terminal amino acid sequence predicted from the open reading frame (768 base pairs) corresponded to that of purified Nat-b. The cloned Nat-b gene was expressed in Escherichia coli. The expressed enzyme (BcNAT) from the recombinant strain was partially purified and characterized. Nat-b from strain 10-L-2 and BcNAT from the recombinant strain were slightly different from each others in substrate specificity and thermo-stability. We examined the biotransformations of 2-aminophenols and phenylenediamines by the whole cells of the recombinant strain. The cells converted these compounds into their corresponding acetanilides. Only one amino group of phenylenediamines was acetylated. The cells utilized 4-nitroacetanilide as an acetyl donor instead of acetyl-CoA. 4-Aminoacetanilide was produced and 4-nitroaniline was released almost stoichiometrically.

Production and accumulation of xylooligosaccharides with long chains by growing culture and xylanase of a mutant strain of Bacillus pumilus X-6-19.

Bacillus pumilus X-6-9 isolated from soil and subsequently identified, produced xylooligosaccharides with long chains from xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increase in the production of the xylooligosaccharides was established, when compared to the original culture conditions of B. pumilus X-6-19. The addition of D-glucose to the culture of the mutant strain U-3 of B. pumilus X-6-9 repressed the synthesis of beta-xylosidase, but not xylanase. Thus, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharides with long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized. The hydrolyzates generated by the purified xylanase contained xylobiose, xylotriose, xylotetraose, and xylopentaose, but not xylose.

Purification, characterization, and gene cloning of Ceriporiopsis sp. strain MD-1 peroxidases that decolorize human hair melanin.

Ceriporiopsis sp. strain MD-1, isolated from forest soil, produced several extracellular enzymes that decolorized human hair melanin. Among them, three enzymes (E1, E2-1, and E2-2) were purified to homogeneity and characterized. The enzymes required hydrogen peroxide in their enzyme reactions and, typical of other fungal peroxidases, oxidized various phenol compounds such as guaiacol, but not 3,4-dimethoxybenzyl alcohol. The spectra of the three enzymes showed an absorption maximum at 406 nm, indicating that they were heme proteins. However, the A(406)/A(280) values of the enzymes were below 0.4, which was lower than those of other peroxidases. E2-1 and E2-2 were similar to each other in their molecular and catalytic properties, and they possibly represent products of posttranslational modifications and/or allelic variants of the same gene, mdcA. The corresponding cDNA was cloned and sequenced; the deduced amino acid sequence showed high identities to the manganese peroxidases from other microorganisms. The specific activities and K(m) values of E2-1 and E2-2 for synthetic and human hair melanins were much higher than those of Phanerochaete chrysosporium manganese peroxidase and lignin peroxidase.

Purification and characterization of two alkaline, thermotolerant alpha-amylases from Bacillus halodurans 38C-2-1 and expression of the cloned gene in Escherichia coli.

A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant alpha-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named alpha-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50-60 degrees C and pH 10-11, and 42 and 38% relative activities at 30 degrees C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding alpha-amylase I was cloned and named amyI. Production of AmyI with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl beta-D(-)-thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.

Cloning and expression of cyclodextrin glycosyltransferase gene from Paenibacillus sp. T16 isolated from hot spring soil in northern Thailand.

Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower K(m) for coupling reaction using cellobiose and cyclodextrins as substrates.

Bacillus cereus strain 10-L-2 produces two arylamine N-acetyltransferases that transform 4-phenylenediamine into 4-aminoacetanilide.

A bacterium, strain 10-L-2, that was isolated from soil and identified as Bacillus cereus grew well on medium containing 4-phenylenediamine and Polypepton. Strain 10-L-2 converted a wide variety of anilines, including 4-phenylenediamine, to their corresponding acetanilides. Growing cells acetylated a single amino group of 4-phenylenediamine to form 4-aminoacetanilide with a 97% molar yield, as shown by mass spectrometry and HPLC. Cell extracts exhibited arylamine N-acetyltransferase (NAT) activity toward 4-phenylenediamine. Two NATs, namely, NAT-a and NAT-b, were separated by DE52 column chromatography and were further purified and characterized. The subunit molecular masses of NAT-a and NAT-b were 31.0 and 27.5 kDa, respectively, as determined by SDS-PAGE analysis. The two enzymes had similar pH- and thermo-stabilities and were similarly affected by pH, temperature, and several reagents. The enzymes showed peak activity toward 5-aminosalicylic acid of the substrates tested, but they differed in substrate specificity. Only NAT-a had activity toward sulfamethazine. Although other wild-type bacterial cultures also synthesize NAT, the ability of strain 10-L-2 to convert and detoxify 4-phenylenediamine is much higher. This report provides the first evidence of two NATs in a eubacterium.

Screening and characterization of bacteria that can utilize ammonium and nitrate ions simultaneously under controlled cultural conditions.

Eighteen bacterial stock cultures were examined for their ability to utilize NH(4)(+) and NO(3)(-) simultaneously in a medium containing NH(4)NO(3) with shaking using a test tube capped with a cotton stopper. Pseudomonas aeruginosa NBRC 12689 utilized 1 mg/ml of NH(4)NO(3) most rapidly of the cultures tested. The bacterium could completely utilize 5 mg/ml of NH(4)NO(3) within 3 d, 6 mg/ml of NH(4)Cl within 3 d, and 20 mg/ml of NaNO(3) within 2 d under optimum conditions. The addition of Fe(2+) to the NH(4)NO(3) medium markedly promoted the utilization of the two ions. When the Pseudomonas strain utilized 5 mg/ml of NH(4)NO(3) completely, the total nitrogen in the culture including its cells decreased to 41% of that of the NH(4)NO(3) originally provided. GC-MS analysis showed that the removed nitrogen was probably denitrified. When the bacterium was incubated in the NH(4)NO(3) medium with shaking in a vial sealed with a rubber stopper, N(2) accumulated, but not N(2)O at the final phase of cultivation. On the other hand, both N(2) and N(2)O were detected in the NaNO(3) medium. We concluded that the bacterium removed NH(4)(+) from NH(4)NO(3) as a nitrogen source for its cell components, together with the denitrification of NO(3)(-) under controlled shaking conditions. In addition, NH(4)(+) promoted the cell growth of the bacterium and denitrification to N(2), preventing the accumulation of N(2)O.

Isolation and characterization of thermotolerant bacterium utilizing ammonium and nitrate ions under aerobic conditions.

A thermotolerant bacterium, identified as Bacillus licheniformis, completely utilized 0.1% (w/v) NH(4)NO(3) at 30 and 50 degrees C under aerobic condition. The addition of 0.5 mM Fe(2+) to the NH(4)NO(3 )medium markedly promoted the utilization of NH (4) (+) and NO (3) (-). At 50 degrees C, of total nitrogen originally provided, 24% was taken up into the cells and 20% remained in the culture supernatant. Residual nitrogen (56%) was probably removed into the atmosphere. The cell extracts contained enzymes involved in denitrification. GC-MS demonstrated that NH (4 ) (15) NO(3) had been converted to (15)N(2)O. These results indicate that the strain has denitrification ability under aerobic condition.

Adaptation of Pseudomonas sp. strain 7-6 to quaternary ammonium compounds and their degradation via dual pathways.

Pseudomonas sp. strain 7-6, isolated from active sludge obtained from a wastewater facility, utilized a quaternary ammonium surfactant, n-dodecyltrimethylammonium chloride (DTAC), as its sole carbon, nitrogen, and energy source. When initially grown in the presence of 10 mM DTAC medium, the isolate was unable to degrade DTAC. The strain was cultivated in gradually increasing concentrations of the surfactant until continuous exposure led to high tolerance and biodegradation of the compound. Based on the identification of five metabolites by gas chromatography-mass spectrometry analysis, two possible pathways for DTAC metabolism were proposed. In pathway 1, DTAC is converted to lauric acid via n-dodecanal with the release of trimethylamine; in pathway 2, DTAC is converted to lauric acid via n-dodecyldimethylamine and then n-dodecanal with the release of dimethylamine. Among the identified metabolites, the strain precultivated on DTAC medium could utilize n-dodecanal and lauric acid as sole carbon sources and trimethylamine and dimethylamine as sole nitrogen sources, but it could not efficiently utilize n-dodecyldimethylamine. These results indicated pathway 1 is the main pathway for the degradation of DTAC.

Metabolism of azo dyes by Lactobacillus casei TISTR 1500 and effects of various factors on decolorization.

Lactobacillus casei TISTR 1500 was isolated from soil of a dairy wastewater treatment plant and selected as the most active azo dye degrader of 19 isolates. Growing cells and freely suspended cells of this strain completely degraded methyl orange, thereby decolorizing the medium. The strain stoichiometrically converted methyl orange to N,N-dimethyl-p-phenylenediamine and 4-aminobenzenesulfonic acid, which were identified by HPLC, GC, and GC-MS analyses. The enzyme activity responsible for the cleavage of the azo bond of methyl orange was localized to the cytoplasm of cells grown on modified MRS medium containing methyl orange. The effect of sugars, oligosaccharides, organic acids, metal ions, pHs, oxygen and temperatures on methyl orange decolorization by freely suspended cells was investigated. The optimal conditions for the decolorization of methyl orange by the Lactobacillus casei TISTR 1500 are incubation at 35 degrees C and pH 6 with sucrose provided as the energy source.