Improvement of the halotolerance of a Bacillus serine protease by protein surface engineering.

A moderately halotolerant serine protease was previously isolated from Bacillus subtilis from salted, fermented food. Eight mutation sites on the protein surface were selected for protein engineering based on sequence and structural comparisons with moderately halotolerant proteases and homologous non-halotolerant proteases. The newly constructed multiple mutants with substituted Asp and Arg residues were compared with the recombinant wild type (rApr) and the previously constructed mAla-8 substituted with Ala to analyze the contribution of protein surface charge to the salt adaptation of the protease. The three mutants showed >1.2-fold greater halotolerance than rApr. In addition, the mutants showed a broader range of pH stability than rApr, retaining >80% of their maximum activity in the pH range 5.0-11. The mutants also retained >75% of their activity after incubation for 1 h at pH 8.0 and 55°C or at pH 11.5 and 25°C. The Asp and Arg residues exchanged by multiple substitution probably played a role in increasing protein surface hydration and solubility in high salt conditions. This study illustrated that increasing a high proportion of the negative or positive charge on the surface of the Bacillus serine protease stably improved the protein's salt adaptation.

Decolorization of Reactive Red 159 by a consortium of photosynthetic bacteria using an anaerobic sequencing batch reactor (AnSBR).

This experiment aimed to decolorize Reactive Red 159 using a high potential of a consortium of purple nonsulfur bacteria (PNSB) with an application of response surface methodology through a central composite design in open system. The three factors of hydraulic retention time (HRT), sludge retention time (SRT) and dye concentration were applied to the design. The decolorization was operated in an anaerobic sequencing batch reactor until the system reached to a pseudosteady state for 30 cycles in each experiment. The optimal condition was 6,500 mg/L of Reactive Red 159 concentration with 20 days of SRT and 8 days of HRT, achieving dye effluent of 142.62 ± 5.35 mg/L, decolorization rate of 264.54 ± 7.13 mg/L/h and decolorization efficiency of 97.68 ± 0.74%. The results revealed that PNSB efficiently decolorized the high concentration of Reactive Red 159 and they were a high potential of microorganisms for dyes contaminated wastewater treatment.

Characterization and mutation analysis of a halotolerant serine protease from a new isolate of Bacillus subtilis.

OBJECTIVES: A bacterial halotolerant enzyme was characterized to understand the molecular mechanism of salt adaptation and to explore its protein engineering potential. RESULTS: Halotolerant serine protease (Apr_No16) from a newly isolated Bacillus subtilis strain no. 16 was characterized. Multiple alignments with previously reported non-halotolerant proteases, including subtilisin Carlsberg, indicated that Apr_No16 has eight acidic or polar amino acid residues that are replaced by nonpolar amino acids in non-halotolerant proteases. Those residues were hypothesized to be one of the primary contributors to salt adaptation. An eightfold mutant substituted with Ala residues exhibited 1.2- and 1.8-fold greater halotolerance at 12.5% (w/v) NaCl than Apr_No16 and Carlsberg, respectively. Amino acid substitution notably shifted the theoretical pI of the eightfold mutant, from 6.33 to 9.23, compared with Apr_No16. The resulting protein better tolerated high salt conditions. CONCLUSIONS: Changing the pI of a bacterial serine protease may be an effective strategy to improve the enzyme's halotolerance.

Extracellular protease derived from lactic acid bacteria stimulates the fermentative lactic acid production from the by-products of rice as a biomass refinery function.

A lactic acid producing bacterium, Lactobacillus rhamnosus M-23, newly isolated from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. This strain has the ability to utilize the non-sterilized rice washing drainage and rice bran as a source of carbohydrate, saccharifying enzymes and nutrients for lactic acid production. Observation of extracellular protease activity in SSF culture broth showed that a higher protease activity was present in strain M-23 than in other isolated lactic acid producing bacteria (LABs). To investigate the structural changes of solid particles of rice washing drainage throughout LAB cultivation, scanning electron microscopic (SEM) observation and Fourier transform infrared-spectroscopy (FT-IR) analysis were performed. The results of the SEM observation showed that the surface material could be removed from solid particles of rice washing drainage treated by culture broth (supernatant) of strain M-23, thus exposing the crystal structure of the starch particle surface. The results of the FT-IR analysis revealed that the specific transmittance decrease of the CC and CO stretching and OH group of the solid particles of the rice washing drainage were highly correlated with the produced lactic acid concentration and extracellular protease activity, respectively. These results demonstrate the high lactic acid producing ability of strain M-23 from a non-sterilized mixture of rice washing drainage and rice bran under the SSF condition due to the removal of proteinaceous material and exposure of the starch particle surface by extracellular protease.

Eco-friendly processing in enzymatic xylooligosaccharides production from corncob: Influence of pretreatment with sonocatalytic-synergistic Fenton reaction and its antioxidant potentials.

Delignification can be considered as a feasible process to pretreat lignocellulosic biomass in xylooligosaccharides production after the performance and efficiency has been improved through a few modifications. This study compared various pretreatment strategies such as Fenton, sonocatalytic, and sonocatalytic-synergistic Fenton employed on corncob in order to expose lignin content and saccharides to enhance the xylooligosaccharides yield by enzymatic hydrolysis. The dissolution of lignin and xylooligosaccharides production of corncob was enhanced by ultrasound assisted TiO2 and Fenton reaction. The corncob pretreated with a sonocatalytic-synergistic Fenton reaction gave the highest release of the lignin concentration level (1.03 g/L), dissolution level (80.25%), and xylooligosaccharides content (46.45 mg/g substrate). A two-step pretreatment processes consisting of the alkali treatment (pretreatment) and sonocatalytic-synergistic Fenton process (posttreatment) illustrated that subsequent enzymatic hydrolysis could be enhanced considerably. The release of the lignin concentration and xylooligosaccharides content were 33.20 g/L and 174.81 mg/g substrate, respectively. The antioxidant potential of xylooligosaccharides showed significant differences regarding the amount of xylooligosaccharides and the phenolic compounds produced.

Treatability of cheese whey for single-cell protein production in nonsterile systems: Part I. Optimal condition for lactic acid fermentation using a microaerobic sequencing batch reactor (microaerobic SBR) with immobilized Lactobacillus plantarum TISTR 2265 and microbial communities.

Cheese whey contains a high organic content and causes serious problems if it is released into the environment when untreated. This study aimed to investigate the optimum condition of lactic acid production using the microaerobic sequencing batch reactor (microaerobic SBR) in a nonsterile system. The high production of lactic acid was achieved by immobilized Lactobacillus plantarum TISTR 2265 to generate an acidic pH condition below 4.5 and then to support single-cell protein (SCP) production in the second aerobic sequencing batch reactor (aerobic SBR). A hydraulic retention time (HRT) of 4 days and a whey concentration of 80% feeding gave a high lactic acid yield of 12.58 g/L, chemical oxygen demand (COD) removal of 62.38%, and lactose utilization of 61.54%. The microbial communities in the nonsterile system were dominated by members of lactic acid bacteria, and it was shown that the inoculum remained in the system up to 330 days.

Treatability of cheese whey for single-cell protein production in nonsterile systems: Part II. The application of aerobic sequencing batch reactor (aerobic SBR) to produce high biomass of Dioszegia sp. TISTR 5792.

This study aimed to investigate the efficiency of an aerobic sequencing batch reactor (aerobic SBR) in a nonsterile system using the application of an experimental design via central composite design (CCD). The acidic whey obtained from lactic acid fermentation by immobilized Lactobacillus plantarum sp. TISTR 2265 was fed into the bioreactor of the aerobic SBR in an appropriate ratio between acidic whey and cheese whey to produce an acidic environment below 4.5 and then was used to support the growth of Dioszegia sp. TISTR 5792 by inhibiting bacterial contamination. At the optimal condition for a high yield of biomass production, the system was run with a hydraulic retention time (HRT) of 4 days, a solid retention time (SRT) of 8.22 days, and an acidic whey concentration of 80% feeding. The chemical oxygen demand (COD) decreased from 25,230 mg/L to 6,928 mg/L, which represented a COD removal of 72.15%. The yield of biomass production and lactose utilization by Dioszegia sp. TISTR 5792 were 13.14 g/L and 33.36%, respectively, with a long run of up to 180 cycles and the pH values of effluent were rose up to 8.32 without any pH adjustment.

Characterization of the native form and the carboxy-terminally truncated halotolerant form of α-amylases from Bacillus subtilis strain FP-133.

Two amylases, amylase I and amylase II from Bacillus subtilis strain FP-133, were purified to homogeneity and characterized. Their stabilities toward temperature, pH, and organic solvents, and their substrate specificities toward polysaccharides and oligosaccharides were similar. Under moderately high salt conditions, both amylases were more stable than commercial B. licheniformis amylase, and amylase I retained higher amylase activity than amylase II. The N-terminal amino acid sequence, genomic southern blot analysis, and MALDI-TOFF-MS analysis indicated that the halotolerant amylase I was produced by limited carboxy-terminal truncation of the amylase II peptide. The deduced amino acid sequence of amylase II was >95% identical to that of previously reported B. subtilis α-amylases, but their carboxy-terminal truncation points differed. Three recombinant amylases--full-length amylase corresponding to amylase II, an artificially truncated amylase corresponding to amylase I, and an amylase with a larger artificial C-terminal truncation--were expressed in B. subtilis. The artificially truncated recombinant amylases had the same high amylase activity as amylase I under moderately high salt conditions. Sequence comparisons indicated that an increased ratio of Asp/Glu residues in the enzyme may be one factor responsible for increasing halotolerance.

Exopolysaccharides from lactic acid bacteria: structural analysis, molecular weight effect on immunomodulation.

Exopolysaccharides (EPS) obtained from the culture medium of Lactobacillus confusus TISTR 1498 were investigated to determine their molecular characteristics and the effect of molecular weight (Mw) on immunomodulatory activity. The EPS mainly consisted of carbohydrates (81.9±2.4%) with only one type of monosaccharide, D-glucose, which was mostly connected by α-(1→6) glycosidic linkages. The EPS itself was unable to stimulate RAW264.7 cells to produce pro-inflammatory mediator nitric oxide (NO) and cytokines. However, considerable stimulation of RAW264.7 cells was observed by the low Mw of EPSs having Mw values≤70×10(3)g/mol. The partially hydrolyzed EPS stimulated RAW264.7 cells to induce considerable NO and various cytokine production such as TNF-α, IL-1ß, IL-6 and IL-10 via up-regulation of their mRNA expression. In addition, the degradation Iκ-B and the phosphorylation of c-Jun NH2-terminal kinase (JNK) were facilitated by BW-30 and MW-40, suggesting that the partially hydrolyzed EPS stimulated RAW264.7 cells through the activation of NF-κB and JNK pathways.

Anticholinesterase and antioxidant activities of fucoxanthin purified from the microalga Phaeodactylum tricornutum.

Hot extraction with acetone was the most efficient method for the extraction of fucoxanthin from Phaeodactylum tricornutum. The purified compound resulted in three main peaks consisted of the trans form along with two isomers. The structure of microalgal fucoxanthin was similar to that of brown seaweed, but the ratio of trans- to cis-form was different. The ratio of the cis-form increased as the extraction temperature increased. Fucoxanthin was unstable at high temperature, in acidic condition, and after long period of storage. Fucoxanthin exhibited strong activity against BChE, with an IC50 value of 1.97 mM and mixed inhibition type, whereas it had weak activity against AChE. The IC50 value on reducing power was 0.01 mM, which was much stronger than those of the positive controls. When the amount of cis-isomer increased by 2%, the scavenging activity against DPPH, hydrogen peroxide, superoxide anion, and reducing power decreased by 21.0, 10.3, 16.0, and 19.7%, respectively. Therefore, fucoxanthin could be a useful approach for Alzheimer's disease treatment.

Strategies to decolorize high concentrations of methyl orange using growing cells of Lactobacillus casei TISTR 1500.

Batch, fed-batch, and continuous fermentation was used in the processing of methyl orange decolorization using growing cells of Lactobacillus casei TISTR 1500. This report presents the optimal conditions for methyl orange decolorization by the strain TISTR 1500 in modified MRS via a central composite design (CCD) experiment. In particular, the highest decolorization efficiencies were obtained with 13.41 g/L of meat extract, and with 10.89 g/L of yeast extract at pH 6.88 at 35 °C. Under the optimal conditions, the rate of decolorization increased to 322% of that obtained for un-optimized MRS medium. The high concentration of methyl orange (5 g/L) was completely degraded within 9 h in batch fermentation. The total methyl orange load with 8.075 g/L was also decolorized in fed-batch fermentation within 13 h, and the biomass of the strain dramatically decreased after an incubation time of 8 h due to a shortage of sucrose. In the continuous system with a dye-loading rate of 600 mg/L/h and a total of loaded azo dye of 7.2 g/L, high efficiency of methyl orange removal was significantly high, at 98%.

Optimization of exopolysaccharide overproduction by Lactobacillus confusus in solid state fermentation under high salinity stress.

It is believed that high concentrations of sodium chloride (NaCl) suppress the biosynthesis of exopolysaccharide (EPS) in lactic acid bacteria (LAB). Nevertheless, overproduction of EPSs due to high salinity stress in solid state fermentation performed on an agar surface was demonstrated in this study using a response surface methodology via a central composite design (CCD). Under optimized conditions with NaCl 4.97% and sucrose 136.5 g/L at 40.79 h of incubation, the EPS yield was 259% (86.36 g/L of EPS), higher than the maximum yield produced with the modified MRS medium containing only 120 g/L of sucrose without NaCl (33.4 g/L of EPS). Biosynthesis of EPS by Lactobacillus confusus TISTR 1498 was independent of biomass production. Our results indicated that high salinity stress can enhance EPS production in solid state fermentation.

Optimization of cellulase-free xylanase production by thermophilic Streptomyces thermovulgaris TISTR1948 through Plackett-Burman and response surface methodological approaches.

Cellulase-free xylanase production by thermophilic Streptomyces thermovulgaris TISTR1948 was cultivated in a basal medium with rice straw as sole source of carbon and as an inducible substrate. Variable medium components were selected in accordance with the Plackett-Burman experimental design. The optimization conditions of physical factors (pH and temperature levels) were then combined in further studies through the response surface methodology approach. Only two significant components, rice straw and yeast extract, were chosen for the optimization studies. A second-order quadratic model was constructed by central composite design (CCD). The model revealed that both pH and temperature levels were significant, and were dependent on xylanase production. Under these experimental designs, the xylanase yield increased from 51.11 to 274.49 U/mL (3,400 to 10,000 U/g of rice straw) or about 537% higher than an unoptimized basal medium. The optimum conditions to achieve maximum yield of xylanase were 27.45 g/L of rice straw and 5.42 g/L of yeast extract under relatively neutral conditions of pH 7.11, 50.03 °C, and a incubation period.

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.

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.