Optimal Secretory Expression of Acetaldehyde Dehydrogenase from Issatchenkia terricola in Bacillus subtilis through a Combined Strategy

Acetaldehyde dehydrogenases are potential enzyme preparations that can be used to detoxify acetaldehyde and other exogenous aldehydes from pharmaceuticals, food, and biofuel production. In this study, we enhanced the expression of acetaldehyde dehydrogenase sourced from Issatchenkia terricola (istALDH) in Bacillus subtilis using a combinatorial strategy for the optimization of signal peptides, promoters, and growth conditions. First, a library of various signal peptides was constructed to identify the optimal signal peptides for efficient istALDH secretion. The signal peptide yqzG achieved the highest extracellular istALDH activity (204.85 ± 3.31 U/mL). Second, the aprE promoter was replaced by a constitutive promoter (i.e., P43) and an inducible promoter (i.e., Pglv), resulting in 12.40% and 19.97% enhanced istALDH, respectively. Furthermore, the tandem promoter P43-Pglv provided a better performance, resulting in 30.96% enhanced istALDH activity. Third, the production of istALDH was optimized by testing one factor at a time. Physical parameters were optimized including the inducer (e.g., maltose) concentrations, incubation temperatures, and inoculation amounts, and the results were 2.0%, 35 °C, and 2.0%, respectively. The optimized medium results were 2.0% glucose, 1.5% peptone, 2.5% yeast extract, 1% NaCl, and 0.5% (NH₄)₂SO₄. The extracellular istALDH activity was 331.19 ± 4.19 U/mL, yielding the highest production reported in the literature to date.     

Production and Purification of Pectinase from Bacillus subtilis 15A-B92 and Its Biotechnological Applications

Enzymes that degrade pectin are called pectinases. Pectinases of microbial origin are used in juice clarification as the process is cost-effective. This study screened a pectinase-producing bacterium isolated from soil and identified as Bacillus subtilis 15A B-92 based on the 16S rRNA molecular technique. The purified pectinase from the isolate showed 99.6 U/mg specific activity and 11.6-fold purity. The molecular weight of the purified bacterial pectinase was 14.41 ± 1 kD. Optimum pectinase activity was found at pH 4.5 and 50 °C, and the enzyme was 100% stable for 3.5 h in these conditions. No enzymatic inhibition or activation effect was seen with Fe²⁺, Ca²⁺, or Mg²⁺. However, a slight inhibition was seen with Cu²⁺, Mn²⁺, and Zn²⁺. Tween 20 and 80 slightly inhibited the pectinase, whereas iodoacetic acid (IAA), ethylenediaminetetraacetate (EDTA), urea, and sodium dodecyl sulfate (SDS) showed potent inhibition. The bacterial pectinase degraded citrus pectin (100%); however, it was inactive in the presence of galactose. With citrus pectin as the substrate, the Km and Vmax were calculated as 1.72 mg/mL and 1609 U/g, respectively. The high affinity of pectinase for its substrate makes the process cost-effective when utilized in food industries. The obtained pectinase was able to clarify orange and apple juices, justifying its application in the food industry.     

Purification of Bioactive Lipopeptides Produced by Bacillus subtilis Strain BIA

Bacillus subtilis strain BIA was used for the production of bioactive lipopeptides. Different extraction and purificationmethods were assayed as liquid–liquid extraction, and acid and ammonium sulfate precipitation followed by TLC, SPE, and gel filtration. Active fractions were further purified using RP-HPLC. The molecular mass of the purified product from HPLC was determined through Tris-Tricine SDS-PAGE and MALDI–TOF-MS. The results revealed that Bacillus subtilis strain BIA produced surfactin and iturin like compounds. Coproduction of surfactin and iturin like compounds by this strain is a remarkable trait for a potential biocontrol agent. This paper also includeds techniques that have been developed for the optimal and convenient extraction of bioactive lipopeptides from microbial origin.

     

Purification and characterization of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis.

Bacillus subtilis pyrrolidone carboxyl peptidase (Pcp) overexpressed in Escherichia coli was purified to homogeneity in less than 12 h using ammonium sulphate precipitation and hydrophobic interaction chromatography. The enzyme, which removes amino-terminal L-pyroglutamic acid from peptides, appears to be a tetramer of 25,200 molecular mass subunits. The protein cross-reacted with polyclonal antibodies raised against Pcp from Streptococcus pyogenes. The overexpressed enzyme exhibits an absolute substrate specificity towards N-terminal pyroglutamyl residues with a Michaelis constant of 1.04 mM for L-pyroglutamyl-beta-naphthylamide. The enzyme could be used for the removal of pyroglutamyl residues that block amino termini of proteins and peptides before performing Edman sequential degradation.     

Excited State Photoreaction between the Indole Side Chain of Tryptophan and Halocompounds Generates New Fluorophores and Unique Modifications

Photoreaction of indole containing compounds with chloroform and other trichlorocompounds generates products with redshifted fluorescence. In proteins, this reaction can be used for the fluorescent detection of proteins. Little characterization of products generated through the photochemical reaction of indoles with halocompounds has been done, yet is fundamental for the development of other fluorophores, protein labeling agents, and bioactive indole derivatives. Here, we have characterized which isomers form in the photoreaction between tryptophan and chloroform using ¹H‐NMR of tryptophan and methylated derivatives to reveal that the two major products that are formed result from modification at the 4‐ and 6‐carbon positions of the indole ring. Reaction at position 6 generates 6‐formyl tryptophan and the reaction at position 4 generates an imine because the formyl derivative that is initially formed reacts further with the tryptophan amine group. The spectroscopic properties and product molecular weights of photoproducts formed from photoreaction of tryptophan with other trihalo and monohalocompounds are also determined. The indole ring of tryptophan can be modified with various additions from halocompounds, including the addition of labels to the indole ring via methylene groups. This opens possibilities for generating novel tryptophan based fluorophores and protein labeling strategies using this photochemistry.     

Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus

The aim of this experimental study was to isolate and partially purify protease enzyme from Bacillus cereus and Bacillus subtilis. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from B. subtilis was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from B. cereus was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.     

Chemical Modifications of Laccase from White-Rot Basidiomycete Cerrena unicolor

Laccases belong to the group of phenol oxidizes and constitute one of the most promising classes of enzymes for future use in various fields. For industrial and biotechnological purposes, laccases were among the first enzymes providing larger-scale applications such as removal of polyphenols or conversion of toxic compounds. The wood-degrading basidiomycete Cerrena unicolor C-139, reported in this study, is one of the high-laccase producers. In order to facilitate novel and more efficient biocatalytic process applications, there is a need for laccases with improved biochemical properties, such as thermostability or stability in broad ranges of pH. In this work, modifications of laccase isoforms by hydrophobization, hydrophilization, and polymerization were performed. The hydrophobized and hydrophilized enzyme showed enhanced surface activity and higher ranges of pH and temperatures in comparison to its native form. However, performed modifications did not appear to noticeably alter enzyme??s native structure possibly due to the formation of coating by particles of saccharides around the molecule. Additionally, surface charge of modified laccase shifted towards the negative charge for the hydrophobized laccase forms. In all tested modifications, the size exclusion method led to average 80?% inhibition removal for hydrophilized samples after an hour of incubation with fluoride ions. Samples that were hydrophilized with lactose and cellobiose showed an additional 90?% reversibility of inhibition by fluoride ions after an hour of concluding the reaction and 40?% after 24?h. The hydrophobized laccase showed higher level of the reversibility after 1?h (above 80?%) and 24?h (above 70?%) incubation with fluoride ions. The addition of ascorbate to laccase solution before a fluoride spike resulted in more efficient reversibility of fluoride inhibitory effect in comparison to the treatments with reagents used in the reversed sequence.     

Characterization of Novel EGs Reconstructed from Bacillus subtilis Endoglucanase

Bacterial cellulases have taken on satisfactory application performance and economic value in detergent industry. Neutral endoglucanase (EG1) gene was cloned from Bacillus subtilis and expressed Pichia pastoris in our previous study. Redesigned endoglucanases enhanced cellulase domain, added and deleted carbohydrate-binding module (CBM), named EG2, EG3, and EG4, respectively, were constructed in this study. The redesigned EG genes were expressed in P. pastoris, and their characters were also discussed. The optimal temperature and pH value of the all EGs was 65 °C and 6.0, respectively, where their enzymatic activities in P. pastoris cultivation supernatant reached 867, 651, 966, and 881 U/mL. EG2 showed 24.9 % enzymatic activity loss compared to natural endoglucanase. EG4 showed specific activity 30 % loss and thermostability decrease compared to EG1, which suggested CBM played an important role in improving the catalytic power and heat stability of cellulase family which attached. The specific activity of EG2 and EG3 showed similar to EG1, which suggested neither enhancement of CD nor CBM to endoglucanase can improve its catalytic power, which might rest with its intact topologic structure.     

Identification of Antifungal Peptides from Bacillus subtilis BS-918

Bacillus subtilis BS-918 shows strong activity against a broad spectrum of plant and postharvest pathogenic fungi. Antifungal compounds produced by BS-918 were isolated by extraction and gel chromatography, and structural identification was performed by electrospray ionization coupled with collision induced dissociation mass spectrometry. Two kinds of precursor ions belonging to iturin and fengycin were revealed. The first precursor ion at m/z 1071.7 was analyzed by collision induced dissociation mass spectrometry and was identified to be iturin A with a C₁₆ β-amino fatty acid chain. Collision induced dissociation mass spectrometry of the second precursor showed the presence of two fengycins. Ions at m/z 1449.5 and m/z 1463.9 were identified to homologs of fengycin A with C₁₅ and C₁₆ β-hydroxy fatty acid chains, whereas ions at m/z 1477.8 and m/z 1491.9 were homologs of fengycin B with C₁₅ and C₁₆ fatty acid chains.     

Partial purification and characterization of protease enzyme from Bacillus subtilis megatherium

Bacteria of genus Bacillus are active producers of extracellular proteases, and characteristics of enzyme production by Bacillus species have been well studied. The aim of this experimental study is isolation and partial purification of protease enzyme from the Bacillus subtilis megatherium bacteria species. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species on suitable media. The partial purification was reali-zed by applying successively ammonium sulfate precipitation, dialysis, DEAE-cellulose ion exchange chromatography to the supernatant. In this study, the effect of substrate concentration, reaction time, the effect of inhibitor and activator on the optimum pH, optimum temperature, pH stability, and temperature stability was determined. Molecular weight of the obtained enzyme was investigated by SDS-PAGE. In this study, the specific activity of the supernatant, which was partially purified from Bacillus subtilis megatherium bacteria, was 10.4 U/mg, specific activity of supernatant was 13.5 U/mg after 80% ammonium sulfate fractionation. The final enzyme preparation was 1.1-fold purer than the crude homogenate. Molecular weight of the protease was determined, and it was found that the weight of enzyme was 45 kDa by using SDS-PAGE.     

定向进化提高枯草芽孢杆菌脂肪酶的活力

通过定向进化的方法, 提高了一个新的源于枯草芽孢杆菌 (Bacillus subtilis) 的脂肪酶 (BSL2) 的活力. 经过两轮易错 PCR 以及高通量筛选, 最终获得了比活力为野生出发酶 4.5 倍的突变体 3-1B2. 基因比对结果表明, 共有两个氨基酸发生了突变. 突变酶的酶学性质研究发现, 与野生酶相比, 它的热稳定性及 pH 稳定性略有增加, 最适温度和最适 pH 值则无太大的变化. 同源模建了 BSL2 与 3-1B2 的结构, 并与底物进行了分子对接. 结果表明, 突变体 3-1B2 的结合能比野生型 BSL2 低 1.29 kcal/mol, 活性中心 Ser77 残基与底物羰基的距离也由 0.319 nm 减小为 0.278 nm, 因而加快了酶反应速度, 提高了酶活力.     

Purification of Alcohol Dehydrogenase from Saccharomyces cerevisiae Using Magnetic Dye-Ligand Affinity Nanostructures

Reactive Green 19 was covalently immobilized onto magnetic nanostructures for purification of alcohol dehydrogenase from Saccharomyces cerevisiae. The Reactive Green 19 immobilized magnetic nanostructures were characterized by Fourier transform infrared spectroscopy, electron spin resonance, atomic force microscope, and energy dispersive X-ray analysis. Particle size of nanostructures was found to be roughly 70 nm. Alcohol dehydrogenase adsorption experiments were investigated under different conditions in batch system (i.e., medium pH, alcohol dehydrogenase concentration, temperature, and ionic strength). Maximum alcohol dehydrogenase adsorption capacity was found to be 176.09 mg/g polymer while nonspecific alcohol dehydrogenase adsorption onto plain magnetic nanostructures was negligible (19.4 mg/g polymer). Alcohol dehydrogenase molecules were desorbed by using 1.0 M NaCl with 98.4 % recovery. Alcohol dehydrogenase from S. cerevisiae was purified 45.63-fold in single step with dye-immobilized magnetic nanostructures, and purity of alcohol dehydrogenase was shown by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

亲和层析分离纯化乳酸脱氢酶

以壳聚糖为载体,戊二醛为交联剂,氨基三乙酸为配基,制备固载铜离子的金属螯合亲和吸附剂,层析分离纯化乳酸脱氢酶(LDH),收得率为23.3％,纯化倍数达3.1.实验结果表明该亲和层析固定相对LDH有较高的分离纯化选择性.     

The Catalytic Property of 3-Hydroxyisobutyrate Dehydrogenase from Bacillus cereus on 3-Hydroxypropionate

The MmsB gene product from Bacillus cereus ATCC14579 exhibits 3-hydroxypropionate dehydrogenase activity. It encodes the 32-kDa enzyme protein composed of 292 amino acids. Recombinant 3-hydroxyisobutyrate dehydrogenase (3-HIBADH) was purified 100-fold from cell extract by ammonium sulfate fractionation and column chromatography. The enzyme catalyzed oxidation of 3-hydroxypropionate (3-HP) between pH?7.0 and 10.0 with optimal activity between 8.8 and 9.0. A Km of 16.8 mM for 3-HP was calculated from a Lineweaver–Burk plot. The semialdehyde as products has been proven by spectrophotometric determination. The dehydrogenase apparently has no metal ion requirement. Kinetic determinations established that 3-HIBADH was more active with NADP⁺ than NAD⁺, which did not show similarity with previously reported 3-HIBADH except that from Thermus thermophilus.     

香紫苏醇侧链的氧化降解

研究了高锰酸钾、高硼酸钠、高碘酸钠等对香紫苏醇侧链的氧化降解，并通过 IR，NMR，MS或元素分析等技术对氧化降解产物的结构进行了表征。