Thermostability Improvement of L-Asparaginase from Acinetobacter soli via Consensus-Designed Cysteine Residue Substitution.

To extend the application range of L-asparaginase in food pre-processing, the thermostability improvement of the enzyme is essential. Herein, two non-conserved cysteine residues with easily oxidized free sulfhydryl groups, Cys8 and Cys283, of Acinetobacter soli L-asparaginase (AsA) were screened out via consensus design. After saturation mutagenesis and combinatorial mutation, the mutant C8Y/C283Q with highly improved thermostability was obtained with a half-life of 361.6 min at 40 °C, an over 34-fold increase compared with that of the wild-type. Its melting temperature (Tm) value reaches 62.3 °C, which is 7.1 °C higher than that of the wild-type. Molecular dynamics simulation and structure analysis revealed the formation of new hydrogen bonds of Gln283 and the aromatic interaction of Tyr8 formed with adjacent residues, resulting in enhanced thermostability. The improvement in the thermostability of L-asparaginase could efficiently enhance its effect on acrylamide inhibition; the contents of acrylamide in potato chips were efficiently reduced by 86.50% after a mutant C8Y/C283Q treatment, which was significantly higher than the 59.05% reduction after the AsA wild-type treatment. In addition, the investigation of the mechanism behind the enhanced thermostability of AsA could further direct the modification of L-asparaginases for expanding their clinical and industrial applications.

Designed symmetrical ß-hairpin peptides for treating multidrug-resistant salmonella typhimurium infections.

The rapid emergence and prevalence of multidrug-resistant salmonellosis lack effective therapies, which causes epidemic health problems and stimulates the development of antimicrobials with novel modes of action. In this research, 10 short symmetrical ß-hairpin peptides are synthesized by combining the ß-turn of Leucocin-A with recurring hydrophobic and cationic amino acid sequences. Those designed peptides exhibited potent antibacterial activities against drug-susceptible and drug-resistant Salmonella. One of the 10 peptides, WK2 ((WK)2CTKSGC(KW)2), displayed best cell selectivity towards Salmonella cells over macrophages and erythrocytes in a co-culture model. Fluorescent measurements and microscopic observations reflected that WK2 exerted its antimicrobial activity through a membrane-lytic mechanism. Moreover, the ß-hairpin peptides can bind to endotoxin (LPS) and suppress the production of LPS-induced proinflammatory cytokines in RAW264.7 cells, indicating as a potent anti-inflammatory activity. The preliminary in vivo studies can also demonstrate that WK2 decreased loads of Salmonella in the liver and spleen, mitigated Salmonella-caused inflammation and maintained the integrity of intestinal mucosal surfaces. Ultimately, the results highlight that WK2 is a promising therapeutic agent to prevent multidrug-resistant S. Typhimurium infections in humans and animals.

Anti-toxicogenic fungi and toxin-reducing effects of bacillomycin D in combination with fungicides.

Mycotoxins are toxic secondary metabolites produced by fungus including Aspergillus and Fusarium. They can contaminate food and cause major health issues. Bacillomycin D (BD) is a natural antimicrobial lipopeptide generated by Bacillus that has excellent antifungal capabilities, but its high price prevents it from being widely used. Chemically produced and essential oil-based fungicides are also currently the most frequent types. In the study, the effects of combining BD with two types of fungicides on the growth of toxicogenic fungi as well as the generation of deoxynivalenol (DON) and fumonisin B1 (FB1) were examined. It was discovered that BD was more effective in suppressing molds than the other two types of fungicides, and it could be combined with synthetic or essential oil-based fungicides to provide a synergistic or additive effect. BD 31.25 µg/mL + Thymol (Thy) 7.81 µg/mL and BD 11.45 µg/mL + Cinnamon oil (Cin) 3.90 µg/mL inhibited F. graminearum, respectively. The combination of BD+Thy and BD+Cin at this concentration considerably reduced 60%-80% spore germination, when DON dropped below 300 ng/L. Furthermore, both combinations suppressed F. moniliforme growth and FB1 synthesis in a dose-dependent manner at lower concentrations. At an action dose of 2 MIC, FB1 production might be reduced to less than 100 ng/L. Our findings indicated that BD might interact synergistically with various fungicides, suggesting that it could be useful in the field of antifungal and toxicity reduction in food.

Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential.

L-asparaginase (E.C.3.5.1.1) is a well-known agent that prevents the formation of acrylamide both in the food industry and against childhood acute lymphoblastic leukemia in clinical settings. The disadvantages of L-asparaginase, which restrict its industrial application, include its narrow range of pH stability and low thermostability. In this study, a novel L-asparaginase from Mycobacterium gordonae (GmASNase) was cloned and expressed in Escherichia coli BL21 (DE3). GmASNase was found to be a tetramer with a monomeric size of 32 kDa, sharing only 32% structural identity with Helicobacter pylori L-asparaginases in the Protein Data Bank database. The purified GmASNase had the highest specific activity of 486.65 IU mg-1 at pH 9.0 and 50 °C. In addition, GmASNase possessed superior properties in terms of stability at a wide pH range of 5.0-11.0 and activity at temperatures below 40 °C. Moreover, GmASNase displayed high substrate specificity towards L-asparagine with Km, kcat, and kcat/Km values of 6.025 mM, 11,864.71 min-1 and 1969.25 mM-1min-1, respectively. To evaluate its ability to mitigate acrylamide, GmASNase was used to treat potato chips prior to frying, where the acrylamide content decreased by 65.09% compared with the untreated control. These results suggest that GmASNase is a potential candidate for applications in the food industry.

Iturin A Induces Resistance and Improves the Quality and Safety of Harvested Cherry Tomato.

The soft rot disease caused by Rhizopus stolonifer is an important disease in cherry tomato fruit. In this study, the effect of iturin A on soft rot of cherry tomato and its influence on the storage quality of cherry tomato fruit were investigated. The results showed that 512 µg/mL of iturin A could effectively inhibit the incidence of soft rot of cherry tomato fruit. It was found that iturin A could induce the activity of resistance-related enzymes including phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), glucanase (GLU), and chitinase (CHI), and active oxygen-related enzymes including ascorbate peroxidases (APX), superoxide dismutases (SOD), catalases (CAT), and glutathione reductase (GR) of cherry tomato fruit. In addition, iturin A treatment could slow down the weight loss of cherry tomato and soften the fruit. These results indicated that iturin A could retard the decay and improve the quality of cherry tomato fruit by both the inhibition growth of R. stolonifera and the inducing the resistance.

Novel Bacillus Milk-Clotting Enzyme Produces Diverse Functional Peptides in Semihard Cheese.

Although rennet is one of the best choices for cheese manufacturing, its production cannot meet the growing demands of the cheese industry. Thus, new milk-clotting enzymes (MCEs) with similar or better properties as/than those of calf chymosin are needed urgently. Here, three MCEs, BY-2, BY-3, and BY-4, were mined by bioinformatic analysis and then expressed in and isolated from Escherichia coli. BY-4 had the highest milk-clotting activity/proteolytic activity (238.76) with enzyme properties similar to those of calf chymosin. BY-4 cheese had a composition, appearance, consistency/texture, and overall acceptability proximate to calf chymosin cheese. The EC50 values of peptides extracted from BY-4 cheese for 2,2-diphenyl-1-picrylhydrazyl inhibition (antioxidant property), angiotensin-converting enzyme inhibition (antihypertensivity), and growth inhibition of liver cancer cells (antitumor property) were found to be 81, 49, and 238 µg/mL, respectively, which were 2.35, 2.59, and 2.12 folds higher than those of calf chymosin cheese. These results indicated the potential of BY-4 as a supplement to calf chymosin in cheese manufacturing, especially for functional and health care purposes.

Biofilm Formation by Shiga Toxin-Producing Escherichia coli on Stainless Steel Coupons as Affected by Temperature and Incubation Time.

Forming biofilm is a strategy utilized by Shiga toxin-producing Escherichia coli (STEC) to survive and persist in food processing environments. We investigated the biofilm-forming potential of STEC strains from 10 clinically important serogroups on stainless steel at 22 °C or 13 °C after 24, 48, and 72 h of incubation. Results from crystal violet staining, plate counts, and scanning electron microscopy (SEM) identified a single isolate from each of the O113, O145, O91, O157, and O121 serogroups that was capable of forming strong or moderate biofilms on stainless steel at 22 °C. However, the biofilm-forming strength of these five strains was reduced when incubation time progressed. Moreover, we found that these strains formed a dense pellicle at the air-liquid interface on stainless steel, which suggests that oxygen was conducive to biofilm formation. At 13 °C, biofilm formation by these strains decreased (P < 0.05), but gradually increased over time. Overall, STEC biofilm formation was most prominent at 22 °C up to 24 h. The findings in this study identify the environmental conditions that may promote STEC biofilm formation in food processing facilities and suggest that the ability of specific strains to form biofilms contributes to their persistence within these environments.

Combined Effects and Cross-Interactions of Different Antibiotics and Polypeptides in Salmonella bredeney.

Salmonella spp. are health-threatening foodborne pathogens. The increasingly common spread of antibiotic-resistant Salmonella spp. is a major public healthcare issue worldwide. In this study, we wished to explore (1) antibiotic or polypeptide combinations to inhibit multidrug-resistant Salmonella bredeney and (2) the regulation of cross-resistance and collateral sensitivity of antibiotics and polypeptides. We undertook a study to select antibiotic combinations. Then, we promoted drug-resistant strains of S. bredeney after 15 types of antibiotic treatment. From each evolving population, the S. bredeney strain was exposed to a particular single drug. Then, we analyzed how the evolved S. bredeney strains acquired resistance or susceptibility to other drugs. A total of 105 combinations were tested against S. bredeney following the protocols of CLSI-2016 and EUCAST-2017. The synergistic interactions between drug pairings were diverse. Notably, polypeptides were more likely to be linked to synergistic combinations: 56% (19/34) of the synergistic pairings were relevant to polypeptides. Simultaneously, macrolides demonstrated antagonism toward polypeptides. The latter were more frequently related to collateral sensitivity than the other drugs because the other 13 drugs sensitized S. bredeney to polypeptides. In an experimental evolution involving 15 drugs, single drug-evolved strains were examined against the other 14 drugs, and the results were compared with the minimal inhibitory concentration of the ancestral strain. Single drug-evolved S. bredeney strains could alter the sensitivity to other drugs, and S. bredeney evolution against antibiotics could sensitize it to polypeptides.

Knockout of rapC Improves the Bacillomycin D Yield Based on De Novo Genome Sequencing of Bacillus amyloliquefaciens fmbJ.

Bacillus amyloliquefaciens, a Gram-positive and soil-dwelling bacterium, could produce secondary metabolites that suppress plant pathogens. In this study, we provided the whole genome sequence results of B. amyloliquefaciens fmbJ, which had one circular chromosome of 4 193 344 bp with 4249 genes, 87 tRNA genes, and 27 rRNA genes. In addition, fmbJ was found to contain several gene clusters of antimicrobial lipopeptides (bacillomycin D, surfactin, and fengycin), and bacillomycin D homologues were further comprehensively identified. To clarify the influence of rapC regulating the synthesis of lipopeptide on the yield of bacillomycin D, rapC gene in fmbJ was successfully deleted by the marker-free method. Finally, it was found that the deletion of rapC gene in fmbJ significantly improved bacillomycin D production from 240.7 ± 18.9 to 360.8 ± 30.7 mg/L, attributed to the increased the expression of bacillomycin D synthesis-related genes through enhancing the transcriptional level of comA, comP, and phrC. These results showed that the production of bacillomycin D in B. amyloliquefaciens fmbJ might be regulated by the RapC-PhrC system. The findings are expected to advance further agricultural application of Bacillus spp. as a promising source of natural bioactive compounds.

The antibacterial activity of LI-F type peptide against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and inhibition of infections in murine scalded epidermis.

LI-F type peptides are a family of cyclic lipodepsipeptide antibiotics isolated from Paenibacillus polymyxa and display potent activities against positive bacteria including methicillin-resistant S. aureus (MRSA). In this study, we investigated the mechanism of action of LI-F type peptide AMP-jsa9 against a MRSA (S. aureus CICC10790), which is resistant to ciprofloxacin, gentamicin, kanamycin, chloramphenicol, methicillin, and tetracycline. It was found that AMP-jsa9 mainly targets the cell membrane of MRSA and is able to inhibit biofilm formation through killing planktonic bacteria cells. Moreover, AMP-jsa9 can bind to DNA in vitro, which represents another pathway for the action on MRSA. Furthermore, in vivo treatment of scalded mice with AMP-jsa9 resulted in inhibiting MRSA infections and healing of the scalded wound. In addition, it was demonstrated that AMP-jsa9 can effectively inhibit MRSA infections in scalded murine epidermis and that inflammatory cytokines including IL-8, IL-6, tumor necrosis factor alpha (TNF-α), and monocyte chemotactic factor-1 (MCP-1) were reduced; moreover, both protein and gene expression levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (e-NOS) were enhanced, which promote neovascularization and proliferation of new granulation tissue.

Identification and characterization of Streptomyces flavogriseus NJ-4 as a novel producer of actinomycin D and holomycin.

This paper is the first public report that Streptomyces flavogriseus can produce both actinomycin D and holomycin. The actinomycete strain NJ-4 isolated from the soil of Nanjing Agricultural University was identified as S. flavogriseus. This S. flavogriseus strain was found for the first time to produce two antimicrobial compounds that were identified as actinomycin D and holomycin. GS medium, CS medium and GSS medium were used for the production experiments. All three media supported the production of actinomycin D, while holomycin was detected only in GS medium and was undetectable by HPLC in the CS and GSS media. The antimicrobial activity against B. pumilus, S. aureus, Escherichia coli, F. moniliforme, F. graminearum and A. niger was tested using the agar well diffusion method. Actinomycin D exhibited strong antagonistic activities against all the indicator strains. Holomycin exhibited strong antagonistic activities against B. pumilus, S. aureus and E. coli and had antifungal activity against F. moniliforme and F. graminearum but had no antifungal activity against A. niger. The cell viability was determined using an MTT assay. Holomycin exhibited cytotoxic activity against A549 lung cancer cells, BGC823 gastric cancer cells and HepG2 hepatocellular carcinoma cells. The yield of actinomycin D from S. flavogriseus NJ-4 was 960 mg/l. S. flavogriseus NJ-4 exhibits a distinct capability and has the industrial potential to produce considerable yields of actinomycin D under unoptimized conditions.

Mechanism of action of AMP-jsa9, a LI-F-type antimicrobial peptide produced by Paenibacillus polymyxa JSa-9, against Fusarium moniliforme.

LI-F type peptides (AMP-jsa9) are a group of cyclic lipodepsipeptides that exhibit broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi. We sought to assess the toxicity of AMP-jsa9 and the mechanism of AMP-jsa9 action against Fusarium moniliforme. AMP-jsa9 exhibited weak hemolytic activity and weak cytotoxicity at antimicrobial concentrations (32µg/ml). Confocal laser microscopy, SEM, and TEM indicated that AMP-jsa9 primarily targets the cell wall, plasma membrane, and cytoskeleton, increases membranepermeability, and enhances cytoplasm leakage (e.g., K+, protein). Quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) detected a total of 162 differentially expressed proteins (59 up-regulated and 103 down-regulated) following treatment of F. moniliforme with AMP-jsa9. AMP-jsa9 treatment also led to reductions in chitin, ergosterol, NADH, NADPH, and ATP levels. Moreover, fumonisin B1 expression and biosynthesis was suppressed in AMP-jsa9-treated F. moniliforme. Our results provide a theoretical basis for the application of AMP-jsa9 as a natural and effective antifungal agent in the agricultural, food, and animal feed industries.

iTRAQ-based proteomic analysis of LI-F type peptides produced by Paenibacillus polymyxa JSa-9 mode of action against Bacillus cereus.

LI-F type peptides (AMP-jsa9) produced by Paenibacillus polymyxa JSa-9 are a group of cyclic lipodepsipeptide antibiotics that exhibit a broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi, especially Bacillus cereus and Fusarium moniliforme. In this study, to better understand the antibacterial mechanism of AMP-jsa9 against B. cereus, the ultrastructure of AMP-jsa9-treated B. cereus cells was observed by both atomic force microscopy and transmission electron microscopy, and quantitative proteomic analysis was performed on proteins extracted from treated and untreated bacterial cells by using isobaric tag for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to access differentially expressed proteins. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis, including determinations of ATP, NAD(+)H, NADP(+)H, reactive oxygen species (ROS), the activities of catalase (CAT) and superoxide dismutase (SOD), and the relative expression of target genes by quantitative real-time PCR. Bacterial cells exposed to AMP-jsa9 showed irregular surfaces with bleb projections and concaves; we hypothesize that AMP-jsa9 penetrated the cell wall and was anchored on the cytoplasmic membrane and that ROS accumulated in the cell membrane after treatment with AMP-jsa9, modulating the bacterial membrane properties and increasing membrane permeability. Consequently, the blebs were formed on the cell wall by the impulsive force of the leakage of intercellular contents. iTRAQ-based proteomic analysis detected a total of 1317 proteins, including 176 differentially expressed proteins (75 upregulated (fold >2) and 101 downregulated (fold <0.5)). Based on proteome analysis, the putative pathways of AMP-jsa9 action against B. cereus can be summarized as: (i) inhibition of bacterial sporulation, thiamine biosynthesis, energy metabolism, DNA transcription and translation, and cell wall biosynthesis, through direct regulation of protein levels; and (ii) indirect effects on the same pathways through the accumulation of ROS and the consequent impairment of cellular functions, resulting from downregulation of antioxidant proteins, especially CAT and SOD. BIOLOGICAL SIGNIFICANCE: The mode of action of LI-F type antimicrobial peptides (AMP-jsa9) against B. cereus was elucidated at the proteomic level. Two pathways of AMP-jsa9 action upon B. cereus cells were identified and the mechanism of bleb formation on the surfaces of bacterial cells was predicted based on the results of ultrastructural observation and proteomic analysis. These results are helpful in understanding the mechanism of LI-F type peptides and in providing the theoretical base for applying AMP-jsa9 or its analogs to combat Gram-positive pathogenic bacteria in the food and feed industries.

Insights into the Antimicrobial Activity and Cytotoxicity of Engineered α-Helical Peptide Amphiphiles.

Antimicrobial peptides (AMPs) have gained increasing attention, as they can overcome recurring microbial invasions. However, their poor antimicrobial activity and potential cytotoxicity remain impediments to their clinical applications as novel therapeutic agents. To enhance the antimicrobial activity and cell selectivity of AMPs, a series of amphiphilic peptides based on leucocin A were designed by substituting noncharged hydrophilic residues with arginine and leucine. Of the engineered peptides, peptide 7 (WRL3) (WLRAFRRLVRRLARGLRR-NH2) exhibited the highest cell selectivity toward bacterial cells over erythrocytes and macrophages. Fluorescent measurements and microscopic observations demonstrated that 7 increased cell membrane permeability and disrupted membrane envelope integrity, and eventually led to whole cell lysis. Additionally, flow cytometry analysis and subcellular localization studies revealed that 7 showed potent cytotoxicity against human hepatoma cells HepG2. In summary, the data indicate that these engineered peptides, in particular 7, have enormous promise for antibacterial and/or antitumor therapeutics.

Control de las tasas de aireación y agitación en la producción de surfactina en un cultivo alimentado (fed-batch) en espuma desbordante con fermentación industrial / Control of agitation and aeration rates in the production of surfactin in foam overflowing fed-batch culture with industrial fermentation

Bacillus amyloliquefaciens fmb50 produces a high yield of surfactin, a lipopeptide-type biosurfactant that has been widely studied and has potential applications in many fields. A foam overflowing culture has been successfully used in the combined production-enrichment fermentation of surfactin. In this study, the agitation and aeration rates were found to have relationships with foam formation and surfactin enrichment. A maximum surfactin concentration of 4.7 g/l of foam was obtained after 21 h of culture with an agitation rate of 150 rpm and an aeration rate of 1 vvm in fed-batch culture. By controlling the foam overflow rate (f out) of a fed-batch culture, surfactin concentration in the foam was continuously maintained above 4 g/l.

Bacillus amyloliquefaciens fmb50 produce gran cantidad de surfactina, un biosurfactante de tipo lipopeptídico que ha sido objeto de estudios pormenorizados y tiene aplicaciones en muchos campos. El cultivo en espuma desbordante se ha utilizado con éxito en la fermentación combinada de producción-enriquecimiento de surfactina. En este estudio, se halló que las tasas de aireación y agitación tienen relación con la formación de espuma y el enriquecimiento de la surfactina. Se obtuvo una concentración máxima de surfactina de 4,7 g/l de espuma después de 21 h de cultivo con una tasa de agitación de 150 rpm y una tasa de aireación de 1 vvm en un cultivo alimentado (fed-batch). Al controlar la tasa de espuma desbordante (f out) de un cultivo fed-batch, la concentración de surfactina en la espuma se mantuvo continua por encima de 4 g/l.

Quantitative proteomic analysis reveals that anti-cancer effects of selenium-binding protein 1 in vivo are associated with metabolic pathways.

Previous studies have shown the tumor-suppressive role of selenium-binding protein 1 (SBP1), but the underlying mechanisms are unclear. In this study, we found that induction of SBP1 showed significant inhibition of colorectal cancer cell growth and metastasis in mice. We further employed isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins that were involved in SBP1-mediated anti-cancer effects in tumor tissues. We identified 132 differentially expressed proteins, among them, 53 proteins were upregulated and 79 proteins were downregulated. Importantly, many of the differentially altered proteins were associated with lipid/glucose metabolism, which were also linked to Glycolysis, MAPK, Wnt, NF-kB, NOTCH and epithelial-mesenchymal transition (EMT) signaling pathways. These results have revealed a novel mechanism that SBP1-mediated cancer inhibition is through altering lipid/glucose metabolic signaling pathways.

Effect of inulin on efficient production and regulatory biosynthesis of bacillomycin D in Bacillus subtilis fmbJ.

The effect of inulin on the production of bacillomycin D and the levels of mRNA of bacillomycin D synthetase genes: bmyA (BYA), bmyB (BYB), bmyC (BYC), the thioesterase gene (TE) and regulating genes: AbrB, ComA, DegU, PhrC, SigmaH and Spo0A in Bacillus subtilis fmbJ were investigated. The production of bacillomycin D was enhanced with the increase of biomass concentration. The maximum production and productivity of bacillomycin D were found to be 1227.49 mg/L and 10.23 mg/L h. Inulin significantly improved the expression of bacillomycin D synthetase genes: bmyA (BYA), bmyB (BYB), bmyC (BYC) and the thioesterase gene (TE). Also, inulin up-regulated ComA, DegU, SigmaH and Spo0A and therefore promoted the high production of bacillomycin D. Our results provided a practical approach for efficient production of bacillomycin D and a meaningful explanation for regulatory mechanism of bacillomycin D biosynthesis.

Mining and evaluation of new specific molecular targets for the PCR detection of Salmonella spp. genome.

The purpose of this study is to find new molecular targets for the detection of Salmonella. With the online BLAST Program, we compared homology of genomic sequences and specificity in GenBank among Salmonella serovars and non-Salmonella strains and found 98 Salmonella specific target sequences. We selected 33 target sequences of Gene ID from 3335000 to 3337003 for the specificity evaluation, and finally 8 specific fragments screened out, they are 3334138, 3335583, 3335471, 3335211, 3335068, 3336466, 3336736 and 3336998. Primer SC8 of gene 3335583 and SC9 of gene 3335471 were the best in specificity and sensitivity among these primers. The detection sensitivity of Primer SC9 was 1.23 fg/µl for DNA templates and 720 cfu/ml for whole cells, while primer SC8's was 12.3 fg/µl and 720 cfu/ml, respectively. Salmonella could be detected successfully by the PCR method developed in this study after 8 h enrichment when the milk samples were artificially contaminated by this organism at 7 cfu per 10 ml milk.

Degradation of triphenylmethane dyes using a temperature and pH stable spore laccase from a novel strain of Bacillus vallismortis.

The characterization of a spore laccase from Bacillus vallismortis fmb-103, isolated from textile industry disposal sites, is described. The activity was 6.5 U/g of dry spore with ABTS as the substrate. The enzyme was quite stable at high temperature. It retained more than 90% of its initial activity after 10h at 70 °C. The enzyme demonstrated broad pH stability in both acidic and alkaline conditions. There was almost no activity loss at pH 3 over an extended period of time, and the relative activity remained at 82% and 38% at pH 7 and pH 9 after 10 days. NaN(3), SDS, L-cysterine, Dithiothreitol, EDTA and NaCl inhibit the enzyme activity. Triphenylmethane dyes, including malachite green, brilliant green and aniline blue were efficiently degraded by the enzyme after 24h in combination with a mediator with efficiencies of 76.84%, 96.56% and 81.17%, respectively. The reusability of spore laccase for decolorization dyes was also examined.

Gene cloning, expression, and characterization of a novel acetaldehyde dehydrogenase from Issatchenkia terricola strain XJ-2.

Acetaldehyde is a known mutagen and carcinogen. Active aldehyde dehydrogenase (ALDH) represents an important mechanism for acetaldehyde detoxification. A yeast strain XJ-2 isolated from grape samples was found to produce acetaldehyde dehydrogenase with a high activity of 2.28 U/mg and identified as Issatchenkia terricola. The enzyme activity was validated by oxidizing acetaldehyde to acetate with NAD(+) as coenzyme based on the headspace gas chromatography analysis. A novel acetaldehyde dehydrogenase gene (ist-ALD) was cloned by combining SiteFinding-PCR and self-formed adaptor PCR. The ist-ALD gene comprised an open reading frame of 1,578 bp and encoded a protein of 525 amino acids. The predicted protein of ist-ALD showed the highest identity (73%) to ALDH from Pichia angusta. The ist-ALD gene was expressed in Escherichia coli, and the gene product (ist-ALDH) presented a productivity of 442.3 U/mL cells. The purified ist-ALDH was a homotetramer of 232 kDa consisting of 57 kDa-subunit according to the SDS-PAGE and native PAGE analysis. Ist-ALDH exhibited the optimal activity at pH 9.0 and 40°C, respectively. The activity of ist-ALDH was enhanced by K(+), NH4(+), dithiothreitol, and 2-mercaptoethanol but strongly inhibited by Ag(+), Hg(2+), Cu(2+), and phenylmethyl sulfonylfluoride. In the presence of NAD(+), ist-ALDH could oxidize many aliphatic, aromatic, and heterocyclic aldehydes, preferably acetaldehyde. Kinetic study revealed that ist-ALDH had a k (cat) value of 27.71/s and a k (cat)/K (m) value of 26.80 × 10(3)/(mol s) on acetaldehyde, demonstrating ist-ALDH, a catalytically active enzyme by comparing with other ALDHs. These studies indicated that ist-ALDH was a potential enzymatic product for acetaldehyde detoxification.