Fermentation of millet bran with Bacillus natto: enhancement of bioactivity levels and the bioactivity of bran extract.

BACKGROUND: Millet bran (MB), a byproduct of millet production, is rich in functional components but it is underutilized. In recent years, researchers have shown that fermentation can improve the biological activity of cereals and their byproducts. This study used Bacillus natto to ferment millet bran to improve its added value and broaden the application of MB. The bioactive component content, physicochemical properties, and functional activity of millet bran extract (MBE) from fermented millet bran were determined. RESULTS: After fermentation, the soluble dietary fiber (SDF) content increased by 92.0%, the ß-glucan content by 164.4%, the polypeptide content by 111.4%, the polyphenol content by 32.5%, the flavone content by 16.4%, and the total amino acid content by 95.4%. Scanning electron microscopy revealed that the microscopic morphology of MBE changed from complete and dense blocks to loosely porous shapes after fermentation. After fermentation, the solubility, water-holding capacity, and viscosity significantly increased and the particle size decreased. Moreover, the glucose adsorption capacity (2.1 mmol g-1), glucose dialysis retardation index (75.3%), and α-glucosidase inhibitory (71.4%, mixed reversible inhibition) activity of the fermented MBE (FMBE) were greater than those of the unfermented MBE (0.99 mmol g-1, 32.1%, and 35.1%, respectively). The FMBE presented better cholesterol and sodium cholate (SC) adsorption properties and the adsorption was considered inhomogeneous surface adsorption. CONCLUSION: Fermentation increased the bioactive component content and improved the physicochemical properties of MBE, thereby improving its hypoglycemic and hypolipidemic properties. This study not only resolves the problem of millet bran waste but also encourages the development of higher value-added application methods for millet bran. © 2024 Society of Chemical Industry.

Rational engineering and insight for a L-glutaminase activity reduced type II L-asparaginase from Bacillus licheniformis and its antileukemic activity in vitro.

Type II L-asparaginase (ASNase) has been approved by the FDA for treating acute lymphoid leukemia (ALL), but its therapeutic effect is limited by low catalytic efficiency and L-glutaminase (L-Gln) activity. This study utilized free energy based molecular dynamics calculations to identify residues associated with substrate binding in Bacillus licheniformis L-asparaginase II (BLASNase) with high catalytical activity. After saturation and combination mutagenesis, the mutant LGT (74 L/75G/111 T) with intensively reduced l-glutamine catalytic activity was generated. The l-glutamine/L-asparagine activity (L-Gln/L-Asn) of LGT was only 6.6 % of parent BLASNase, whereas the L-asparagine (L-Asn) activity was preserved >90 %. Furthermore, structural comparison and molecular dynamics calculations indicated that the mutant LGT had reduced binding ability and affinity towards l-glutamine. To evaluate its effect on acute leukemic cells, LGT was supplied in treating MOLT-4 cells. The experimental results demonstrated that LGT was more cytotoxic and promoted apoptosis compared with commercial Escherichia coli ASNase. Overall, our findings firstly provide insights into reducing l-glutamine activity without impacting L-asparagine activity for BLASNase to possess remarkable potential for anti-leukemia therapy.

Thermal Stability Enhancement of L-Asparaginase from Corynebacterium glutamicum Based on a Semi-Rational Design and Its Effect on Acrylamide Mitigation Capacity in Biscuits.

Acrylamide is present in thermally processed foods, and it possesses toxic and carcinogenic properties. L-asparaginases could effectively regulate the formation of acrylamide at the source. However, current L-asparaginases have drawbacks such as poor thermal stability, low catalytic activity, and poor substrate specificity, thereby restricting their utility in the food industry. To address this issue, this study employed consensus design to predict the crucial residues influencing the thermal stability of Corynebacterium glutamicum L-asparaginase (CgASNase). Subsequently, a combination of site-point saturating mutation and combinatorial mutation techniques was applied to generate the double-mutant enzyme L42T/S213N. Remarkably, L42T/S213N displayed significantly enhanced thermal stability without a substantial impact on its enzymatic activity. Notably, its half-life at 40 °C reached an impressive 13.29 ± 0.91 min, surpassing that of CgASNase (3.24 ± 0.23 min). Moreover, the enhanced thermal stability of L42T/S213N can be attributed to an increased positive surface charge and a more symmetrical positive potential, as revealed by three-dimensional structural simulations and structure comparison analyses. To assess the impact of L42T/S213N on acrylamide removal in biscuits, the optimal treatment conditions for acrylamide removal were determined through a combination of one-way and orthogonal tests, with an enzyme dosage of 300 IU/kg flour, an enzyme reaction temperature of 40 °C, and an enzyme reaction time of 30 min. Under these conditions, compared to the control (464.74 ± 6.68 µg/kg), the acrylamide reduction in double-mutant-enzyme-treated biscuits was 85.31%, while the reduction in wild-type-treated biscuits was 68.78%. These results suggest that L42T/S213N is a promising candidate for industrial applications of L-asparaginase.

Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design.

l-Asparaginase has gained much attention for effectively treating acute lymphoblastic leukemia (ALL) and mitigating carcinogenic acrylamide in fried foods. Due to high-dose dependence for clinical treatment and low mitigation efficiency for thermal food processes caused by poor thermal stability, a method to achieve thermostable l-asparaginase has become a critical bottleneck. In this study, a rational design including free energy combined with structural and conservative analyses was applied to engineer the thermostability of l-asparaginase from Bacillus licheniformis (BlAsnase). Two enhanced thermostability mutants D172W and E207A were screened out by site-directed saturation mutagenesis. The double mutant D172W/E207A exhibited highly remarkable thermostability with a 65.8-fold longer half-life at 55 °C and 5 °C higher optimum reaction temperature and melting temperature (Tm) than those of wild-type BlAsnase. Further, secondary structure, sequence, molecular dynamics (MD), and 3D-structure analysis revealed that the excellent thermostability of the mutant D172W/E207A was on account of increased hydrophobicity and decreased flexibility, highly rigid structure, hydrophobic interactions, and favorable electrostatic potential. As the first report of rationally designing l-asparaginase with improved thermostability from B. licheniformis, this study offers a facile and efficient process to improve the thermostability of l-asparaginase for industrial applications.

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.

Characterization of a novel and glutaminase-free type II L-asparaginase from Corynebacterium glutamicum and its acrylamide alleviation efficiency in potato chips.

Type II L-asparaginase as a pivotal enzyme agent has been applied to treating for acute lymphoblastic leukemia (ALL) and efficient mitigation of acrylamide formed in fried and baked foods. However, low activity, narrow range of pH stability, as well as undesirable glutaminase activity hinder the applications of this enzyme. In our work, A novel type II L-asparaginase (CgASNase) from Corynebacterium glutamicum with molecular mass of about 35 kDa was chosen to express in E. coli. CgASNase shared only 27 % structural identity with the reported L-asparaginase from Helicobacter pylori. The purified CgASNase showed the highest specific activity of 1979.08 IU mg-1 to L-asparagine, compared with reported type II ASNases in the literature. CgASNase displayed superior stability at a wide pH range from 5.0 to 11.0, and retained about 76 % of its activity at 30 °C for 30 min. The kinetic parameters Km (Michaelis constant), kcat (turnover number), and kcat/Km (catalytic efficiency) values of 4.66 mM, 79,697.40 min-1, and 17,102.45 mM-1 min-1, respectively. More importantly, CgASNase exhibited strict substrate specificity towards L-asparagine, no detectable activity to l-glutamine. To explore its ability to catalyze L-asparagine, CgASNase was supplied in frying potato chips, which produced the fries with 84 % less acrylamide content compared with no supply. These findings suggest that CgASNase presents excellent properties for chemotherapy against diseases and great potential in the food processing industry.

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.

Non-classical secretion of a type I L-asparaginase in Bacillus subtilis.

L-asparaginase (EC 3.5.1.1) showed great commercial value owing to its effective treatment of acute lymphoblastic leukemia (ALL), lymphoid system malignancies and Hodgkin disease, and also to its use in the prevention of acrylamide formation in fried and baked foods. In this study, a type I L-asparaginase gene from Bacillus licheniformis Z-1 (BlAase) was cloned and expressed in Bacillus subtilis RIK 1285. Results showed that even without the mediation of any N-terminal signal peptides, BlAase can efficiently secrete into the medium. Further investigation indicated that the secretion of the BlAase was via neither Sec- nor Tat-dependent secretion pathway, and both the N- and C-terminal regions of the BlAase were essential for its expression and secretion, implying that BlAase might be secreted via a non-classical secretion pathway. To explore its secretion ability, BlAase was used as a signal peptide to direct the secretion of various heterologous proteins, where two of five proteins were successfully secreted with the mediation of BlAase. To the best of our knowledge, this is the first time to achieve extracellular expression of L-asparaginase via non-classical protein secretion pathway in B. subtilis, and provide a potential tool for secretion of recombinant proteins expressed in B. subtilis using BlAase as a signal peptide.

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.

Minimally invasive perventricular versus open surgical ventricular septal defect closure in infants and children: a randomised clinical trial.

BACKGROUND: Robust evidence is lacking regarding the clinical efficacy, safety and cardiopulmonary performance of perventricular closure. This study investigated the perioperative efficacy, safety and cardiorespiratory performance of perventricular closure of perimembranous ventricular septal defects (pmVSDs). METHODS: Operation-naïve infants and young children aged 5-60 months with isolated pmVSDs were randomised to receive either standard open surgical or minimally invasive perventricular closure via direct entry into the ventricle with a catheter from a subxiphoid incision. The primary outcomes included complete closure at discharge, major and minor adverse events and the changes in perioperative cardiorespiratory performance from baseline. Complete closure was mainly analysed in the modified intention-to-treat (mITT) population, with sensitivity analyses for the ITT, per-protocol (PP) and as-treated (AT) populations (non-inferiority margin -5.0%). RESULTS: We recruited 200 patients with pmVSDs for this study (mean age 24.38 months, range 7-58 months, 104 girls), of whom 100 were randomly allocated to one of the study groups. The non-inferiority of perventricular to surgical closure regarding complete closure at discharge was not shown in the ITT (absolute difference -0.010 (95% CI -0.078 to 0.058)) and mITT populations (-0.010 (95% CI -0.069 to 0.048)), but was shown in the PP (0.010 (95% CI -0.043 to 0.062)) and AT populations (0.048 (95% CI -0.009 to 0.106)). Perventricular closure reduced the rate of compromising cardiac haemodynamics, electrophysiological responses, cardiomyocyte viability, respiratory mechanics, ventilatory and gas exchange function and oxygenation and tissue perfusion compared with surgical closure (all between-group P<0.05). CONCLUSIONS: For infants and young children with pmVSD, perventricular closure reduced the rate of postoperative cardiorespiratory compromise compared with surgical closure, but the non-inferiority regarding complete closure should be interpreted in the context of the specific population. TRIAL REGISTRATION NUMBER: NCT02794584 ;Results.

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.

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.

Surgical management of apical muscular ventricular septal defects using the sandwich technique.

OBJECTIVE: We performed closure of apical muscular ventricular septal defects (VSDs) using the sandwich technique and assessed its role in the treatment of the defects. METHODS: Twenty-one patients (nine males and 12 females) underwent VSD closure at a mean age of 15.4 months (range, 1 month to 6 years) and a mean weight of 8.3 kg (range, 4 to 20 kg). Associated cardiac malformations were present in all of the patients. VSDs were exposed through the tricuspid valve and also from the left ventricular (LV) side through a coexisting large perimembranous VSD or through the mitral valve through an interatrial septostomy. All the apical muscular VSDs were closed using the sandwich technique. RESULTS: There were no hospital deaths, and the postoperative course was uneventful in all patients. There was no residual shunt in 13 patients, and a minimal residual shunt (diameter ≤2 mm) was observed in four patients. Mild residual shunt (diameter ≤4 mm) was observed in two patients. At the latest follow-up, all the residual shunts had disappeared except in one patient. The wall motion of the interventricular septum and cardiac function were normal in all the patients one month after surgery. All patients were free of cardiac medications. CONCLUSIONS: We conclude that the sandwich technique is safe and reliable. Even in cases when a residual shunt is present, the shunt tends to decrease with time. Further experience and longer follow-up of these patients are necessary to conclude whether this technique is applicable to neonates and young infants.

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.

The influence of deep hypothermic global brain ischemia on EEG in a new rat model.

OBJECTIVES: Neurological complications following deep hypothermic circulatory arrest (DHCA) occur between t 4% ≈ 25%. However, the cerebral injury mechanisms are still not well understood due to a lack of a practical and simple animal model. We aimed to establish a rodent deep hypothermic global brain ischemia (DHGBI) model, which can be used to elucidate these mechanisms in future studies. DESIGN: 30 Sprague-Dawley rats were divided randomly into three groups: the carotid occlusion DHGBI group, the internal carotid shunt DHGBI group, and the sham operation group. We validated the model in terms of electroencephalogram (EEG) and regional cerebral blood flow (rCBF). All rats were sacrificed for analysis of brain moisture capacity after 24 hours. RESULTS: In the internal carotid shunt DHGBI group the EEG activity was suppressed to "flat-line" and the relative power of the α and θ frequency bands was decreased (p < 0.05). However, in the carotid occlusion DHGBI group we only observed the relative power of the α frequency band depressed (p < 0.05). The rCBF was significantly decreased in all groups. In the internal carotid shunt DHGBI group the rCBF was significantly reduced to 4.27 ± 2.75%, and was lower than the other two groups (p < 0.05). The result of brain moisture capacity was consistent with the EEG and rCBF observations. CONCLUSIONS: The current study presents a novel cerebral recovery model of DHCA in the rat. This experimental model may be suitable to further elucidate the mechanisms associated with adverse cerebral outcomes after DHCA and to investigate potential neuroprotective strategies.

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.

[Cloning and expression of organic solvent tolerant lipase gene from Staphylococcus saprophyticus M36].

Lipases are important biocatalysts that are widely used in food processing and bio-diesel production. However, organic solvents could inactivate some lipases during applications. Therefore, the efficient cloning and expression of the organic solvent-tolerant lipase is important to its application. In this work, we first found out an organic solvent-tolerant lipase from Staphylococcus saprophyticus M36 and amplified the 741 bp Lipase gene lip3 (GenBank Accession No. FJ979867), by PCR, which encoded a 31.6 kD polypeptide of 247 amino acid residues. But the lipase shared 83% identity with tentative lip3 gene of Staphylococcus saprophyticus (GenBank Accession No. AP008934). We connected the gene with expression vector pET-DsbA, transformed it into Escherichia coli BL21 (DE3), and obtained the recombinant pET-DsbA-lip3. With the induction by 0.4 mmol/L of isopropyl beta-D-thiogalactopyranoside at pH 8.0, OD600 1.0, 25 degrees C for 12 h, the lipase activity reached up to 25.8 U/mL. The lipase expressed was stable in the presence of methanol, n-hexane, and isooctane, n-heptane.

[Media optimization for the novel antimicrobial peptide by Bacillus sp. fmbJ224].

The novel antimicrobial peptide in submerged fermentation by Bacillus sp. fmbJ224 is strongly influenced by many internal and external factors, namely medium constituents and fermentation conditions. In this study, Plackett-Burman design was undertaken to evaluate the effects of the seventeen factors. By the statistical regression analysis, the significant factors affecting the novel antimicrobial peptide in submerged fermentation by Bacillus sp. fmbJ224 were determined as follows: glucose, NH4NO3, glutamic acid, CaCl2, MnSO4. In the second phase of the optimization process, a response surface methodology (RSM) was used to optimize the above critical internal factors, and to find out the optimization concentraction levels and the relationships between these factors. By solving the quadratic regression model equation using appropriate statistic methods, the optimal concentration of the variables were determined as: 8.13 g/L glucose, 6.14 g/L NH4NO3, 4.2 g/L glutamic acid, 3.98 mg/L CaCl2, 4.87 mg/L MnSO4. The content of the novel antimicrobial peptide was increased from 1304.21 microg/mL to 1487.58 microg/mL. The experimental data under various conditions have validated the theoretical values.