Haizhen Zhao

Purification and Characterization of Plantaricin 163, a Novel Bacteriocin Produced by Lactobacillus plantarum 163 Isolated from Traditional Chinese Fermented Vegetables

Presumptive lactic acid bacteria (LAB) strains isolated from traditional Chinese fermented vegetables were screened for bacteriocin production. A novel bacteriocin-producing strain, Lactobacillus plantarum 163, was identified on the basis of its physiobiochemical characteristics and characterized by 16S rDNA sequencing. The novel bacteriocin, plantaricin 163, produced by Lb. plantarum 163 was purified by salt precipitation, gel filtration, and reverse-phase high-performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of plantaricin 163 revealed the molecular weight to be 3553.2 Da. The complete amino acid sequence showed VFHAYSARGNYYGNCPANWPSCRNNYKSAGGK, and no similarity to known bacteriocins was found. Plantaricin 163 was highly thermostable (20 min, 121 °C), active in the presence of acidic pH (3-5), sensitive to protease, and exhibited broad-spectrum antimicrobial activity against LAB and other tested Gram-positive and Gram-negative bacteria. The results suggest that plantaricin 163 may be employed as a biopreservative in the food industry.

Antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer

The antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer were investigated. The MIC (minimal inhibitory concentration) of fengycin without commercial surfactin added was 0.4 mg/ml while the MIC of fengycin with commercial surfactin added was 2.0 mg/ml. Fengycin acted on cell membrane and cellular organs and inhibited DNA synthesis. The antifungal effect of fengycin was reduced after commercial surfactin was added. All these results suggest that the fungal cell membrane may be the primary target of fengycin action and commercial surfactin may reduce the antifungal activity of fengycin.

Purification and characterization of a temperature- and pH-stable laccase from the spores of Bacillus vallismortis fmb-103 and its application in the degradation of malachite green.

Malachite green residue can affect aquaculture food safety. Bioremediation of contaminated water by enzyme treatment is an environmentally friendly and economical way to remove contaminating substances. In the present study, a temperature- and pH-stable laccase was purified from the spores of Bacillus ballismortis fmb-103 and was used to degrade malachite green. The laccase from fmb-103 (fmb-L103) was purified 15.2-fold to homogeneity (389.9 mU/mg protein with respect to ABTS as a substrate) by precipitation with 30-80% (NH4)2SO4, DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-100 chromatography. fmb-L103 is a nonblue laccase with a molecular weight of 55.0 kDa and Cu content of 2.5 (mol:mol). fmb-L103 retained more than 50% activity after 10 h at 70 °C and demonstrated broad pH stability in both acidic and alkaline conditions. The effects of inhibitors and metal ions on fmb-L103 activity were also examined. A kinetic study revealed that ABTS was a suitable substrate with a Km of 22.7 μmol and a Vmax of 3.32 μmol/mL/min. fmb-L103 can efficiently degrade malachite green after a 48 h treatment period in combination with a mediator, without the appearance of leucomalachite green.

Identification of LI-F type antibiotics and di-n-butyl phthalate produced by Paenibacillus polymyxa

Paenibacillus polymyxa strain JSa-9, a soil isolate that displayed antibacterial and antifungal activities in vitro, had been found to produce two types of antimicrobial substances. The two compounds were extracted from the fermentation broth of JSa-9 using ethyl acetate and subsequently purified by high performance liquid chromatography. By means of liquid chromatography-mass spectrometry and tandem mass spectrometry analysis, one of two antagonistic compounds was determined as di-n-butyl phthalate. And another was characterized as a mixture of related peptides of molecular masses of 883, 897, 911, 947, and 961Da, with the most likely structure of them determined to be a cyclic depsipeptide with an unusual 15-guanidino-3-hydroxypentadecanoic acid moiety bound to a free amino group. These peptides were therefore members of the LI-F group of cyclic depsipeptides.

Antagonistic Action of Bacillus subtilis Strain fmbj on the Postharvest Pathogen Rhizopus stolonifer

The antagonistic activities of Bacillus subtilis fmbj against the Rhizopus stolonifer pathogen causing peach soft rot disease were studied in this paper. Bacillus subtilis fmbj exhibited a high antifungal effect on the mycelium growth, sportulation, and germ tube elongation of R. stolonifer with the inhibition rate of 86.5% and 97.42%, respectively, spore germination rate of 20% under antagonist strain concentration of 10⁸ CFU/mL. By using of scanning electron microscope (SEM) and transmission electron microscope (TEM), it was observed that B. subtilis fmbj strain strongly induced morphological abnormalities of R. stolonifer and destroyed structure of hypha and spore. When hypha cell wall of R. stolonifer was damaged, the organelles and cytoplasms inside cell would exude, which led to cell death.

Synthesis and characterisation of galactosyl glycerol by β-galactosidase catalysed reverse hydrolysis of galactose and glycerol

A study of an enzymatic method for the production of galactosylglycerol is described. The effects of enzyme sources, enzyme amount, reaction temperature, reaction time, substrate ratio of glycerol to galactose, buffer content and pH on galactosylglycerol yield (mg/ml) were investigated. Under the optimum reaction conditions of β-galactosidases from Kluyveromyces lactis 240 U/ml, temperature 40 °C, time 24 h, buffer amount 60% (percent volume of buffer to that of substrates, pH 6.5), and the substrate molar ratio of 10 (glycerol16 mmol:galactose 1.6 mmol), the yield of galactosylglycerol was up to 116.47 mg/ml (galactose conversion 55.88%). The product was purified by activated charcoal and Sephadex G-15 column chromatography, up to 96%. The purified galactosylglycerol was fully characterised by MS and NMR, and identified as a mixture of (2R)- and (2S)- 3-O-β-D-galactopyranosyl-glycerol.

Engineering of a thermostable β‐1,3‐1,4‐glucanase from Bacillus altitudinis YC‐9 to improve its catalytic efficiency

BACKGROUND Error-prone polymerase chain reaction (PCR) is frequently used in directed evolution of enzymes to modify their quality. In this study, error-prone PCR was used to improve the catalytic efficiency of β-1,3-1,4-glucanase from Bacillus altitudinis YC-9. RESULTS By screening, the mutant Glu-3060 with higher activity was selected among 5000 transformants. After induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), the activity of the mutant Glu-3060 reached 474.6 U mL(-1), resulting in a 48.6% increment of the parent enzyme activity. Research on the characterization of the mutated enzyme showed the optimal pH of the mutated enzyme to be 5.0, which is lower than the parent enzyme, but thermal stability was almost the same between them. Sequence analysis of the mutated enzyme revealed that three amino acids were changed compared with the parent enzyme, including K142N, Q203L and N214D. CONCLUSION The three-dimensional structure predicted by SWISS-MODEL of the mutated enzyme Glu-3060 showed that the substitution of three amino acids had an effect on the catalytic activity, stability and optimal pH of the enzyme, through changing the charge properties or electron density, forming secondary keys, the acidity of the amino acids and the side chain group. The sum effects of all the factors were increased activity of the mutated enzyme and decreased optimal pH, while the same thermostability was maintained, thereby increasing the suitability of the enzyme for industrial use.

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.

Influence of surfactin on physical and oxidative stability of microemulsions with docosahexaenoic acid.

Docosahexaenoic acid (DHA), one of the most important omega-3 polyunsaturated fatty acids (PUFAs), shows significant health benefits for human beings. In order to stabilize nutrients like DHA, microemulsion is normally used through the addition of surfactant, and surfactin as a natural peptide biosurfactant shows strong surface activity. In this study, we investigated the effects of surfactin on the stability of docosahexaenoic acid single cell oil (DHASCO) microemulsions. The microemusion region was significantly increased with elevated surfactin concentration from 0 to 0.2mmol/L, and reached a maximum. The o/w region of DHASCO microemulsion could significantly increase and the diameters of microemulsion particles were reduced from 140 to 15nm after addition of 0.2mmol/L surfactin into emulsion system. Generally, the physical and anti-oxidation stability of the o/w DHASCO microemulsion with surfactin was highly enhanced. DHA oxidation in microemulsion with surfaction was significantly reduced even stored at 37°C for 60days as compared to non-surfactin. The excellent properties of microemulsion with surfactin could be useful in functional food and medicine.

Expression of a novel bacteriocin-the plantaricin Pln1-in Escherichia coli and its functional analysis.

A potential bacteriocin gene was isolated from 18575 ORFs by bioinformatics methods. It was named pln1, and cloned into pET32a. Then, it was expressed as a thioredoxin-Pln1 fusion protein in Escherichia coli BL21 (DE3). The fusion protein was purified by Ni-NTA, and thioredoxin was removed by enterokinase. Finally, Pln1 was purified using a cation affinity column. The yields of fused and cleaved Pln1 peptides were 100-110 mg/l and 9-11 mg/l, respectively. Pln1 was stable in an acidic environment and at temperatures below 60 °C, but was easily degraded under alkaline conditions and by protease treatment. The cleaved and purified Pln1 showed strong antimicrobial activity against gram-positive bacteria such as Micrococcus luteus CMCC 63202, Staphylococcus epidermidis, Lactococcus lactis NZ3900, Lactobacillus paracasei CICC 20241, and Listeria innocua CICC 10417. In particular, Pln1 had a better activity against methicillin-resistant S. epidermidis (MRSE) than nisin, thereby offering an attractive approach to counter bacterial antibiotic resistance.