Xiaomei Bie

Production of γ-aminobutyric acid by Streptococcus salivarius subsp. thermophilus Y2 under submerged fermentation

Summary.γ-Aminobutyric acid (GABA), a major inhibitory neurotransmitter in the central nervous system, has several well-known physiological functions and has been applied to the production of many drugs and functional foods. The technology of GABA production via submerged fermentation by Streptococcus salivarius subsp. thermophilus Y2 was investigated in this paper. It indicated that the GABA production was related to the biochemical characteristics of glutamate decarboxylase (GAD) of S. salivarius subsp. thermophilus Y2. After 24 h of fermentation at 37 °C, which is the suitable culture conditions for GAD-production, then the culture condition were adjusted to the optimal temperature (40 °C) and pH (4.5) for the GAD reaction activity in biotransformation of cells and pyridoxal 5′-phosphate (0.02 mmol/l) were added to the broth at the 48 h, the GABA production was increased up to 1.76-fold, reaching 7984.75 ± 293.33 mg/l. The strain shows great potential use as a starter for GABA-containing yoghurt, cheese and other functional fermented food productions.

A Newly Isolated Organic Solvent Tolerant Staphylococcus saprophyticus M36 Produced Organic Solvent-Stable Lipase

Thirty-eight high lipase activity strains were isolated from soil, seawater, and Brassica napus. Among them, a novel organic solvent tolerant bacterium (strain M36) was isolated from the seawater in Jiangsu, China. Isolate M36 was able to grow at high concentration of benzene or toluene up to 40% (vol/vol), and later identified as Staphylococcus saprophyticus by biochemical test and 16s ribosomal DNA sequence. No work on Staphylococcus producing lipase with organic solvent tolerance has been reported so far. The lipase of strain M36 whose activity in liquid medium was 42 U mL−1 at 24-h incubation time was stable in the presence of 25% (vol/vol) p-xylene, benzene, toluene, and hexane.

Identification of fengycin homologues from Bacillus subtilis with ESI-MS/CID

Bacillus subtilis fmbJ (CGMCC no.0943) was used to produce lipopeptides antibiotics fengycin. The fengycin mixture was precipitated with 6N HCl and extracted by methanol. Electrospray ionization mass spectrometry/collision-induced dissociation (ESI-MS/CID) were used to analyze the fengycin mixture. In the ESI-MS/CID spectra of fengycin molecular ions, production ions (m/z 1080, m/z 966, m/z 1108 and m/z 994) were always detected. Therefore, these production ions (m/z 1080, m/z 966, m/z 1108 and m/z 994) could be used as fingerprints to quickly detect fengycin A and fengycin B. By the method of ESI-MS/CID, six homologues of fengycin A with C14 to 19 and four homologues of fengycin B with C14 to 17 were characterized. The method will be useful for rapidly determining whether a strain produces fengycin homologues.

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.

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.

Purification and characterization of a novel anticoagulant and fibrinolytic enzyme produced by endophytic bacterium Paenibacillus polymyxa EJS-3

INTRODUCTION Endophytes may become a new source of thrombolytic agents for thrombosis treatment. MATERIALS AND METHODS A novel fibrinolytic enzyme from Paenibacillus polymyxa EJS-3 (PPFE-I) was purified with ammonium sulfate precipitation, hydrophobic chromatography, ion exchange and gel filtration chromatography. The characterization of the enzyme was investigated by means of fibrinolysis plate, hydrolysis of fibrinogen and anticoagulant effect in vitro. RESULTS The fibrinolytic enzyme is purified to homogeneity with a purification of 14.5 fold and a recovery of 3.3%. The enzyme was shown to have a molecular mass of 63.3kDa by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The optimum temperature and pH value were 37°C and 7.5, respectively. Results from the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the Aα-chain of fibrinogen, followed by the Bβ-chains. It also hydrolyzed the γ-chains, but more slowly. It was activated by metal ions such as Zn(2+), Mg(2+), and Fe(2+), but inhibited by Ca(2+) and Cu(2+). Furthermore, PPFE-I activity was inhibited strongly by PMSF, and it was found to exhibit a higher specificity for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA for chymotrypsin, indicating that the enzyme is a chymotrypsin-like serine protease. Additionly, PPFE-I showed a significant anticoagulant effect in vitro. CONCLUSION The fibrinolytic enzyme PPFE-I from endophytic bacterium Paenibacillu polymyxa EJS-3 exhibits a profound fibrinolytic activity.

Isolation and Identification of an Endophytic Strain EJS-3 Producing Novel Fibrinolytic Enzymes

An endophytic strain EJS-3, which produces a novel fibrinolytic enzyme, was screened from root tissue of Stemona japonica (Blume) Miq, a chinese traditional medicine. This strain was identified as Paenibacillus polymyxa (DQ120522) by morphological, physiological, and biochemical tests and 16S rRNA gene sequence analysis. Two serine-type fibrinolytic enzymes with a relative molecular weight about 118 and 49 kDa, respectively, which are larger than known fibrinolytic enzymes, were found by the SDS–fibrin zymogram or by fibrin-inhibitor zymography gels. No work on P. polymyxa-producing fibrinolytic enzymes has been reported.

Isolation and characterization of peptide antibiotics LI-F04 and polymyxin B6 produced by Paenibacillus polymyxa strain JSa-9.

Paenibacillus polymyxa JSa-9 had been found to produce five cyclic LI-F type antibiotics which were released into culture medium in accordance with our previous report. In this study, another three kinds of antagonistic compounds were extracted from P. polymyxa JSa-9 cell pellets and (or) spores by methanol. Using high performance liquid chromatography (HPLC) method, two antagonistic fractions were separated and collected from the methanol extract. One showed inhibition against Escherichia coli and Staphylococcus aureus, while the other was active against Aspergillus niger and S. aureus. By means of electrospray ionization mass spectroscopy (ESI-MS), infrared spectroscopy (IR), and amino acid analysis, two kinds of compounds from fraction B with molecular masses of 901 and 915Da were characterized as the linear lipopeptide analogs of antibiotics LI-F04a and LI-F04b, respectively. Another antimicrobial substance from fraction A could be attributed to polymyxin B(6).

Genome shuffling of Bacillus amyloliquefaciens for improving antimicrobial lipopeptide production and an analysis of relative gene expression using FQ RT-PCR

Genome shuffling is an efficient approach for the rapid improvement of the yield of secondary metabolites. This study was undertaken to enhance the yield of surfactin produced by Bacillus amyloliquefaciens ES-2-4 using genome shuffling and to examine changes in SrfA expression of the improved phenotype at the transcriptional level. Six strains with subtle improvements in lipopeptide yield were obtained from populations generated by ultraviolet irradiation, nitrosoguanidine, and ion beam mutagenesis. These strains were then subjected to recursive protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both ultraviolet irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant F2-38 strain that exhibited 3.5- and 10.3-fold increases in surfactin production in shake flask and fermenter respectively, was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR. Delta CT (threshold cycle) relative quantitation analysis revealed that surfactin synthetase gene (srfA) expression at the transcriptional level in the F2-38 strain was 15.7-fold greater than in the ES-2-4 wild-type. The shuffled strain has a potential application in food and pharmaceutical industries. At the same time, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering.

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.