Efficient extracellular recombinant production and purification of a Bacillus cyclodextrin glucanotransferase in Escherichia coli.

BACKGROUND: Cyclodextrin glucanotransferases (CGTases) catalyze the synthesis of cyclodextrins, cyclic oligosaccharides composed of glucose monomers that find applications in the pharmaceutical, food, and cosmetic industries. An economic application of these industrially important enzymes requires their efficient production and recovery. In this study, the effect of Sec-type signal peptides on the recombinant expression of a CGTase derived from Bacillus sp. G825-6 was investigated in Escherichia coli BL21(DE3) using a codon-adapted gene. In addition, a novel purification method for the CGTase using starch adsorption was developed. RESULTS: Expression vectors encoding N-terminal PelB, DacD, and the native Bacillus sp. G825-6 CGTase signal peptides (SP) were constructed for the recombinant CGTase. With the DacD SP derived from E. coli, a 3.9- and 3.1-fold increase in total enzyme activity was obtained compared to using the PelB and the native CGTase SP, respectively. DacD enabled a 7.3-fold increase of activity in the extracellular fraction after induction for 24 h compared to the native CGTase SP. After induction for 48 h, 75% of the total activity was detected in the extracellular fraction. By a batch wise adsorption to starch, the extracellular produced CGTase could be purified to homogeneity with a yield of 46.5% and a specific activity of 1637 U/mg. CONCLUSIONS: The signal peptide DacD promoted the high-level heterologous extracellular expression of a recombinant CGTase from Bacillus sp. G825-6 with a pET20b(+) vector in E. coli BL21(DE3). A protocol based on starch adsorption enabled a fast and efficient purification of the recombinant enzyme.

Surfactant Protein-G in Wildtype and 3xTg-AD Mice: Localization in the Forebrain, Age-Dependent Hippocampal Dot-like Deposits and Brain Content.

The classic surfactant proteins (SPs) A, B, C, and D were discovered in the lungs, where they contribute to host defense and regulate the alveolar surface tension during breathing. Their additional importance for brain physiology was discovered decades later. SP-G, a novel amphiphilic SP, was then identified in the lungs and is mostly linked to inflammation. In the brain, it is also present and significantly elevated after hemorrhage in premature infants and in distinct conditions affecting the cerebrospinal fluid circulation of adults. However, current knowledge on SP-G-expression is limited to ependymal cells and some neurons in the subventricular and superficial cortex. Therefore, we primarily focused on the distribution of SP-G-immunoreactivity (ir) and its spatial relationships with components of the neurovascular unit in murine forebrains. Triple fluorescence labeling elucidated SP-G-co-expressing neurons in the habenula, infundibulum, and hypothalamus. Exploring whether SP-G might play a role in Alzheimer's disease (AD), 3xTg-AD mice were investigated and displayed age-dependent hippocampal deposits of ß-amyloid and hyperphosphorylated tau separately from clustered, SP-G-containing dots with additional Reelin-ir-which was used as established marker for disease progression in this specific context. Semi-quantification of those dots, together with immunoassay-based quantification of intra- and extracellular SP-G, revealed a significant elevation in old 3xTg mice when compared to age-matched wildtype animals. This suggests a role of SP-G for the pathophysiology of AD, but a confirmation with human samples is required.

Antimicrobial lavandulylated flavonoids from a sponge-derived actinomycete.

Analysis of an antimicrobial culture broth extract of the sponge-derived actinomycete Streptomyces sp. (strain G246) led to the isolation of two new lavandulylated flavonoids, 6-lavandulyl-7-methoxy-5,2',4'-trihydroxylflavanone (1) and 5'-lavandulyl-4'-methoxy-2,4,2',6'-tetrahydroxylchalcone (2), along with eight known compounds 3-10. Their structures were established by spectral data analysis, including MS, 1D, 2D-NMR and CD. The absolute configurations of 1 and 2 were suggested by comparison of their experimental and calculated electronic circular dichroism spectra. All the isolated compounds were evaluated for their antimicrobial activity against a panel of clinically significant microorganisms. Compounds 1 and 2 had a broad-spectrum of antimicrobial activity. Additionally, except the strain Escherichia coli, compound 2 exhibited remarkable inhibitory activity against Pseudomonas aeruginosa, Salmonella enterica, Enterococcus faecalis, Staphylococcus aureus, Bacillus cereus, and Candida albicans strains.

Antimicrobial metabolites from a marine-derived Actinomycete Streptomyces sp. G278.

Analysis of an antimicrobial extract prepared from culture broth of the marine-derived actinomycete Streptomyces sp. G278 led to the isolation of ten compounds, 1-10. Two compounds, 2,5-Bis(5-tert-butyl-2-benzoxazolyl)thiophene (1), and 3-hydroxyl-2-methylpyridine (2) were isolated from a natural source for the first time. The structures of the isolated compounds were established by their spectral data analysis, including mass spectrometry, 1D-NMR, 2D-NMR, and X-ray crystallographic analysis in case of compound 3. All isolated compounds were evaluated for their antimicrobial activity against a panel of clinically significant microorganisms. Compounds 1 and 3 selectively inhibited Enterococcus faecalis (MIC: 256 µg/mL). Compound 2 was found to have antibacterial and antifungal activity against Escherichia coli (MIC: 64 µg/mL), Salmonella enterica (MIC: 256 µg/mL), Staphylococcus aureus (MIC: 256 µg/mL), Enterococcus faecalis (MIC: 256 µg/mL), and Candida albicans (MIC: 64 µg/mL). Except for compounds 9 and 10, the other known metabolites (4-8) also exhibited antimicrobial activity.

Screening of potential probiotic lactic acid bacteria and production of amylase and its partial purification.

Probiotics are the healthy living bacteria when administered in adequate amounts confers health benefits in the host. The main objective of present study was to screen the bacteria for potential probiotic characters and enzyme production. The probiotic characters like tolerance to low pH, bile salts, antibiotic sensitivity, hydrophobicity and auto-aggregation properties were evaluated. Among all isolates Lactobacillus fermentum and Lactobacillus sp G3_4_1TO2 showed maximum potential probiotic characters and produced amylase enzyme by observing the halo zone around the colonies with the diameter 0.9 mm and 1.23 mm. Lactobacillus sp G3_4_1TO2 produced maximum amylase when compared with Lb. fermentum. The protein yield was 55.4% with the specific activity of 88.9 U/mg and obtained 40.8% purification fold. The molecular weight of amylase enzyme determined by SDS PAGE was 95,000 Da. From the present study it was considered that Lactobacillus sp G3_4_1TO2 was a potential probiotic bacteria producing maximum amylase enzyme.

Enhancing digestibility of Miscanthus using lignocellulolytic enzyme produced by Bacillus.

In this study an effective bacterial pretreatment method was developed to improve digestibility of Miscanthus. Seven new bacterial isolates, which showed excellent xylanase production ability using Miscanthus as carbon source, were used to perform the pretreatment experiments. After pretreatment, the hemicellulose content and crystallinity index of Miscanthus were decreased, while the reducing sugars released from Miscanthus were significantly increased by 30.8-87.8% after enzymatic hydrolysis. Bacillus sp. G0 was selected to optimize the pretreatment parameters via response surface methodology due to its high reducing sugars released from Miscanthus. According to the optimal model, the pretreatment parameters were set as citrate buffer/G0 fermentation broth ratio at 0.34, pretreatment time at 100h and Tween-20 concentration at 1.73%. The reducing sugars released from Miscanthus pretreated by optimal parameters were 305mgg-1 dry biomass. The results suggested our bacterial pretreatment approaches have great potential to increase digestibility of bioenergy crops.

Rapid biodegradation of organophosphorus pesticides by Stenotrophomonas sp. G1.

Organophosphorus insecticides have been widely used, which are highly poisonous and cause serious concerns over food safety and environmental pollution. A bacterial strain being capable of degrading O,O-dialkyl phosphorothioate and O,O-dialkyl phosphate insecticides, designated as G1, was isolated from sludge collected at the drain outlet of a chlorpyrifos manufacture plant. Physiological and biochemical characteristics and 16S rDNA gene sequence analysis suggested that strain G1 belongs to the genus Stenotrophomonas. At an initial concentration of 50 mg/L, strain G1 degraded 100% of methyl parathion, methyl paraoxon, diazinon, and phoxim, 95% of parathion, 63% of chlorpyrifos, 38% of profenofos, and 34% of triazophos in 24 h. Orthogonal experiments showed that the optimum conditions were an inoculum volume of 20% (v/v), a substrate concentration of 50 mg/L, and an incubation temperature in 40 °C. p-Nitrophenol was detected as the metabolite of methyl parathion, for which intracellular methyl parathion hydrolase was responsible. Strain G1 can efficiently degrade eight organophosphorus pesticides (OPs) and is a very excellent candidate for applications in OP pollution remediation.

Freezing/thawing pretreatment coupled with biological process of thermophilic Geobacillus sp. G1: Acceleration on waste activated sludge hydrolysis and acidification.

A novel pretreatment method combining freezing/thawing with Geobacillus sp. G1 was employed to pretreat waste activated sludge (WAS) for enhancing the WAS hydrolysis and subsequent short-chain fatty acids (SCFAs) production. Results showed that freezing/thawing combined with Geobacillus sp. G1 pretreatment achieved the maximal concentrations of soluble protein from 40±6mg COD/L (non-pretreated) to 1226±24mg COD/L (pretreated), and accumulated SCFAs concentration increased from 248±81mg COD/L to 3032±53mg COD/L. Excitation emission matrix (EEM) fluorescence spectroscopy revealed the highest fluorescence intensity (FI) of protein-like substances, which was the dominant fluorescent organic matters, indicating the synergistic effect of freezing/thawing and Geobacillus sp. G1 pretreatment on organics hydrolysis. High-throughput pyrosequencing analysis investigated that the abundance of bacteria responsible for WAS hydrolysis (such as Clostridium and Caloramator) and SCFAs production (such as Parabacteroides and Bacterodies) was greatly enhanced due to the novel pretreatment method used.