Statistical optimization of l-leucine amino peptidase production from Streptomyces gedanensis IFO 13427 under submerged fermentation using response surface methodology

Response surface methodology (RSM) employing the central composite design (CCD) was used to optimize the fermentation medium for the production of l-leucine amino peptidase (LAP) from Streptomyces gedanensis IFO13427 under submerged fermentation. The design was employed by selecting substrate concentration, NaCl concentration and initial pH as model factors by ‘one variable at a time’ experiment. A second-order quadratic model and response surface method showed that the optimum conditions (soy bean 0.3%, NaCl, 0.03 M, and initial pH 7) resulted in the improvement of LAP production (25.69 IU/ml) as compared to the initial level (12.17 ± 0.23 IU/ml) after 72 h of fermentation, whereas its value predicted by the quadratic model was 24.56 IU/ml. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.9799, ensuring a satisfactory adjustment of the quadratic model with the experimental data. This is first report on LAP production by S. gedanensis using statistical experimental design and response surface methodology in submerged fermentation.     

IL-6 protects enterocytes from hypoxia-induced apoptosis by induction of bcl-2 mRNA and reduction of fas mRNA

Interleukin-6 (IL-6) has been shown to rescue enterocytes from hypoxia-induced apoptosis when given orally following hemorrhagic shock. In vitro models using an intestinal epithelial cell line (IEC-6) cultured with lipopolysaccharide (LPS) under low O2 conditions, to mimic intestinal conditions, show that these cells also undergo apoptosis, which can be reduced by subsequent culture with IL-6. To examine further the mechanisms of rescue, we cultured normal rat intestinal epithelial cells (IEC-6) under both normoxic and hypoxic conditions and analyzed their responses to LPS and IL-6. We showed that IEC-6 expressed IL-6 receptor on its surface. Further, IEC-6 cells could be rescued from hypoxia-induced apoptosis by co-culture with IL-6. RNase protection assay (RPA) examination revealed that under hypoxic conditions, IEC-6 cells that were resistant to apoptosis showed reduced fas expression and increased bcl-2 expression after co-culture with LPS+IL-6.     

Molecular cloning,overexpression and characterization of the raw-starch-digesting <Emphasis Type="Italic">α</Emphasis>-amylase of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

Raw starch is the most abundant source of glucose in the world. Therefore, finding enzymes capable of digesting raw starch would find high industrial demand. The α-amylase gene of Bacillus amyloliquefaciens ATCC 23842 was amplified, cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme was purified to apparent homogeneity using ion exchange and gel filtration chromatography. The raw-starch digestibility of the purified enzyme was characterized by studying the hydrolysis and adsorption rate on a variety of raw starches (potato, cassava, corn, wheat and rice). The raw-starch digestion was further confirmed by scanning electron microscopy studies, which revealed an effective rate of hydrolysis. The kinetic studies revealed a relatively low K _m of 2.76 mg/mL, exhibiting high affinity towards the soluble starch as the most preferred substrate and the inhibition kinetic studies revealed a high K _i value (350 mM).     

Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the l-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more l-glutamate and l-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM l-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM l-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.     

Calcium/calmodulin dependent protein kinase II bound to NMDA receptor 2B subunit exhibits increased ATP affinity and attenuated dephosphorylation

Calcium/calmodulin dependent protein kinase II (CaMKII) is implicated to play a key role in learning and memory. NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) is a high affinity binding partner of CaMKII at the postsynaptic membrane. NR2B binds to the T-site of CaMKII and modulates its catalysis. By direct measurement using isothermal titration calorimetry (ITC), we show that NR2B binding causes about 11 fold increase in the affinity of CaMKII for ATPγS, an analogue of ATP. ITC data is also consistent with an ordered binding mechanism for CaMKII with ATP binding the catalytic site first followed by peptide substrate. We also show that dephosphorylation of phospho-Thr(286)-α-CaMKII is attenuated when NR2B is bound to CaMKII. This favors the persistence of Thr(286) autophosphorylated state of CaMKII in a CaMKII/phosphatase conjugate system in vitro. Overall our data indicate that the NR2B- bound state of CaMKII attains unique biochemical properties which could help in the efficient functioning of the proposed molecular switch supporting synaptic memory.     

Exposition of antitumour activity of a chemically characterized exopolysaccharide from a probiotic Lactobacillus plantarum MTCC 9510

As majority of chemical compounds identified as anti-cancerous are toxic to normal cells, the discovery and identification of new safe drugs is a necessity in the biomedical field. The antioxidant, antitumour and immunomodulating properties of an exopolysaccharide of sequence -α-d-glucose, α-d-mannose and β-d-glucose-, purified from a probiotic Lactobacillus plantarum was studied. The immunostimulation of the compound in human lymphocytes was seen at a concentration of 0.1 mg/mL with 20% proliferation rate. The antitumour studies by morphological apoptosis determination and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay on breast adenocarcinoma cell line (MCF-7) exhibited an IC₅₀ of 10 mg/mL for the compound.     

The PERK Arm of the Unfolded Protein Response Regulates Mitochondrial Morphology during Acute Endoplasmic Reticulum Stress

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