Samir Bejar

Application of a statistical design to the optimization of parameters and culture medium for α-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product)

The production optimization of alpha-amylase (E.C.3.2.1.1) from Aspergillus oryzae CBS 819.72 fungus, using a by-product of wheat grinding (gruel) as sole carbon source, was performed with statistical methodology based on three experimental designs. The optimisation of temperature, agitation and inoculum size was attempted using a Box-Behnken design under the response surface methodology. The screening of nineteen nutrients for their influence on alpha-amylase production was achieved using a Plackett-Burman design. KH(2)PO(4), urea, glycerol, (NH(4))(2)SO(4), CoCl(2), casein hydrolysate, soybean meal hydrolysate, MgSO(4) were selected based on their positive influence on enzyme formation. The optimized nutrients concentration was obtained using a Taguchi experimental design and the analysis of the data predicts a theoretical increase in the alpha-amylase expression of 73.2% (from 40.1 to 151.1 U/ml). These conditions were validated experimentally and revealed an enhanced alpha-amylase yield of 72.7%.

Purification and characterization of a thermostable keratinolytic serine alkaline proteinase from Streptomyces sp. strain AB1 with high stability in organic solvents

A keratinolytic alkaline proteinase (KERAB) was isolated from Streptomyces sp. strain AB1. Based on MALDI-TOF mass spectrometry analysis, the purified enzyme is a monomer with a molecular mass of 29850.17Da. The NH(2)-terminal sequence of the enzyme was determined to be TQANPPSWGLDDIDQTAL. This keratinase was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DIFP), which suggests that it belongs to the serine protease family. Using keratin azure as a substrate, the optimum pH and temperature values for keratinase activity were pH 11.5 and 75 degrees C, respectively. This keratinase was stable between 30 and 60 degrees C and pH 4 and 11 for 4 and 96 h, respectively, and thermoactivity and thermostability were enhanced in the presence of 5 mM Mg(2+). Its catalytic efficiency was higher than those of SAPB-L31I/T33S/N99Y, nattokinase and subtilisin Carlsberg. KERAB exhibited stability to detergents and high resistance against organic solvents and was able to degrade feathers completely. These properties make KERAB a potential candidate for future applications in detergent formulations, dehairing during leather processing, and non-aqueous peptide biocatalysis.

Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis.

The serine alkaline protease, SAPB, from Bacillus pumilus CBS is characterized by its high thermoactivity, pH stability and high catalytic efficiency (k(cat)/K(m)) as well as its excellent stability and compatibility with an alkaline environment under harsh washing conditions. Based on sequence alignments and homology-modeling studies, the present study identified five amino acids Leu31, Thr33, Asn99, Phe159 and Gly182 being putatively important for the enzymatic behaviour of SAPB. To corroborate the role of these residues, 12 mutants were constructed by site-directed mutagenesis and then purified and characterized. The findings demonstrate that the single mutants F159T, F159S and G182S and combined double substitutions were implicated in the decrease of the optimum pH and temperature to 8.0-9.0 and 50 degrees C, respectively, and that mutant F159T/S clearly affected substrate affinity and catalytic efficiency. With regards to the single L31I, T33S and N99Y and combined double and triple mutations, the N99Y mutation strongly improved the half-life times at 50 degrees C and 60 degrees C to 660 and 295 min from of 220 and 80 min for the wild-type enzyme, respectively. More interestingly, this mutation also shifted the optimum temperature from 65 degrees C to 75 degrees C and caused a prominent 31-fold increase in k(cat)/K(m) with N-succinyl-l-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF). The L31I and T33S mutants were observed to improve mainly the optimum pH from 11.0 to 11.5 and from 11.0 to 12.0, respectively. Kinetic studies of double and triple mutants showed that the cumulative effect of polar uncharged substitutions had a synergistic effect on the P1 position preference using synthetic peptide substrates, which confirms the implication of these amino acids in substrate recognition and catalytic efficiency.

A new Lactobacillus plantarum strain, TN8, from the gastro intestinal tract of poultry induces high cytokine production.

This study aimed to determine the probiotic potential of 100 strains of Lactic Acid Bacteria (LAB) isolated from different intestinal segments of indigenous poultry in Tunisia. The strains were submitted to a battery of standard tests and criteria commonly used for determining their probiotic properties and attributes. The findings revealed that 19 of the isolates exhibited antimicrobial activity against 4 pathogenic bacteria, and that 4 (TN1, TN8, TN7, and TN13) showed good resistance to pH 3 and 5% bovine bile. Three isolates, namely TN1, TN8, and TN13, showed sensitivity to several antibiotics and were, therefore, selected for further enzymatic activity assays. Two isolates, namely TN1 and TN8, showed high efficacy of adhesion to chicken enterocytes. The cytokines released after stimulation by the two isolates showed high anti-inflammatory profiles, with an increased rate of Interleukin-10 (IL-10) production for the TN8 strain. Showing the highest performance, TN8 was submitted to 16S rRNA gene sequencing, which revealed that the strain was of the species Lactobacillus plantarum. Overall, the findings indicate that the Lactobacilli from poultry intestine has a number of promising properties that make it candidate for application as a probiotic additive in poultry industry.

Rational design of Bacillus stearothermophilus US100 l-arabinose isomerase: Potential applications for d-tagatose production

L-arabinose isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.

Production of D-tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase

Lactobacillusdelbrueckii subsp. bulgaricus and Streptococcus thermophilus are used for the biotransformation of milk in yoghurt. During milk fermentation, these lactic acid bacteria (LAB) hydrolyze lactose producing a glucose moiety that is further metabolized and a galactose moiety that they are enable to metabolize. We investigated the ability of L. bulgaricus and S. thermophilus strains expressing a heterologous L-arabinose isomerase to convert residual D-galactose to D-tagatose. The Bacillus stearothermophilus US100l-arabinose isomerase (US100l-AI) was expressed in both LAB, using a new shuttle vector where the araA US100 gene is under the control of the strong and constitutive promoter of the L. bulgaricus ATCC 11842 hlbA gene. The production of L-AI by these LAB allowed the bioconversion of D-galactose to D-tagatose during fermentation in laboratory media and milk. We also established that the addition of L-AI to milk also allowed the conversion of D-galactose into D-tagatose during the fermentation process.

Exploring the acidotolerance of β-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus: an attractive enzyme for lactose bioconversion

The LacZ gene encoding beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 (L. bulgaricus) was cloned, sequenced and expressed in Escherichia coli, followed by purification and characterization of the protein. The recombinant enzyme was shown to be a homotetramer and could be distinguished from homologues by its relatively low and broad optimal temperature range, from 35 to 50 degrees C, coupled with an optimal pH of 5.0-5.5. Remarkably, the E491A mutant showed the same optimal temperature, but displayed an optimal pH at 6.5-7.0. Whilst these beta-galactosidases are inhibited by Cu(2+) they require only 1mM Mn(2+) and 1mM Co(2+) for optimal activity and thermostability. The wild-type enzyme was remarkably stable at acid pH values when compared to mutant E491A. Kinetic studies demonstrated that the E491A mutation affected catalysis rather than enzyme affinity. Furthermore, the wild-type protein efficiently cleaved lactose extracted from whey; however, in milk the E491A mutant showed the highest lactose bioconversion rate. Thus, these enzymes are interesting at the industrial level for hydrolysis of lactose extracted from whey or milk, and thus could contribute to overcoming the lactose intolerance problem generated by milk products.