Statistical Optimization and Partial Characterization of Xylanases Produced by Streptomyces sp. S1M3I Using Olive Pomace as a Fermentation Substrate.

Xylanase production by Streptomyces sp. S1M3I was optimized by response surface methodology (RSM), followed by a partial characterization of these enzymes. Olive pomace was used as a substrate for growing Streptomyces sp. S1M3I in submerged fermentation. Effects of incubation time, pH, temperature, carbon source, nitrogen source, and inoculum size on xylanase production were studied, through the one-factor-at-a-time method. Then, a 33-factorial experimental design with RSM and the Box-Behnken design was investigated for the major influence factors. Maximum xylanase production (11.28 U/mL) was obtained when the strain was grown in mineral medium supplemented with 3% (w/v) of olive pomace powder and 0.3% (w/v) of ammonium sulfate, at a pH 7.4 and an incubation temperature of 40 °C. The xylanases in the supernatant degraded all tested substrates, with higher activity for the low-viscosity wheat arabinoxylan substrate. Two xylanases with close molecular masses were detected by zymogram analysis: Xyl-1 and Xyl-2 with molecular masses of 24.14 kDa and 27 kDa, respectively. The optimization of enzyme production parameters of Streptomyces sp. S1M3I and the characterization of these enzymes are prerequisites to enhancing xylanase production yield, which is crucial for further biotechnological processes.

The Mechanisms of Alpha-Amylase Inhibition by Flavan-3-Ols and the Possible Impacts of Drinking Green Tea on Starch Digestion.

Many studies have shown that flavan-3-ols inhibit mammalian alpha-amylases but the published IC50 and Ki values vary up to a thousand times. We therefore tested the effects of 6 pure flavan-3-ols-abundant in green tea-on the activity of pure porcine pancreatic alpha-amylase (PPA) under steady-state kinetic conditions. We used both amylose and maltopentaose as substrates, along with spectrophotometry and chromatography as analytical tools, respectively. A Docking approach was also used to probe the interaction between PPA and each flavan-3-ol. The results showed that the 6 flavan-3-ols inhibit amylose hydrolysis with Ki comprised between 7 and 34 µM, according to a mixed inhibition profile for gallocatechin gallate, and a competitive inhibition profile for the 5 other flavanols. Only the galloyl-containing flavan-3-ols inhibited the maltopentaose hydrolysis with a Ki of about 30 µM according to a noncompetitive profile. We conclude that dietary flavan-3-ols could inhibit starch digestion nonnegligibly. The results of the docking trials were concordant with the kinetic data and have noticeably revealed that the cis-flavan-3-ols epigallocatechin gallate and epicatechin gallate bind similarly to PPA, involving π-stacking with Trp59.

Selective Isolation and Screening of Actinobacteria Strains Producing Lignocellulolytic Enzymes Using Olive Pomace as Substrate.

BACKGROUND: Olive pomace, as the main by-product of the olive oil industry, is recently recycled as fermentation substrate for enzyme production. OBJECTIVES: Actinobacteria isolates were separated from an Algerian soil under olive pomace cultivation and were evaluated for their lignocellulolytic enzymes production. MATERIALS AND METHODS: Isolates of Actinobacteria were separated from soils around oil mills using four isolation media, among them three were enriched by olive pomace. The isolates were screened for their cellulolytic, xylanolytic and ligninolytic activities. Isolates with potential of producing lignocellulose-degrading enzymes were selected under submerged fermentation based olive pomace. RESULTS: Ninety isolates of Actinobacteria were separated from soil samples. M3 medium (raw pomace autoclaved alone) was the best isolation medium (68 strains), whereas, the soil from oil mill with continuous system (S1) led to separation of 52 strains. Among the 90 isolates, 82 were shown promising enzyme activity, 19 isolates were presented the largest zone diameter (<30 mm). S1M3I and S1M3II isolates were exhibited the highest values. CONCLUSIONS: Olive pomace with medium low cost and high titers of enzymes can be valorized by culture of Actinobacteria to produce lignocellulolytic enzymes for industrial applications.

Functional characterization of a vacuolar invertase from Solanum lycopersicum: post-translational regulation by N-glycosylation and a proteinaceous inhibitor.

Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves.

GH10 xylanase D from Penicillium funiculosum: biochemical studies and xylooligosaccharide production.

BACKGROUND: The filamentous fungus Penicillium funiculosum produces a range of glycoside hydrolases (GH). The XynD gene, encoding the sole P. funiculosum GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast Pichia pastoris, in order to compare the results obtained with the P. funiculosum GH11 xylanases data. RESULTS: High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L-1. The protein was purified to homogeneity using one purification step. The apparent size on SDS-PAGE was around 64 kDa and was 46 kDa by mass spectrometry thus higher than the expected molecular mass of 41 kDa. The recombinant protein was N- and O-glycosylated, as demonstrated using glycoprotein staining and deglycosylation reactions, which explained the discrepancy in molecular mass. Enzyme-catalysed hydrolysis of low viscosity arabinoxylan (LVAX) was maximal at pH 5.0 with Km(app) and kcat/Km(app) of 3.7 ± 0.2 (mg.mL-1) and 132 (s-1mg-1.mL), respectively. The activity of XynD was optimal at 80°C and the recombinant enzyme has shown an interesting high thermal stability at 70°C for at least 180 min without loss of activity. The enzyme had an endo-mode of action on xylan forming mainly xylobiose and short-chain xylooligosaccharides (XOS). The initial rate data from the hydrolysis of short XOS indicated that the catalytic efficiency increased slightly with increasing their chain length with a small difference of the XynD catalytic efficiency against the different XOS. CONCLUSION: Because of its attractive properties XynD might be considered for biotechnological applications. Moreover, XOS hydrolysis suggested that XynD possess four catalytic subsites with a high energy of interaction with the substrate and a fifth subsite with a small energy of interaction, according to the GH10 xylanase literature data.

Proteomic of lipid rafts in the exocrine pancreas from diet-induced obese rats.

In the present work, we induced obesity in rats with high-energy-starch diet and studied exocrine pancreas response. The zymogen granule (ZG) or purified plasma membrane (PM) from the exocrine pancreas was used for the isolation of the detergent-resistant membranes (DRMs). Based on high content of cholesterol, GM1, the bile salt dependent lipase (BSDL), and GP2 enrichment, the low-density fractions were defined as lipid rafts. Additionally, the rafts vesicles were determined by immunogold labeling with anti BSDL. By combining MALDI-TOF/MS and nano-LC ESI Q-TOF MS/MS proteomic identification we have selected 33 proteins from the lipid rafts which were classified into at least four functional families. Our data suggest that the acinar PM from the diet-induced obesity rats may be organized into lipid rafts, and characterization of rafts proteome can contribute to improve our understanding of food digestion under obesity.

Potential physiological role of plant glycosidase inhibitors.

Carbohydrate-active enzymes including glycosidases, transglycosidases, glycosyltransferases, polysaccharide lyases and carbohydrate esterases are responsible for the enzymatic processing of carbohydrates in plants. A number of carbohydrate-active enzymes are produced by microbial pathogens and insects responsible of severe crop losses. Plants have evolved proteinaceous inhibitors to modulate the activity of several of these enzymes. The continuing discovery of new inhibitors indicates that this research area is still unexplored and may lead to new exciting developments. To date, the role of the inhibitors is not completely understood. Here we review recent results obtained on the best characterised inhibitors, pointing to their possible biological role in vivo. Results recently obtained with plant transformation technology indicate that this class of inhibitors has potential biotechnological applications.

Porcine pancreatic alpha-amylase inhibition by the kidney bean (Phaseolus vulgaris) inhibitor (alpha-AI1) and structural changes in the alpha-amylase inhibitor complex.

Porcine pancreatic alpha-amylase (PPA) is inhibited by the red kidney bean (Phaseolus vulgaris) inhibitor alpha-AI1 [Eur. J. Biochem. 265 (1999) 20]. Inhibition kinetics were carried out using DP 4900-amylose and maltopentaose as substrate. As shown by graphical and statistical analysis of the kinetic data, the inhibitory mode is of the mixed noncompetitive type whatever the substrate thus involving the EI, EI2, ESI and ESI2 complexes. This contrast with the E2I complex obtained in the crystal and with biophysical studies. Such difference very likely depends on the [I]/[E] ratio. At low ratio, the E2I complex is favoured; at high ratio the EI, ESI and EI2 complexes are formed. The inhibition model also differs from those previously proposed for acarbose [Eur. J. Biochem. 241 (1996) 787 and Eur. J. Biochem. 252 (1998) 100]. In particular, with alpha-AI1, the inhibition takes place only when PPA and alpha-AI are preincubated together before adding the substrate. This indicates that the abortive PPA-alphaAI1 complex is formed during the preincubation period. One additional carbohydrate binding site is also demonstrated yielding the ESI complex. Also, a second protein binding site is found in EI2 and ESI2 abortive complexes. Conformational changes undergone by PPA upon alpha-AI1 binding are shown by higher sensitivity to subtilisin attack. From X-ray analysis of the alpha-AI1-PPA complex (E2I), the major interaction occurs with two hairpin loops L1 (residues 29-46) and L2 (residues 171-189) of alpha-AI1 protruding into the V-shaped active site of PPA. The hydrolysis of alpha-AI1 that accounts for the inhibitory activity is reported.

On the mechanism of alpha-amylase.

Two inhibitors, acarbose and cyclodextrins (CD), were used to investigate the active site structure and function of barley alpha-amylase isozymes, AMY1 and AMY2. The hydrolysis of DP 4900-amylose, reduced (r) DP18-maltodextrin and maltoheptaose (catalysed by AMY1 and AMY2) was followed in the absence and in the presence of inhibitor. Without inhibitor, the highest activity was obtained with amylose, kcat/Km decreased 103-fold using rDP18-maltodextrin and 10(5) to 10(6)-fold using maltoheptaose as substrate. Acarbose is an uncompetitive inhibitor with inhibition constant (L1i) for amylose and maltodextrin in the micromolar range. Acarbose did not bind to the active site of the enzyme, but to a secondary site to give an abortive ESI complex. Only AMY2 has a second secondary binding site corresponding to an ESI2 complex. In contrast, acarbose is a mixed noncompetitive inhibitor of maltoheptaose hydrolysis. Consequently, in the presence of this oligosaccharide substrate, acarbose bound both to the active site and to a secondary binding site. alpha-CD inhibited the AMY1 and AMY2 catalysed hydrolysis of amylose, but was a very weak inhibitor compared to acarbose.beta- and gamma-CD are not inhibitors. These results are different from those obtained previously with PPA. However in AMY1, as already shown for amylases of animal and bacterial origin, in addition to the active site, one secondary carbohydrate binding site (s1) was necessary for activity whereas two secondary sites (s1 and s2) were required for the AMY2 activity. The first secondary site in both AMY1 and AMY2 was only functional when substrate was bound in the active site. This appears to be a general feature of the alpha-amylase family.