Mamdouh Ben Ali

Thermostability enhancement and change in starch hydrolysis profile of the maltohexaose-forming amylase of Bacillus stearothermophilus US100 strain

The implications of Asn315 and Val450 in the atypical starch hydrolysis profile of Bacillus stearothermophilus Amy (a-amylase) US100 have been suggested previously [Ben Ali, Mhiri, Mezghani and Bejar (2001) Enzyme Microb. Tech. 28, 537-542]. In order to confirm this hypothesis, three mutants were generated. Of these two have a single mutation, N315D or V450G, whereas the third contains both mutations. Analysis of the starch breakdown-profile of these three mutants, as well as of the wild-type, allowed us to conclude that each single mutation induces a small variation in the hydrolysis product. However, the major end product produced by the double mutant shifts from maltopentaose/maltohexaose to maltose/maltotriose, confirming the involvement of these two residues in starch hydrolysis. The superimposition of AmyUS100 model with that of Bacillus licheniformis shows in AmyUS100 an additional loop containing residues Ile214 and Gly215. Remarkably, the deletion of these two residues increases the half-life at 100 degrees C from 15 min to approx. 70 min. Moreover, this engineered amylase requires less calcium, 25 p.p.m. instead of 100 p.p.m., to reach maximal thermostability.

Purification and sequence analysis of the atypical maltohexaose-forming α-amylase of the B. stearothermophilus US100

The maltohexaose-forming alpha-amylase, of B. stearothermophilus US100, was purified to homogeneity by a combination of osmotic shock, starch adsorption and anion exchange chromatography. This enzyme has a relative molecular mass of 59 kDa. The analysis of the nucleotide sequence, of the corresponding gene, allowed the identification of a single open reading frame encoding a 549 amino acid protein, exhibiting a large homology to the other B. stearothermophilus alpha-amylases. This homology reaches a maximum with those of DY-5 and DN1792 strains with respectively 3 and 4 aa different over 549. The relatively small differences, between Amy US100 and that of DN1792 strain, take in more importance since we have demonstrated that these enzymes differ essentially by their starch hydrolysis pattern.

A thermostable α-amylase producing maltohexaose from a new isolated Bacillus sp. US100 : study of activity and molecular cloning of the corresponding gene

A Bacillus sp. US100 strain was isolated from Tunisian hot spring soil. This strain secretes a thermoactive amylase having an optimal temperature of 82°C and a remarkable thermostability with a half time of 40 min at 110°C in the presence of 20% (w/v) of substrate. The main end products of its action on starch are maltohexaose and maltopentaose which is the smallest substrate hydrolysed by this enzyme. The molecular cloning, in E. coli, of a 3-kb DNA fragment encoding for this activity is also reported.

Engineering of the α‐amylase from Geobacillus stearothermophilus US100 for detergent incorporation

AmyUS100ΔIG is a variant of the most thermoactive and thermostable maltohexaose forming α‐amylase produced by Geobacillus stearothermophilus sp.US100. This enzyme which was designed to improve the thermostability of the wild‐type enzyme has acquired a very high resistance to chelator agents. According to modeling structural studies and with the aim of enhancing its resistance towards chemical oxidation, a mutant (AmyUS100ΔIG/M197A) was created by substituting methionine 197 to alanine. The catalytic proprieties of the resulting mutant show alterations in the specific activity and the profile of starch hydrolysis. Interestingly, AmyUS100ΔIG/M197A displayed the highest resistance to oxidation compared to the AmyUS100ΔIG and to Termamyl300®, the well‐known commercial amylase used in detergent. Further, performance of the engineered α‐amylase was estimated in the presence of commonly used detergent compounds and a wide range of commercial detergent (liquid and solid). These studies indicated a high compatibility and performance of AmyUS100ΔIG/M197A, suggesting its potential application in detergent industry. Biotechnol. Bioeng. 2009;102: 380–389.

Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436Arg substitutions

Based on sequence alignments and homology modeling, Gly 312 and Lys 436 of the maltogenic amylase from Bacillus sp. US149 (MAUS149) were selected as targets for site-directed mutagenesis to improve the thermostability of the enzyme. Variants of MAUS149 with amino acid substitutions G312A, K436R and G312A-K436R had substrate specificities, kinetic parameters and pH optima similar to those of the wild-type enzyme; however, the enzymes with substitutions K436R and G312A-K436R, had an optimal temperature of 45 °C instead of the 40 °C for the wild-type enzyme. The half-life time at 55 °C increased from 15 to 25 min for the double mutant. Molecular modeling suggests that the increase in thermostability was due to new hydrophobic interactions and the formation of a salt bridge and hydrogen bond in the G312A and K436R variants, respectively. The double mutant could be a potential candidate for application in the bread industry.

The importance of an extra loop in the B-domain of an α-amylase from B. stearothermophilus US100

To provide insight into the potential role of a loop in domain B of several bacterial alpha-amylases, molecular and structural investigation of Bacillus stearothermophilus alpha-amylase (Amy US100) was used as a model. Combination deletion mutants of G(213), I(214) and G(215), described as a loop-forming on the surface bacterial amylases, were subjected to biochemical and structural investigation. Thermoactivity, thermostability as well calcium requirement were studied for each mutant. Thus, deletion of one residue differently affects only the thermostability. Shortening the loop by deletion of G(213)-I(214) or I(214)-G(215) improved the thermostability and reduces calcium requirement. However, the deletion of three residues has a negative effect on thermostability and reduces the optimal temperature by 17 degrees C. The structural investigation showed that stabilizing deletions contribute to reinforce the architecture of domain B and the active site conformation. The deletion of three residues reduces the flexibility of this region and abolishes a denser hydrogen bond network.

Crucial role of Pro 257 in the thermostability of Bacillus phytases: biochemical and structural investigation.

We have previously cloned and characterized the thermostable phytase (PHY US417) from Bacillus subtilis US417. It differs with PhyC from B. subtilis VTTE-68013 by the R257P substitution. PHY US417 was shown to be more thermostable than PhyC. To elucidate the mechanism of how the Pro 257 changes the thermostability of Bacillus phytases, this residue was mutated to Arg and Ala. The experimental results revealed that the thermostability of the P257A mutants and especially P257R was significantly decreased. The P257R and P257A mutants recovered, respectively, 64.4 and 81.5% of the wild-type activity after incubation at 75 °C for 30 min in the presence of 5mM CaCl(2). The P257R mutation also led to a severe reduction in the specific activity and catalytic efficiency of the enzyme. Structural investigation, by molecular modeling of PHY US417 and PhyC focused on the region of the 257 residue, revealed that this residue was present in a surface loop connecting two of the six characteristic β sheets. The P257 residue is presumed to reduce the local thermal flexibility of the loop, thus generating a higher thermostability.

Molecular cloning, structural analysis and modelling of the AcAFP antifungal peptide from Aspergillus clavatus.

An abundantly secreted thermostable peptide (designed AcAFP) with a molecular mass of 5777 Da was isolated and purified in a previous work from a local strain of A. clavatus (VR1). Based on the N-terminal amino acid (aa) sequence of the AcAFP peptide, an oligonucleotide probe was derived and allowed the amplification of the encoding cDNA by RT-PCR. This cDNA fragment encodes a pre-pro-protein of 94 aa which appears to be processed to a mature product of 51 aa cys-rich protein. The deduced aa sequence of the pre-pro-sequence reveals high similarity with ascomycetes antifungal peptide. Comparison of the nucleotide sequence of the genomic fragment and the cDNA clone revealed the presence of an open reading frame of 282 bp interrupted by two small introns of 89 and 56 bp with conserved splice site. The three-dimensional (3D) structure modeling of AcAFP exhibits a compact structure consisting of five anti-parallel beta barrel stabilized by four internal disulfide bridges. The folding pattern revealed also a cationic site and spatially adjacent hydrophobic stretch. The antifungal mechanism was investigated by transmission and confocal microscopy. AcAFP cause cell wall altering in a dose-dependent manner against the phytopathogenic fungus Fusarium oxysporum.

Genomic organization of a polygalacturonase gene from a hyperpectinolytic mutant strain of Penicillium occitanis.

The pga1 gene encoding an endopolygalacturonase was isolated from a hyperpectinolytic mutant strain of Penicillium occitanis. It consists of an ORF of 1.155 kbp encoding a putative protein of 346 amino acids with a predicted molecular mass of 39 kDa, belonging to the family 28 of glycosyl hydrolases. The deduced amino acid sequence comprises a putative 38 N terminal amino acids of the prepropeptide. The nature and position of amino acids comprising the active site as well as the overall three-dimensional structure were well conserved between the P. occitanis pga1 and all polygalacturonases. The coding region of the pga1 gene is interrupted by three short introns of 57, 53 and 65 bp in length. In addition to the determination of the transcription start site, the promoter sequence from the pga1 gene was analysed. It showed the conservation of known response elements for CreA and Hap2-3-4 factors. Southern blot analysis at high stringency shows that the isolated polygalacturonase gene exists as a single copy in the fungus genome. Northern blot analysis confirmed the constitutive hyperpectinolytic nature of the hyperpectinolytic CT1 mutation as high levels of pga1 mRNA were observed either on pectin or on glucose-grown cells.

Acute and delayed responses of C-reactive protein, malondialdehyde and antioxidant markers after resistance training session in elite weightlifters: Effect of time of day.

The aim of this study was to investigate the effect of an Olympic-Weightlifting-session followed by 48-h recovery period on the oxidative and antioxidant parameters’ diurnal variation. Nine weightlifters (21 ± 0.5 years) performed, in randomized order, three Olympic-Weightlifting-sessions at 08 h:00, 14 h:00 and 18 h:00. Blood samples were collected: at rest and 3 min and 48 h after each session. C-reactive protein (CRP), rate of lipid peroxidation and antioxidant activities were assessed. At rest, analysis of variance showed a significant time of day (TOD) effect (p < 0.05) for uric acid, catalase and glutathione peroxidase with higher values at 14 h:00 and 18 h:00 compared with 08 h:00. However, no significant TOD effect for malondialdehyde, total bilirubin and CRP was observed. Given the profound changes (p < 0.001) in the post-training session values, these diurnal variations have been altered immediately and even 48 h after the training sessions. Despite the significant decreases in the post-training values after the 48-h recovery period (p < 0.05), levels of lipid peroxidation and enzymatic defense remained elevated (p < 0.05) 48 h after the morning training session. However, after the afternoon and evening sessions, the same period was sufficient to return values to the baseline levels. In conclusion, the morning session seems to generate the most important acute and delayed lipid peroxidation responses. Therefore, weightlifting coaches should avoid scheduling their training sessions in the morning-hours.