Hajer Ben Hlima

Biocatalysts: application and engineering for industrial purposes

Abstract Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application.

Probing the crucial role of Leu31 and Thr33 of the Bacillus pumilus CBS alkaline protease in substrate recognition and enzymatic depilation of animal hide.

The sapB gene, encoding Bacillus pumilus CBS protease, and seven mutated genes (sapB-L31I, sapB-T33S, sapB-N99Y, sapB-L31I/T33S, sapB-L31I/N99Y, sapB-T33S/N99Y, and sapB-L31I/T33S/N99Y) were overexpressed in protease-deficient Bacillus subtilis DB430 and purified to homogeneity. SAPB-N99Y and rSAPB displayed the highest levels of keratinolytic activity, hydrolysis efficiency, and enzymatic depilation. Interestingly, and at the semi-industrial scale, rSAPB efficiently removed the hair of goat hides within a short time interval of 8 h, thus offering a promising opportunity for the attainment of a lime and sulphide-free depilation process. The efficacy of the process was supported by submitting depilated pelts and dyed crusts to scanning electron microscopic analysis, and the results showed well opened fibre bundles and no apparent damage to the collagen layer. The findings also revealed better physico-chemical properties and less effluent loads, which further confirmed the potential candidacy of the rSAPB enzyme for application in the leather industry to attain an ecofriendly process of animal hide depilation. More interestingly, the findings on the substrate specificity and kinetic properties of the enzyme using the synthetic peptide para-nitroanilide revealed strong preferences for an aliphatic amino-acid (valine) at position P1 for keratinases and an aromatic amino-acid (phenylalanine) at positions P1/P4 for subtilisins. Molecular modeling suggested the potential involvement of a Leu31 residue in a network of hydrophobic interactions, which could have shaped the S4 substrate binding site. The latter could be enlarged by mutating L31I, fitting more easily in position P4 than a phenylalanine residue. The molecular modeling of SAPB-T33S showed a potential S2 subside widening by a T33S mutation, thus suggesting its importance in substrate specificity.

Improvement of Trichoderma reesei xylanase II thermal stability by serine to threonine surface mutations.

Three simple mutants, S80T, S146T, and S149T, and a double mutant, S80T-S149T, were constructed and expressed in Escherichia coli to replace Serine on the surface of the Trichoderma reesei xylanase protein with Threonine residues. While the Wild-type (WT) xylanase showed a half-life time (t1/2) of 20 min at 55 °C, the double mutant was more thermostable exhibiting a t1/2 value of 37 min, followed by the S80T and S149T mutants whose t1/2 values were 25 and 23 min, respectively. At 55 °C, the S146T mutant showed a decrease in thermostability with a t1/2 value of 3 min. While the WT enzyme retained only 32% of residual activity after incubation for 5 min at 60°C, the S80T, S149T, and the S80T-S149T mutant enzymes retained 45%, 41%, and 60%, respectively. Molecular modeling attributed the increase in the thermostability of the S80T and S149T mutants to a new hydrogen bond formation and a packing effect, respectively.

Thermostability improvement of maltogenic amylase MAUS149 by error prone PCR

The thermostability of maltogenic amylase from Bacillus sp. US149 (MAUS149) was improved by random mutagenesis using error prone PCR. The library constructed for the mutants obtained was subjected to screening, leading to the selection of a thermostable mutant enzyme named MA-A27. The latter was noted to contain four single mutations, namely D46V, P78L, V145A, and K548E. The half-life times recorded for MA-A27 at 50°C and 55°C were 70 min and 25 min, compared to 30 min and 13 min for the wild type, respectively. The results from molecular modeling attributed the increase in thermostability observed for MA-A27 to P78L and K548E substitutions that led to new hydrogen bond and salt bridge formations. Further site-directed mutagenesis studies showed that the P78L and K548E single mutations underwent an increase in thermostability, thus confirming the joint contribution of both substitutions to the increase in thermostability observed for MA-A27.

Expression of A. niger US368 xylanase in E. coli: purification, characterization and copper activation.

The XAn11 cDNA was cloned in pET-28a(+) and the recombinant plasmid was transformed in Escherichia coli. The His-tagged r-XAn11 was purified using Ni-NTA affinity and anion exchange chromatography. The enzyme showed a specific activity of 415.1 U mg(-1) and a molecular mass of 25 kDa. It had an optimal activity at pH 5 and 50°C. It was stable in a wide range of pH and in the presence of some detergents and organic solvents. In the presence of 3mM Cu2+, the relative activity of the His-tagged r-XAn11 was enhanced by 54%. This is the first work reporting that copper is a strong activator for xylanase activity making this enzyme very attractive for future industrial applications. Molecular modeling suggests that the contact region between the catalytic site and the N-terminal His-tag fusion peptide could be responsible for the different behavior of the native and recombinant enzyme toward copper.

A trimeric and thermostable lichenase from B. pumilus US570 strain: Biochemical and molecular characterization

New β-1,3;1,4-glucanase (GluUS570) was purified from a newly isolated Bacillus pumilus US570 strain. The enzyme was active in a wide range of pH and temperature and displayed a great thermostability with a half-life of 30min at 80°C. The enzyme was demonstrated to be a lichenase since it was only active toward glucan containing β-1,3;1,4- linkages. The analysis of the enzyme in native and denaturing conditions suggests that it has a trimeric form (75kDa). This is the first report on the purification and characterization of a bacterial lichenase with a trimeric structure. β-1,3;1,4-glucanase encoding gene was amplified, cloned and sequenced showing an open reading frame of 732bp encoding 243 amino acids. The GluUS570 enzyme showed 97% homology with glucanase from Bacillus lichenoformis. The 3D model of GluUS570 in trimeric form was generated and showed that a region named R2 was involved in the oligomerization of the enzyme.

Changes in the catalytic properties and substrate specificity of Bacillus sp. US149 maltogenic amylase by mutagenesis of residue 46.

Maltogenic amylase from Bacillus sp. US149 (MAUS149) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of residue Asp46 in the specificity and catalytic properties of MAUS149 by using site-directed mutagenesis. Three mutated enzymes (D46V, D46G and D46N) were constructed and studied. The three mutants were found to be similar to the wild-type MAUS149 regarding thermoactivity, thermostability and pH profile. Nevertheless, the kinetic parameters for all the substrates of the mutant enzymes D46V and D46G were altered enormously as compared with those of the wild type. Indeed, the Km values of MAUS149/D46G for all substrates were strongly increased. Nevertheless, the affinity and catalytic efficiency of MAUS149/D46V toward β-CD were increased fivefold as compared with those of MAUS149. Molecular modelling suggests that residue D46 forms a salt bridge with residue K282. This bond would maintain the arrangement of side chains of residues Y45 and W47 in a particular orientation that promotes access to the catalytic site and maintains the substrate therein. Hence, any replacement with uncharged amino acids influenced the flexibility of the gate wall at the substrate binding cleft resulting in changes in substrate selectivity.

Modelling Tetraselmis sp. growth-kinetics and optimizing bioactive-compound production through environmental conditions

The aim of this study is to predict Tetraselmis cells growth-kinetic and to induce the synthesis of bioactive compounds (chlorophylls, carotenoids and starch) with high potential for biotechnological applications. Using the statistical criteria, the Baranyi-Roberts model has been selected to estimate the microalgae growth-kinetic values. The simultaneous effects of salinity, light intensity and pH of culture medium were investigated to maximize the production of total chlorophylls, carotenoids and starch. The optimal culture conditions for the production of these compounds were found using Box-Behnken Design. Results have shown that total chlorophyll and carotenoids were attained 21.6mg·g-1DW and 0.042mg·g-1DW, respectively. In addition, the highest starch content of 0.624g·g-1DW has been obtained at neutral pH with high irradiance (182μmolphotonsm-2 s-1) and low salinity (20). A highly correlation (R2 = 0.884) has been found between the gravimetric and flow cytometric measurements of chlorophyll content.

Proteomic Analysis of the Chlorophyta Dunaliella New Strain AL-1 Revealed Global Changes of Metabolism during High Carotenoid Production

The green microalgae Dunaliella genus is known for the production of high added value molecules. In this study, strain AL-1 was isolated from the Sebkha of Sidi El Hani (Sousse, Tunisia). This isolate was identified both morphologically and genetically via 18S rRNA gene sequence as a member of the genus Dunaliella. Strain AL-1 was found to be closely related to Dunaliella salina, Dunaliella quartolecta and Dunaliella polymorpha with more than 97% similarity. Response surface methodology was used to maximize carotenoid production by strain AL-1 by optimizing its growth conditions. The highest carotenoid content was obtained at salinity: 51, light intensity: 189.89 μmol photons·m−2·s−1, and nitrogen: 60 mg·L−1. Proteomic profiling, using two-dimensional gel electrophoresis, was performed from standard and optimized cultures. We detected 127 protein spots which were significantly differentially expressed between standard and optimized cultures. Among them 16 protein spots were identified with mass spectrometry and grouped into different functional categories using KEGG (Kyoto Encyclopedia of Genes and Genomes) such as photosynthetic Calvin cycle, regulation/defense, energy metabolism, glycolysis, and cellular processes. The current study could be of great interest in providing information on the effect of stressful conditions in microalgae carotenoid production.

Identification and characterization of single nucleotide polymorphism markers in FADS2 gene associated with olive oil fatty acids composition

BackgroundGenotyping of the FAD2.1 and FAD2.3 polymorphisms in the fatty acid desaturase 2 gene (FADS2) shows that they are associated with the fatty acids composition of olive oil samples. However, these associations require further confirmation in the Tunisian olive oil cultivars, and little is known about the effect of polymorphisms in fatty acid-related genes on olive oil mono- and poly- unsaturated fatty acids distribution.MethodsA set of olive oils from 12 Tunisian cultivars was chosen. The fatty acid composition of each olive oil sample was determined by gas chromatography. Statistical and modeling Bayesian analyses were used to assess whether the FAD2.1 and FAD2.3 genotypes were associated with fatty acids composition.ResultsThe TT-FAD2.1 and the GG-FAD2.3 genotypes were found to be associated with a lower proportion of oleic acid (C18:1) (r = −0.778, p = 0.003; r = −0.781, p= 0.003) as well as higher proportion of linoleic (C18:2) (r = 0.693, p = 0.012; r = −0.759, p= 0.004) and palmitic acids (C16:0) (r = 0.643, p = 0.024; r = −0.503, p= 0.095), making varieties with this haplotype (i.e. Chemlali Sfax and Meski) producing more saturated (C16: 0) and polyunsaturated acids than oleic acid. The latter plays a major role in preventing several diseases.ConclusionThe two associations FADS2 FAD2.1 and FADS2 FAD2.3 with the fatty acid compositions of olive oil samples were identified among the studied olive cultivars. These associations differed between studied cultivars, which might explain variability in lipidic composition among them and consequently reflecting genetic diversity through differences in gene expression and biochemical pathways. FADS2 locus would constitute thus a good marker for detecting interesting lipidic chemotypes among commercial olive oils.