Neodymium adsorption from aqueous solution by ß-cyclodextrin nanosponges and a polymer valorized from potato peels waste: experiments and conventional and statistical physics interpretations.

Using organic waste and residue streams to be turned into valuable and greener materials for various applications has proven an efficient and suitable strategy. In this work, two green materials (nanosponges and a polymer) were synthesized using potato peels and applied for the first time to adsorb and recover Neodymium (Nd3+) from aqueous solutions. The recovery of Nd3+ that belongs to the rare earth elements has attracted important interest due to its/their importance in several industrial and technological applications. The fine potato peel waste (FPPW) polymer presented an irregular shape and porous surface. At the same time, the ß-cyclodextrin (ß-CD) nanosponges had uniform distribution with regular and smooth shapes. ß-CD nanosponges exhibited a much higher total carboxyl content (4.02 mmol g-1) than FPPW (2.50 mmol g-1), which could impact the Nd3+ adsorption performance because carboxyl groups can interact with cations. The adsorption capacity increased with the increase of the pH, reaching its maximum at pHs 6-7 for ß-CD nanosponges and 4-7 for FPPW polymer. The kinetic and equilibrium data were well-fitted by General order and Liu models. ß-CD nanosponges attained adsorption capacity near 100 mg Nd per gram of adsorbent. Thermodynamic and statistical physical results corroborated that the adsorption mechanism was due to electrostatic interaction/complexation and that the carboxyl groups were important in the interactions. ß-CD nanosponges (three cycles of use) were more effective than FPPW (one cycle of use) in the regeneration. Finally, ß-CD nanosponges could be considered an eco-friendly adsorbent to recover Nd3+ from aqueous matrices.

Superoxide dismutase (SOD) family in durum wheat: promising candidates for improving crop resilience.

The SOD family has been extensively analyzed at genome wide level in several crops. However, little is known about this family in durum wheat. In this study, a total of 14 TdSOD genes were identified in whole durum wheat genome including 8 TdCu-ZnSODs, 2 TdMnSODs, and 4 TdFeSODs. In silico analysis evinced that TdSOD family members displayed a closer evolutionary relationship, similar gene structure and protein features with their homologs from other plant species. Furthermore, the analysis of their promoter regions revealed the presence of a great number of cis-regulatory elements related to plant development, abiotic and biotic stresses, phytohormones, and several potential binding sites for transcription factors. Interestingly, 3D structure analysis revealed that TdCu-ZnSOD2A-2 and TdCu-ZnSOD2B-2, belonging to the Cu-Zn group, were modeled as copper chaperone for SOD like their homologs from rice and Arabidopsis. The expression profile of eight TdSOD candidate genes was investigated under salt, drought, cold, and ABA treatments. Notably, TdCu-ZnSOD2A-1, TdFeSOD4A-1, and TdFeSOD7A-1 were significantly up-regulated under all stress treatments. On the other hand, TdCu-ZnSOD7B and TdMnSOD2B were strongly expressed in roots and leaves under cold stress and TdCu-ZnSOD2B-2 was particularly up-regulated in leaves under ABA treatment. Ultimately, these findings provide valuable information for the identification of attractive candidate genes to improve wheat resilience.

Identification and molecular characterization of a novel non-specific lipid transfer protein (TdLTP2) from durum wheat.

Non-specific lipid transfer proteins (nsLTPs) are small, cysteine-rich proteins, a part of the pathogenesis-related protein family, and numerous of them act as positive regulators during plant disease resistance, growth, and reproduction. These proteins are involved also in the intracellular transfer of lipids, as well as in plant immune responses. Besides their differences in sequences, they show similar features in their structure. However, they show distinct lipid-binding specificities signifying their various biological roles that dictate further structural study. This study reports the identification, in silico characterization and purification of a novel member of the nsLTP2 protein family from durum wheat, TdLTP2. It was generated and purified using the combination of gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its identity was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MALDI-TOF). TdLTP2 had been expressed in different stress to detect its localization; therefore, fluor-immunolocalization studies accomplished this data. In this approach, to assess the allergenicity of TdLTP2, thirty patients with baker's asthma were enrolled and ELISA to detect the presence of specific IgE antibodies tested their sera. Moreover, the lipid-binding properties of TdLTP2 were examined in vitro and validated using a molecular docking study. In summary, our results demonstrate a new addition of member in plant nsLTPs family, TdLTP2, which can develop a better understanding about its biological functions and shed light on future applications.

Highlight on mutations affecting the US132 cyclodextrin glucanotransferase binding specificity, thermal stability, and anti-staling activity.

We have already reported that the triple mutant (K47E-S382P-N655S of Paenibacillus pabuli US132 cyclodextrin glucanotransferase US132 (CGTase)) altered the CGTase specificity. In the current study, the single (K47E, S382P and N655S) and double (K47E+S382P, K47E+N655S, and S382P+N655S) mutants were constructed to elucidate the synergic or antagonist substitutions effect on the enzyme behavior. For the six generated mutants, an improvement of the dextrinization/cyclization ratio from 4.4 to 6-fold was observed when compared to the wild-type enzyme. The mutations effect on enzyme specificity was not attributed to synergy modulation since the single mutant N655S had the highest ratio enhancement. Moreover, the mutant N655S revealed the highest ß-cyclodextrin binding affinity with a high amount of hydrophobic bonds which might be contributed to the apparent decrease in the cyclization activity. On the other hand, mutations N655S, K47E, and (K47E-N655S) showed the same positive effect on thermal activity. The highest stability was attained at 70 °C by N655S to be 3.6-fold higher than the wild-type. The addition of N655S to wheat flour induced a decrease of dough and bread hardness and led to an increase in dough and bread cohesiveness and a rise in bread masticability values compared to the control. This mutant addition also corrected the dough elasticity decrease engendered by the wild-type CGTase indicating that N655S-CGTase could be an alternative anti-staling agent.

Make it clean, make it safe: A review on virus elimination via adsorption.

Recently, the potential dangers of viral infection transmission through water and air have become the focus of worldwide attention, via the spread of COVID-19 pandemic. The occurrence of large-scale outbreaks of dangerous infections caused by unknown pathogens and the isolation of new pandemic strains require the development of improved methods of viruses' inactivation. Viruses are not stable self-sustaining living organisms and are rapidly inactivated on isolated surfaces. However, water resources and air can participate in the pathogens' diffusion, stabilization, and transmission. Viruses inactivation and elimination by adsorption are relevant since they can represent an effective and low-cost method to treat fluids, and hence limit the spread of pathogen agents. This review analyzed the interaction between viruses and carbon-based, oxide-based, porous materials and biological materials (e.g., sulfated polysaccharides and cyclodextrins). It will be shown that these adsorbents can play a relevant role in the viruses removal where water and air purification mostly occurring via electrostatic interactions. However, a clear systematic vision of the correlation between the surface potential and the adsorption capacity of the different filters is still lacking and should be provided to achieve a better comprehension of the global phenomenon. The rationalization of the adsorption capacity may be achieved through a proper physico-chemical characterization of new adsorbents, including molecular modeling and simulations, also considering the adsorption of virus-like particles on their surface. As a most timely perspective, the results on this review present potential solutions to investigate coronaviruses and specifically SARS-CoV-2, responsible of the COVID-19 pandemic, whose spread can be limited by the efficient disinfection and purification of closed-spaces air and urban waters.

A thermophilic and thermostable xylanase from Caldicoprobacter algeriensis: Recombinant expression, characterization and application in paper biobleaching.

A novel xylanase gene xynBCA, encoding a polypeptide of 439 residues (XynBCA), was cloned from Caldicoprobacter algeriensis genome and recombinantly expressed in Escherichia coli BL21(DE3). The amino acid sequence analysis showed that XynBCA belongs to the glycoside hydrolase family 10. The purified recombinant enzyme has a monomeric structure of 52 kDa. It is active and stable in a wide range of pH from 3 to 10 with a maximum activity at 6.5. Interestingly, XynBCA was highly thermoactive with an optimum temperature of 80 °C, thermostable with a half-life of 20 min at 80 °C. The specific activity was 117 U mg-1, while the Km and Vmax were 1.247 mg ml-1, and 114.7 µmol min-1 mg-1, respectively. The investigation of XynBCA in kraft pulp biobleaching experiments showed effectiveness in releasing reducing sugars and chromophores, with best achievements at 100 U g-1 of pulp and 1 h of incubation. The comparative molecular modeling studies with the less thermostable Xylanase B from Clostridium stercorarium, revealed extra charged residues at the surface of XynBCA potentially participating in the formation of intermolecular hydrogen bonds with solvent molecules or generating salt bridges, therefore contributing to the higher thermal stability.

Highlight on the expression and the function of a novel MnSOD from diploid wheat (T. monococcum) in response to abiotic stress and heavy metal toxicity.

Superoxide dismutases (SODs) play a pivotal role in improving abiotic stress tolerance in plant cells. A novel manganese superoxide dismutase gene, denoted as TmMnSOD, was identified from Triticum monococcum. The encoded protein displayed high sequence identity with MnSOD family members and was highly homologous to TdMnSOD from durum wheat. Furthermore, the 3D structure analysis revealed that TmMnSOD displayed homotetramer subunit organization, incorporating four Mn2+ ions. Notably, TmMnSOD structure contains predominantly alpha helices with three beta sheets. On the other hand, under stress conditions, TmMnSOD transcript level was significantly up-regulated by salt, oxidative and heavy metal stresses. At the functional level, TmMnSOD imparts tolerance of yeast and E. coli cells under diverse stresses. Promoter analysis of TmMnSOD gene showed the presence of a great number of salt and pathogen-responsive cis-regulatory elements, highlighting the interest of this gene in breeding programs towards improved tolerance to salt stress in wheat.

Localization and expression analysis of a novel catalase from Triticum monococcum TmCAT1 involved in response to different environmental stresses.

Catalase proteins play a crucial role in detoxifying hydrogen peroxide, generated during plant growth, and in response to various environmental stresses. Despite their importance, little is known about their localization and expression in wheat. In this study, we identified and characterized a novel peroxisomal catalase gene from Triticum monococcum, designated as TmCAT1. Phylogenetic analysis revealed that TmCAT1 shared high identity with TdCAT1 and other plant catalases belonging to subfamily 1. We predicted the 3D structure model and the oligomerization arrangement of TmCAT1. Besides, we displayed an arrangement in asymmetric unit, which involved interactions including, mainly, residues from N-terminal domain. Interestingly, sequence analysis indicated that TmCAT1, like TdCAT1, had the peroxisomal targeting signal (PTS1) around its C-terminus. Transient expression of TmCAT1-GFP and TdCAT1-GFP in tobacco leaves revealed that the two fused proteins are targeted into peroxisomes. However, the truncated forms lacking the tripeptide QKL remained in the cytosol. Concerning the expression profile analysis, TmCAT1 is expressed especially in leaves in normal condition. On the other hand, it is up-regulated by different stress incorporating salt, osmotic, oxidative, heavy metal and hormones stresses. Functional analysis by heterologous expression in yeast cells showed that TmCAT1 improved tolerance to multiple abiotic stresses. The presence of important cis-regulatory elements in the promoter region of TmCAT1 strongly reinforces the interest of this gene in plant adaptation to various stresses.

Molecular and structural characterization of a novel Cry1D toxin from Bacillus thuringiensis with high toxicity to Spodoptera littoralis (Lepidoptera: Noctuidae).

The investigation of new Bacillus thuringiensis (Bt) insecticidal proteins (Cry) with specific toxicity is one of the alternative measures used for Lepidopteran pest control. In the present study, a new Cry toxin was identified from a promising Bt strain BLB250 which was previously selected for its high toxicity against Spodoptera littoralis. The corresponding gene, designated cry1D-250, was cloned. It showed an ORF of 3498bp, encoding a protein of 1165 amino acid residues with a putative molecular mass of 132kDa which was confirmed by SDS-PAGE and Western blot analyses. The corresponding toxin named Cry1D-250 showed a higher insecticidal activity towards S. littoralis than Cry1D-133 (LC50 of 224.4ngcm-2) with an LC50 of only 166ngcm-2. Besides to the 65kDa active toxin, proteolysis activation of Cry1D-133 protein with S. littoralis midgut juice generated an extra form of 56kDa, which was the result of a second cleavage. Via activation study and 3D structure analysis, novel substitutions found in the Cry1D-250 protein compared to Cry1D-133 toxin were shown to be involved in the protein stability and toxicity. Therefore, the Cry1D-250 toxin can be considered to be an effective alternative for the control of S. littoralis.

Modifing Aspergillus Oryzae S2 amylase substrate specificity and thermostability through its tetramerisation using biochemical and in silico studies and stabilization.

We previously reported that Aspergillus oryzae S2 had produced an amylase called AmyC formed by a tetramer of AmyB subunits under solid state fermentation. In this work, we demonstrated that the half-life time of AmyC at 75 °C and 80 °C were remarkably enhanced to reach 53 min and 41 min compared to 6 min and 4 min for AmyB. The Km values of AmyC for maltoheptaose, maltopentaose, and maltotetraose were 2-fold lower than AmyB. AmyC showed a 6.5 fold higher exo-type activity and hydrolyzed the short oligosaccharides more efficiently than AmyB. The AmyC-3D model was generated and showed that a region named T1 was involved in the oligomerization process. The subunits and the RING network interactions insight suggested that AmyC sub-units were bounded by 20 hydrogen bonds, 4 electrostatic interactions, 16 nodes and 836 edges leading to a higher thermal stability. The disordered (ß3-ß4) and (ß7-ß8) loops contained in the AmyC active cleft were presumed to be the recognition sites of the non-reducing end substrate. The docking studies strongly suggested that AmyC easily accommodated the short substrates as it was exhibited in vitro and seemed to look like maltogenic amylases. The Box-Behnken Response Surface Methodology was applied for Amy C immobilization for efficient use. An optimum condition of an aluminum oxide content of 0.25 g, a carrageenan content of 0.1 g, and a glutaraldehyde content of 0.5%/g of carrier resulted in 76.2% of covalent immobilization yield. The immobilized AmyC kept its total activity for three cycles, shifted the optimum temperature from 60 °C to 65 °C, and had two-fold half-life at 85 °C compared to the free enzyme.

A novel Vip3Aa16-Cry1Ac chimera toxin: Enhancement of toxicity against Ephestia kuehniella, structural study and molecular docking.

Bacillus thuringiensis Vip3A protein has been widely used for crop protection and for delay resistance to existing insecticidal Cry toxins. During current study, a fusion between vip3Aa16 and the toxic core sequence of cry1Ac was constructed in pHT Blue plasmid. Vip3Aa16-Cry1Ac protein was expressed in the supernatant of B. thuringiensis with a size of about 150 kDa. Bioassays tested on Ephestia kuehniella showed that the use of the chimera toxin as biopesticide improved the toxicity to reach 90% ±â€¯2 with an enhancement of 20% compared to the single Vip3Aa16 protein. The findings indicated that the fusion protein design opens new ways to enhance Vip3A toxicity against lepidopteran species and could avoiding insect tolerance of B. thuringiensis delta-endotoxins. Through computational study, we have predicted for the first time the whole 3D structure of a Vip3A toxin. We showed that Vip3Aa16 structure is composed by three domains like Cry toxins: an N-terminal domain containing hemolysin like fold as well as two others Carbohydrate Binding Module (CBM)-like domains. Molecular docking analysis of the chimera toxin and the single Vip3Aa16 protein against specific insect receptors revealed that residues of CBM like domains are clearly involved in the binding of the toxin to receptors.

Molecular characterization of Cry1D-133 toxin from Bacillus thuringiensis strain HD133 and its toxicity against Spodoptera littoralis.

Bacillus thuringiensis subsp. aizawai strain HD133, known by its effectiveness against Spodoptera species, produces bipyramidal crystals encompassing the insecticidal proteins Cry1Ab, Cry1Ca and Cry1D-133 in the proportions 60:37:3, respectively. In this study, we dealt with the relevance of the low rate of Cry1D-133. The cry1D-133 gene from HD133 was cloned and sequenced. Both nucleotide and amino acid sequence similarity analyses with the cry1D genes available in the GenBank database revealed that cry1D-133 is a new variant of cry1Da-type genes with 99% identity with cry1Da1. Molecular modeling of the Cry1D-133 toxin showed that its higher toxicity is correlated to a higher number of toxin-receptor interactions. Optimal culture conditions of 4 h post-induction time, 160 rpm agitation and 37 °C post-induction temperature were determined and adopted to overproduce Cry1D-133 toxin at adequate amounts to carryout bioassays. A gradual increase of the proportion of Cry1D-133 to the HD133 insecticidal proteins forming the crystal (Cry1D-133, Cry1Ca and Cry1Ab) showed an improvement of the toxicity against Spodoptera littoralis larvae. Therefore, the potential of Cry1D-133 to enhance HD133 toxicity could promote its combination with other B. thuringiensis insecticidal proteins toxins in order to increase target range or to delay the emergence of resistance.

Localization and in silico study of the vegetative insecticidal proteins Vip2S-Vip1S of Bacillus thuringiensis.

The Bacillus thuringiensis S1/4 strain was previously found to harbour vip1S, vip2S, and vip3 genes. Its plasmid curing led to the obtaining of four partially cured strains S1/4-2, S1/4-3, S1/4-7, and S1/4-9 (vip2S-vip1S (-), vip3 (+)), one strain S1/4-4 (vip2S-vip1S (+), vip3 (-)), and S1/4-0 strain lacking the three genes. Using these derivative strains as templates, PCR amplification and southern blot assay revealed that vip2S-vip1S operon and vip3 gene were localized on two different large plasmids. Bioinformatics studies showed that vip2S (1.356 kb), and vip1S (2.637 kb) genes are encoding by an operon consisting of two ORFs separated by an intergenic spacer of 4bp. Using the InterPro tool, Vip2S was found to belong to the family of Binary exotoxin A and Vip1S to bacterial exotoxin B. In silico modeling indicated that the 3D structure of Vip2S is a mixed α/ß protein and proposed 3D-model of Vip1S. Bioassays of the partially cured strains supernatants showed a weak toxicity of S1/4-4 to the lepidopteran Spodoptera littoralis comparing to a better effect of S1/4-2, S1/4-3, S1/4-7, and S1/4-9, suggesting its eventual contribution to the toxicity. Nevertheless, the concentrated supernatant of S1/4-4 strain was not toxic against the coleopteran Tribolium castaneum.

US132 Cyclodextrin Glucanotransferase Engineering by Random Mutagenesis for an Anti-Staling Purpose.

The use of the cyclodextrin glucanotransferase (CGTase) of the US132 strain, which is an effective anti-staling agent, has been hampered by its high cyclization activity. Since that random mutagenesis using error-prone PCR is nowadays a method of choice for enzymes engineering, we have optimized this method by adjusting manganese concentration in order to obtain a high percentage of active CGTase mutants. Therefore, the amplification of the gene encoding the US132 CGTase was performed using a MnCl2 concentration ranging between 0 and 0.5 mM. The finding showed that a manganese concentration of 0.04 mM allowed for 90 % of active mutants. A simple method to rapidly screen the obtained mutants was also developed. After the examination of a small library (of less than 1000 clones), the active mutant named MJ13 was selected for a significant decrease in the cyclization activity, thereby showing a remarkable change in the enzyme specificity towards starch dextrinizing. Sequence analysis showed that MJ13 is a triple mutant with two mutations in the catalytic domain (K47E and S382P) and one substitution in the starch binding domain (N655S).

Aspergillus Oryzae S2 α-Amylase Domain C Involvement in Activity and Specificity: In Vivo Proteolysis, Molecular and Docking Studies.

We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α-amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α-amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino-acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α-amylases termed AmyB1 and AmyB2 reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (ß/α)8 barrel domain and the domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility.

Biocatalysts: application and engineering for industrial purposes.

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.

Secretion of cyclodextrin glucanotransferase in E. coli using Bacillus subtilis lipase signal peptide and optimization of culture medium.

The cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132 was fused to the secretive lipase signal peptide of B. subtilis. This leads to an efficient secretion of the recombinant enzyme into the culture medium of E. coli as an active and soluble form contrasting with the native construction leading to a periplasmic production. In order to enhance the yield of CGTase production, an experimental design methodology was applied for the optimization of the culture composition. Hence, the media components were submitted to preliminary screening using a Plakett-Burman design. The concentrations of the major operating ones were then optimized to enhance the secretion of CGTase using response surface methodology. The findings revealed that concentrations of 0.5% potato starch, 3% yeast extract, 3% tryptone, 1.5% casein hydrolysate, 0.5% NaCl, 0.2% KH2PO4, and 0.02% MgSO4 were the optimal conditions for CGTase production. The experimental value (9.43 U/mL) obtained for CGTase activity was very close to the predicted value (9.27 U/mL).

The cyclodextrin glycosyltransferase of Paenibacillus pabuli US132 strain: molecular characterization and overproduction of the recombinant enzyme.

The gene encoding the cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34 residues. The enzyme exhibited the highest identity (94%) to the beta-CGTase of Bacillus circulans no. 8. The production of the recombinant CGTase, as active form, was very low (about 1 U/mL) in shake flasks at 37 degrees C. This production reached 22 U/mL after 22 hours of induction by mainly shifting the postinduction temperature from 37 to 19 degrees C and using 2TY instead of LB medium. High enzyme production (35 U/mL) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask. The recombinant enzyme showed V(max) and K(m) values of 253 +/- 36 mumol of beta-cyclodextrin/mg/min and 0.36 +/- 0.18 g/L, respectively.

Heterologous expression, secretion and characterization of the Geobacillus thermoleovorans US105 type I pullulanase.

Pullulanase type I of Geobacillus thermoleovorans US105 strain (PUL US105) was produced and secreted efficiently in the E. coli periplasmic or extracellular fraction using two different signal peptides. Hence, the open reading frame was connected downstream of the lipase A signal peptide of Bacillus subtilis strain leading to an efficient secretion of an active form enzyme on the periplasmic fraction. In addition, pul US105 was fused to the alpha-amylase signal sequence of the Bacillus stearothermophilus US100 strain. The monitoring of the pullulanase activity and Western blot analysis for this last construction showed that the most activity was found in the supernatant culture, proving the efficient secretion of this natively cytoplasmic enzyme as an active form. The PUL US105 was purified to homogeneity from the periplasmic fraction, using heat treatment, size exclusion, and anion-exchange chromatography. The native pullulanase has a molecular mass of 160 kDa and is composed of two identical subunits of 80 kDa each. It was independent for metallic ions for its activity, while its thermostability was obviously improved in presence of only 0.1 mM CaCl2.

Cloning and sequencing of an original gene encoding a maltogenic amylase from Bacillus sp. US149 strain and characterization of the recombinant activity.

A gene encoding maltogenic amylase from acidic Bacillus sp. US149 (maUS149) was cloned, sequenced and over-expressed in Escherichia coli. The nucleotide sequence analysis revealed an open reading frame (ORF) of 1749 bp encoding a protein of 582 residues. The alignment of deduced amino acid sequence revealed a relatively low homology with the already reported maltogenic amylases. In fact, its highest identity, of only 60%, was found with the maltogenic amylase of Thermus sp. IM6501. The recombinant enzyme (MAUS149) was found to be intracellular and was purified to homogeneity from the cell crude extract with a yield of 23%. According to PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of 135 kDa and is composed of two identical subunits of 67.5 kDa each. The maximum activity was obtained at 40 degrees C and pH 6.5. MAUS149 could be classified as a maltogenic amylase since it produces mainly maltose from starch, maltose and glucose from beta-cyclodextrin, and panose from pullulan.