Optimization of newly isolated Bacillus cereus α-amylase production using orange peels and crab shells and application in wastewater treatment.

A newly isolated amylolytic strain was identified as Bacillus cereus spH1 based on 16S and 16-23S gene sequencing (Accession numbers OP811441.1 and OP819558, respectively), optimization strategies, using one variable at time (OVAT) and Plackett-Burman design, were employed to improve the alpha-amylase (α-amylase) production. Condition inferred revealed that the optimal physical parameters for maximum enzyme production were 30 °C, pH 7.5, and 12 h of incubation, using tryptone, malt extract, orange (Citrus sinensis) peels, crab (Portunus segnis) shells, calcium, and sodium chloride (NaCl) as culture medium. The full factorial design (FFD) model was observed to possess a predicted R2 and adjusted R2 values of 0.9788 and 0.9862, respectively, and it can effectively predict the response variables (p = 0). Following such efforts, α-amylase activity was increased 141.6-folds, ranging from 0.06 to 8.5 U/mL. The ideal temperature and pH for the crude enzyme activity were 65 °C and 7.5, respectively. The enzyme exhibited significant stability, with residual activity over 90% at 55 °C. The maltose was the only product generated during the starch hydrolysis. Moreover, the Bacillus cereus spH1 strain and its α-amylase were used in the treatment of effluents from the pasta industry. Germination index percentages of 143% and 139% were achieved when using the treated effluent with α-amylase and the strain, respectively. This work proposes the valorization of agro-industrial residues to improve enzyme production and to develop a green and sustainable approach that holds great promise for environmental and economic challenges.

Optimization using response surface methodology of phospholipase C production from Bacillus cereus suitable for soybean oil degumming.

This work deals with the optimization of an extracellular phospholipase C production by Bacillus cereus (PLCBc) using Response Surface Methodology (RMS) and Box-Behnken design. In fact, after optimization, a maximum phospholipase activity (51 U/ml) was obtained after 6 h of cultivation on tryptone (10 g/L), yeast extract (10 g/L), NaCl (8.125 g/L), pH 7.5 with initial OD (0.15). The PLCBc activity, esteemed by the model (51 U) was very approximate to activity gutted experimentally (50 U). The PLCBc can be considered as thermoactive phospholipase since it showed a maximal activity of 50 U/mL at 60 °C using egg yolk or egg phosphatidylcholine (PC) as substrate. In addition, the enzyme was active at pH 7 and is stable after incubation at 55 °C for 30 min. The application of B. cereus phospholipase C in soybean oil degumming was investigated. Our results showed that when using enzymatic degumming, the residual phosphorus decrease more than with water degumming, indeed, it passes from 718 ppm in soybean crude oil to 100 ppm and 52 ppm by degumming using water and enzymatic process, respectively. The diacylgycerol (DAG) yield showed an increase of 1.2% with enzymatic degumming compared to soybean crude oil. This makes our enzyme a potential candidate for food industrial applications such as enzymatic degumming of vegetable oils.

Expression of a Copper Activated Xylanase in Yeast: Location of the His-Tag in the Protein Significantly Affects the Enzymatic Properties.

A copper activated xylanase produced by E. coli BL21 was expressed in Pichia pastoris using the pGAPZαB expression vector. Two recombinant GH11 xylanase forms were obtained (N-His-rXAn11 and N-C-His-rXAn11). The findings revealed that the two recombinant xylanases displayed different behaviors toward the copper. In the presence of 3-mM Cu2+, the relative activity of the N-His-rXAn11 was enhanced by about 52%. However, the xylanase activity of the N- and C-terminal tagged one (N-C-His-rXAn11) was strongly inhibited by copper. In the presence of 3-mM Cu2+, the N-His-rXAn11 revealed to be thermostable at 60 °C with a half-life of 10 min. However, the N-C-His-rXAn11 was noted to be unstable since it was inactivated after 15 min of incubation at 55 °C. 3D models of the two recombinant forms showed that the created copper site in the N-His-rXAn11 was loosed in the C-terminal tagged protein. The C-terminal tag could trigger some structural changes with a notable displacement of secondary structures leading to great hindrance of the active site due to high fluctuations and probably new interactions among the N- and C-terminal amino acids.

The optimized production, purification, characterization, and application in the bread making industry of three acid-stable alpha-amylases isoforms from a new isolated Bacillus subtilis strain US586.

A new alpha-amylase-producing strain was assigned as Bacillus subtilis US586. The used statistical methodology indicated that amylase production was enhanced by 5.3 folds. The crude enzyme analysis proved the presence of three amylases isoforms Amy1, Amy2, and Amy3 called Amy586. The purified amylases had molecular masses of 48, 52, and 68 kDa with a total specific activity of 2,133 U/mg. Amy586 generated maltose, maltotriose, and maltopentaose as main final products after starch hydrolysis. It exhibited a large 4-6 optimal pH, a 60°C temperature activity, and a moderate thermostability. Amy586 displayed a high pH stability ranging from 3.5 to 6. The addition of Amy586 to weak wheat flour decreased its P/L ratio from 1.9 to 1.2 and increased its dough baking strength (W) from 138 × 10-4 to 172 × 10-4  J. Amy586 also improved the bread texture parameters by reducing its firmness and boosting the cohesion and elasticity values. PRACTICAL APPLICATIONS: Bacterial alpha-amylases with novel properties have been the major extent of recent research. In this paper, we managed to demonstrate that the addition of a purified amylolytic extract from the new isolated Bacillus subtilis strain US586 to weak local flour improves dough rheological proprieties and bread quality. Therefore, Amy586 can be considered as a bread making improver.

Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: production, purification and novel properties.

A synthetic cDNA-AmyA gene was cloned and successfully expressed in Pichia pastoris as a His-tagged enzyme under the methanol inducible AOX1 promoter. High level of extracellular amylase production of 72 U/mL was obtained after a 72 h induction by methanol. As expected, the recombinant strain produced only the AmyA isoform since the host is a protease deficient strain. Besides, the purified r-AmyA showed a molecular mass of 54 kDa, the same pH optimum equal to 5.6 but a higher thermoactivity of 60 °C against 50 °C for the native enzyme. Unlike AmyA which maintained 50% of its activity after a 10-min incubation at 60 °C, r-AmyA reached 45 min. The higher thermoactivity and thermostability could be related to the N-glycosylation. The r-AmyA activity was enhanced by 46% and 45% respectively in the presence of 4 mM Fe2+ and Mg2+ ions. This enzyme was more efficient in bread-making since such ions were reported to have a positive impact on the nutriment quality and the rheological characteristics of the wheat flour dough. The thermoactivity/thermostability as well as the iron and magnesium activations could also be ascribed to the presence of an additional C-terminal loop containing the His tag.

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.

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 K(m) values of MAUS149/D46G for all substrates were strongly increased. Nevertheless, the affinity and catalytic efficiency of MAUS149/D46V toward ß-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.

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 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.