Rational design of Bacillus stearothermophilus US100 l-arabinose isomerase: Potential applications for d-tagatose production

l-Arabinose isomerases catalyze the bioconversion of d-galactose into d-tagatose. With the aim of producing an enzyme optimized for d-tagatose production, three Bacillus stearothermophilus US100 l-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 °C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0–7.0 and an optimal activity around 50–65 °C, temperatures at which the enzyme was stable without addition of metal ions.     

Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis

The serine alkaline protease, SAPB, from Bacillus pumilus CBS is characterized by its high thermoactivity, pH stability and high catalytic efficiency (k_cat/K_m) as well as its excellent stability and compatibility with an alkaline environment under harsh washing conditions. Based on sequence alignments and homology-modeling studies, the present study identified five amino acids Leu31, Thr33, Asn99, Phe159 and Gly182 being putatively important for the enzymatic behaviour of SAPB. To corroborate the role of these residues, 12 mutants were constructed by site-directed mutagenesis and then purified and characterized. The findings demonstrate that the single mutants F159T, F159S and G182S and combined double substitutions were implicated in the decrease of the optimum pH and temperature to 8.0–9.0 and 50 °C, respectively, and that mutant F159T/S clearly affected substrate affinity and catalytic efficiency. With regards to the single L31I, T33S and N99Y and combined double and triple mutations, the N99Y mutation strongly improved the half-life times at 50 °C and 60 °C to 660 and 295 min from of 220 and 80 min for the wild-type enzyme, respectively. More interestingly, this mutation also shifted the optimum temperature from 65 °C to 75 °C and caused a prominent 31-fold increase in k_cat/K_m with N-succinyl-l-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF). The L31I and T33S mutants were observed to improve mainly the optimum pH from 11.0 to 11.5 and from 11.0 to 12.0, respectively. Kinetic studies of double and triple mutants showed that the cumulative effect of polar uncharged substitutions had a synergistic effect on the P1 position preference using synthetic peptide substrates, which confirms the implication of these amino acids in substrate recognition and catalytic efficiency.     

Biochemical and Molecular Characterization of a Serine Keratinase from Brevibacillus brevis US575 with Promising Keratin-Biodegradation and Hide-Dehairing Activities

Dehairing is one of the highly polluting operations in the leather industry. The conventional lime-sulfide process used for dehairing produces large amounts of sulfide, which poses serious toxicity and disposal problems. This operation also involves hair destruction, a process that leads to increased chemical oxygen demand (COD), biological oxygen demand (BOD), and total suspended solid (TSS) loads in the effluent. With these concerns in mind, enzyme-assisted dehairing has often been proposed as an alternative method. The main enzyme preparations so far used involved keratinases. The present paper reports on the purification of an extracellular keratinase (KERUS) newly isolated from Brevibacillus brevis strain US575. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 29121.11 Da. The sequence of the 27 N-terminal residues of KERUS showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 8 and 40°C. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Ca²⁺. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. KERUS displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than NUE 12 MG and KOROPON® MK EG keratinases. The enzyme also exhibited powerful keratinolytic activity that made it able to accomplish the entire feather-biodegradation process on its own. The kerUS gene encoding KERUS was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rKERUS) were similar to those of native KERUS. Overall, the findings provide strong support for the potential candidacy of this enzyme as an effective and eco-friendly alternative to the conventional chemicals used for the dehairing of rabbit, goat, sheep and bovine hides in the leather processing industry.     

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 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.     

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.     

Dietary Transforming Growth Factor-Beta 2 (TGF-��2) Supplementation Reduces Methotrexate-Induced Intestinal Mucosal Injury in a Rat

Background/Aims

Dietary supplementation with transforming growth factor-beta (TGF-β) has been proven to minimize intestinal damage and facilitate regeneration after mucosal injury. In the present study, we evaluated the effects of oral TGF-β2 supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat and in a cell culture model.

Methods

Caco-2 cells were treated with MTX and were incubated with increasing concentrations of TGF-β2. Cell apoptosis was assessed using FACS analysis by annexin staining and cell viability was monitored using Trypan Blue assay. Male rats were divided into four experimental groups: Control rats, CONTR- TGF-β rats were treated with diet enriched with TGF-β2, MTX rats were treated with a single dose of methotrexate, and MTX- TGF-β rats were treated with diet enriched with TGF-β2. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined at sacrifice. Real Time PCR and Western blot were used to determine bax and bcl-2 mRNA, p-ERK, β-catenin, IL-1B and bax protein expression.

Results

Treatment of MTX-pretreated Caco-2 cells with TGF-B2 resulted in increased cell viability and decreased cell apoptosis. Treatment of MTX-rats with TGF-β2 resulted in a significant increase in bowel and mucosal weight, DNA and protein content, villus-height (ileum), crypt-depth (jejunum), decreased intestinal-injury score, decreased level of apoptosis and increased cell proliferation in jejunum and ileum compared to the untreated MTX group. MTX-TGF-β2 rats demonstrated a lower bax mRNA and protein levels as well as increased bcl-2 mRNA levels in jejunum and ileum compared to MTX group. Treatment with TGF-β2 also led to increased pERK, IL-1B and β-catenin protein levels in intestinal mucosa.

Conclusions

Treatment with TGF-β2 prevents mucosal-injury, enhances p-ERK and β-catenin induced enterocyte proliferation, inhibits enterocyte apoptosis and improves intestinal recovery following MTX-induced intestinal-mucositis in rats.     

PDGF-α stimulates intestinal epithelial cell turnover after massive small bowel resection in a rat

Numerous cytokines have been shown to affect epithelial cell differentiation and proliferation through epithelial-mesenchymal interaction. Growing evidence suggests that platelet-derived growth factor (PDGF) signaling is an important mediator of these interactions. The purpose of this study was to evaluate the effect of PDGF-α on enterocyte turnover in a rat model of short bowel syndrome (SBS). Male rats were divided into four groups: Sham rats underwent bowel transection, Sham-PDGF-α rats underwent bowel transection and were treated with PDGF-α, SBS rats underwent a 75% bowel resection, and SBS-PDGF-α rats underwent bowel resection and were treated with PDGF-α. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined at euthanasia. Illumina's Digital Gene Expression analysis was used to determine PDGF-related gene expression profiling. PDGF-α and PDGF-α receptor (PDGFR-α) expression was determined by real-time PCR. Western blotting was used to determine p-ERK, Akt1/2/3, bax, and bcl-2 protein levels. SBS rats demonstrated a significant increase in PDGF-α and PDGFR-α expression in jejunum and ileum compared with sham animals. SBS-PDGF-α rats demonstrated a significant increase in bowel and mucosal weight, villus height, and crypt depth in jejunum and ileum compared with SBS animals. PDGF-α receptor expression in crypts increased in SBS rats (vs. sham) and was accompanied by an increased cell proliferation following PDGF-α administration. A significant decrease in cell apoptosis in this group was correlated with lower bax protein levels. In conclusion, in a rat model of SBS, PDGF-α stimulates enterocyte turnover, which is correlated with upregulated PDGF-α receptor expression in the remaining small intestine.     

Heterologous expression,secretion and characterization of the <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis> 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 α-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.     

Purification and sequence analysis of the atypical maltohexaose-forming alpha-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.     

Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive

Extremophiles - A thermostable extracellular alkaline protease (called SAPA) was produced (4600&nbsp;U/mL) by Anoxybacillus kamchatkensis M1V, purified to homogeneity, and biochemically...