Cell immobilization for enhanced milk clotting enzyme production from Bacillus amyloliquefacien and cheese quality.

BACKGROUND: Milk clotting enzymes, essential for milk coagulation in cheese production, are obtained from the stomach of young ruminants, an expensive and limited source. This study was accomplished by finding a suitable alternative. Bacterial isolates recovered from honey were screened for milk clotting enzyme activity. and further, by immobilization of the microorganisms to enhance stability and facilitate their repeated use. RESULT: The most effective enzyme was produced by a microbe identified as Bacillus amyloliquefaciens based on 16 S rRNA sequencing. The cells were encapsulated in Ca2+ alginate beads. These beads retained complete enzyme production after being used five times. Glucose and Soybean were selected as the most favorable carbon and nitrogen sources, respectively. The optimum temperature for activity was 35 ℃ for both free and immobilized cells but as the temperature was increased to 55 °C and above, the encapsulated form retained more activity than the free cells. The pH optimum shifted from 6.5 to 7 for the free cells to 7-7.5 for the immobilized cells. The immobilization process decreased the activation energy for enzyme production and activity, prolonged the enzyme half-life, and increased the deactivation energy. Enzyme produced by immobilized cells generated a more compact cheese. CONCLUSIONS: The finding of this study was to identify a less expensive source of milk-clotting enzymes and confirm the success of cell immobilization in improving cell rigidity and stability. Also, immobilization of this B. amyloliquefaciens strain offers an enzyme source of value for industrial production of cheese.

Novel Pyridothienopyrimidine Derivatives: Design, Synthesis and Biological Evaluation as Antimicrobial and Anticancer Agents.

The growing risk of antimicrobial resistance besides the continuous increase in the number of cancer patients represents a great threat to global health, which requires intensified efforts to discover new bioactive compounds to use as antimicrobial and anticancer agents. Thus, a new set of pyridothienopyrimidine derivatives 2a,b-9a,b was synthesized via cyclization reactions of 3-amino-thieno[2,3-b]pyridine-2-carboxamides 1a,b with different reagents. All new compounds were evaluated against five bacterial and five fungal strains. Many of the target compounds showed significant antimicrobial activity. In addition, the new derivatives were further subjected to cytotoxicity evaluation against HepG-2 and MCF-7 cancer cell lines. The most potent cytotoxic candidates (3a, 4a, 5a, 6b, 8b and 9b) were examined as EGFR kinase inhibitors. Molecular docking study was also performed to explore the binding modes of these derivatives at the active site of EGFR-PK. Compounds 3a, 5a and 9b displayed broad spectrum antimicrobial activity with MIC ranges of 4-16 µg/mL and potent cytotoxic activity with IC50 ranges of 1.17-2.79 µM. In addition, they provided suppressing activity against EGFR with IC50 ranges of 7.27-17.29 nM, higher than that of erlotinib, IC50 = 27.01 nM.

Synthesis, Docking Studies, and In Vitro Evaluation of Some Novel Thienopyridines and Fused Thienopyridine-Quinolines as Antibacterial Agents and DNA Gyrase Inhibitors.

A series of novel thienopyridines and pyridothienoquinolines (3a,b-14) was synthesized, starting with 2-thioxo-1,2-dihydropyridine-3-carbonitriles 1a and 1b. All compounds were evaluated for their in vitro antimicrobial activity against six bacterial strains. Compounds 3a,b, 4a, 5b, 6a,b, 7a, 9b, 12b, and 14 showed significant growth inhibition activity against both Gram-positive and Gram-negative bacteria compared with the reference drug. The most active compounds (4a, 7a, 9b, and 12b) against Staphylococcus aureus were also tested for their in vitro inhibitory action on methicillin-resistant Staphylococcus aureus (MRSA). The tested compounds showed promising inhibition activity, with the performance of 12b being equal to gentamicin and that of 7a exceeding it. Moreover, the most promising compounds were also screened for their Escherichia coli DNA gyrase inhibitory activity, compared with novobiocin as a reference DNA gyrase inhibitor. The results revealed that compounds (3a, 3b, 4a, 9b, and 12b) had the highest inhibitory capacity, with IC50 values of 2.26-5.87 µM (that of novobiocin is equal to 4.17 µM). Docking studies were performed to identify the mode of binding of the tested compounds to the active site of E. coli DNA gyrase B.

Cardenolides and pentacyclic triterpenes isolated from Acokanthera oblongifolia leaves: their biological activities with molecular docking study.

Pentacyclic triterpenes and cardenolides were isolated from Acokanthera oblongifolia leaves. Their chemical structures were determined based on comprehensive 1D and 2D NMR spectroscopy. Their MIC was determined against 12 microorganisms. Their exerted cytotoxicity on the immortalized normal cells, hTERT-RPE1 was assessed by the sulforhodamine-B assay. The viral inhibitory effects of compounds against Newcastle disease virus (NDV) and H5N1 influenza virus IV were evaluated. Four in vitro antioxidant assays were performed in comparison with BHT and trolox and a weak activity was exhibited. Acovenoside A was with potent against H5N1-IV and NDV with IC50 ≤ 3.2 and ≤ 2.1 µg/ml and SI values of 93.75 and 95.23%, respectively, in comparison to ribavirin. Its CC50 record on Vero cells was > 400 and 200 µg/ml, respectively. Acobioside A was the most active compound against a broad range of microbes while Pseudomonas aeruginosa was the most sensitive. Its MIC (0.07 µg/ml) was 1/100-fold of the recorded CC50 (7.1 µg/ml/72 h) against hTERT-RPE1. The molecular docking of compounds on human DNA topoisomerase I (Top1-DNA) and IV glycoprotein hemagglutinin were studied using MOE program. This study has introduced the cardenolides rather than triterpenoids with the best docking score and binding interaction with the active site of the studied proteins.

Production of a novel α-amylase by Bacillus atrophaeus NRC1 isolated from honey: Purification and characterization.

Different bacterial isolates with amylolytic activity were insulated from various honey samples. The most active isolate was identified by the molecular 16SrRNA sequence technique as Bacillus atrophaeus NRC1. The bacterium showed maximum amylase production under optimum culture conditions at pH 6.0, 40 °C and after 24 h incubation. Two amylase isoenzymes (AmyI and AmyII) from Bacillus atrophaeus NRC1 have been purified to homogeneity by using ammonium sulfate precipitation, Sephacryl S-200 and DEAE-Sepharose chromatography. The major isoenzyme, AmyI, had a specific activity 4635 U/mg proteins with molecular weight of 61 kDa using SDS-PAGE electrophoresis. The maximum activity of AmyI against starch was determined at pH 6.0 and 50 °C. AmyI was stable up to 50 °C after incubation for 30 min, retained 65 and 23% of its activity at 60 and 70 °C, respectively. Pre-incubation with Ca2+, Mg2+ and Ba2+ cations for 30 min enhanced the enzyme activity; while it was completely inhibited by Hg2+. Varied inhibition degree of the enzyme activity was determined with K+, Ni2+, Zn2+, Na2+ and Cu2+ ions. AmyI was inhibited by EDTA, PMSF and SDS, while it was activated by l-Cysteine-HCl and DTT. AmyI had the ability to degrade starch, amylopectin, glycogen, amylose and lacked the affinity towards ß-1,4-linked xyloses.

Synthesis and Biological Evaluation of New Pyridothienopyrimidine Derivatives as Antibacterial Agents and Escherichia coli Topoisomerase II Inhibitors.

The growing resistance of bacteria to many antibiotics that have been in use for several decades has generated the need to discover new antibacterial agents with structural features qualifying them to overcome the resistance mechanisms. Thus, novel pyridothienopyrimidine derivatives (2a,b-a,b) were synthesized by a series of various reactions, starting with 3-aminothieno[2,3-b]pyridine-2-carboxamides (1a,b). Condensation of compounds 1a,b with cyclohexanone gave 1'H-spiro[cyclohexane-1,2'-pyrido[3',2':4,5]thieno[3,2-d]pyrimidin]-4'(3'H)-ones (2a,b), which in turn were utilized to afford the target 4-substituted derivatives (3a,b-8a,b). In vitro antibacterial activity evaluations of all the new compounds (2a,b-8a,b) were performed against six strains of Gram-negative and Gram-positive bacteria. The target compounds showed significant antibacterial activity, especially against Gram-negative strains. Moreover, the compounds (2a,b; 3a,b; 4a,b; and 5a,b) that exhibited potent activity against Escherichia coli were selected to screen their inhibitory activity against Escherichia coli topoisomerase II (DNA gyrase and topoisomerase IV) enzymes. Compounds 4a and 4b showed potent dual inhibition of the two enzymes with IC50 values of 3.44 µΜ and 5.77 µΜ against DNA gyrase and 14.46 µΜ and 14.89 µΜ against topoisomerase IV, respectively. In addition, docking studies were carried out to give insight into the binding mode of the tested compounds within the E. coli DNA gyrase B active site compared with novobiocin.

Optimization of pectinase immobilization on grafted alginate-agar gel beads by 24 full factorial CCD and thermodynamic profiling for evaluating of operational covalent immobilization.

Pectinase produced by a honey derived from the fungus Aspergillus awamori KX943614 was covalently immobilized onto gel beads made of alginate and agar. Polyethyleneimine, glutaraldehyde, loading time and enzyme's units were optimized by 24 full factorial central composite design (CCD). The immobilization process increased the optimal working pH for the free pectinase from 5 to a broader range of pH4.5-5.5 and the optimum operational temperature from 55°C to a higher temperature, of 60°C, which is favored to reduce the enzyme's microbial contamination. The thermodynamics studies showed a thermal stability enhancement against high temperature for the immobilized formula. Moreover, an increase in half-lives and D-values was achieved. The thermodynamic studies proved that immobilization of pectinase made a remarkable increase in enthalpy and free energy because of enzyme stability enhancement. The reusability test revealed that 60% of pectinase's original activity was retained after 8 successive cycles. This gel formula may be convenient for immobilization of other industrial enzymes.

Production, immobilization and thermodynamic studies of free and immobilized Aspergillus awamori amylase.

Enzyme cost, stability and its thermodynamic characteristics are the main criteria for industrial use. In this study, Aspergillus awamori amylase was constitutively produced using various agro-industrial wastes. Olive oil cake gave the highest activity (230U/g). The amylase was partially purified to 2.81-fold purification. Immobilization was achieved using different carriers by covalent binding. The novel carrier Ca+2 alginate (Alg) starch (St)/polyethyleneimine (PEI)/glutaraldehyde (GA), showed the highest operational stability and was selected for further studies. The optimum temperature for the free and immobilized form was 50°C and 55-60°C, respectively. The immobilization process had a major role in improving enzyme thermal stability. In comparison to free enzyme, the immobilized form showed the highest optimum temperature, activation energy (Ea) and deactivation rate constants (kd). Also, t1/2, D-values (decimal reduction time), change in enthalpy (ΔH° kJmol-1), and Gibbs free energy (ΔG°) increased and was higher than the native enzyme within 50-80°C. The magnitude of negative value of entropy (ΔS° kJmol-1) for immobilized enzyme was negative for the free and immobilized enzymes revealing that native form of enzyme was in more ordered state. Km and Vmax values were slightly affected by the temperature variations 40-70°C.