Purification of lipase from Burkholderia metallica fermentation broth in a column chromatography using polymer impregnated resins.

In this work, porous glass beads grafted with polyethylene glycol (PEG) were used as an adsorbent to purify lipase from Burkholderia metallica in column chromatography. The purification parameters viz. salt stability, types and concentrations of PEG and salt, pH of the binding solution, and flow rate were studied to determine the performance of the purification system in an XK16/20 column. The crude lipase was mixed with different types and concentrations of salts 1-5% (w/w) (sodium citrate, potassium citrate, and sodium acetate) and subjected to the column containing the polymeric glass bead. One-variable-at-a-time experimentation revealed that 20% (w/w) PEG 6000 g/mol impregnated glass beads with a binding solution of 5% sodium citrate at pH 7.7, a flow rate of 1.0 mL/min and extraction time of 10 min resulted in the highest purification factor and recovery yield at 3.67 and 88%, respectively. The purified lipase has 55 ∼ 60 kDa molecular mass. The outcome of the study showed PEG could be applied to modify the inert glass beads into polymeric form, providing a biocompatible and mild separation condition for lipase. Thus, PEG could be successfully applied for the purification of lipase from B. metallica fermentation broth using column chromatography.

Synthesis of uniform submicron poly(lactic acid)-based particles/capsules by radical precipitation polymerization.

Poly(l-lactic acid) (PLLA) is a well-known biopolymer, usually synthesized via step-growth or ring-opening polymerization from lactic acid or a lactide monomer, respectively. PLLA microspherical particles are produced by dispersion polymerization with a ring-opening lactide monomer using a particular copolymer chain as a stabilizer. This is not easy to achieve when dehydration is needed. Here, a robust and simple synthesis of a nearly monodisperse, submicron PLLA-based particle/capsule was proposed via radical precipitation polymerization without the use of surfactant. A commercial PLLA was first glycolyzed with ethylene glycol to obtain a low molecular weight glycolyzed PLLA (GPLLA). Then, the GPLLA was copolymerized with methacrylic acid and ethylene glycol dimethacrylate monomers using a benzoyl peroxide initiator. Active sites on the GPLLA backbone were generated by hydrogen abstraction of benzoyloxy radicals that further copolymerized before self-assembly to form the polymer particles. Uniform particle size of about 580 nm with a low polydispersity index (PDI) of 0.012 was obtained. This method was also implemented to produce nearly monodisperse capsules containing linalool. The particle size of PLLA-based capsules was about 280 nm with narrow particle size distribution (PDI of 0.120). The PLLA-based capsules effectively inhibited microbial growth of Staphylococcus aureus, Escherichia coli and Candida albicans and were not toxic to human cells.

Isolation and characterization of Enterococcus faecium DSM 20477 with ability to secrete antimicrobial substance for the inhibition of oral pathogen Streptococcus mutans UKMCC 1019.

Streptococcus mutans and Candida albicans are the main oral pathogens which contribute to dental caries that affects all ages of human being. OBJECTIVES: This study focuses on the potential of crude cell free supernatant (CCFS) from lactic acid bacteria (LAB) to inhibit of the growth of S. mutans UKMCC 1019. DESIGN: A total of 61 CCFS from LAB strains were screened for their inhibitory ability against S. mutans UKMCC 1019 by broth microdilution method. The selected LAB with highest antimicrobial activity was identified and its CCFS was characterized for pH stability, temperature tolerance, enzyme sensitivity, metabolism of carbohydrates, enzymatic activities and antimicrobial activity against S. mutans UKMCC 1019 and C. albicans UKMCC 3001 by well diffusion assay. The effect of CCFS on cell structure of S. mutans UKMCC 1019 was observed under transmission electron microscopy (TEM). RESULTS: The CCFS from isolate CC2 from Kimchi showed the highest inhibition against S. mutans UKMCC 1019, which was 76.46 % or 4406.08 mm2/mL and it was identified to be most closely related to Enterococcus faecium DSM 20477 based on 16 s rRNA sequencing. The CCFS of E. faecium DSM 20477 had high tolerance to acidic and alkaline environment as well as high temperature. It also shows high antifungal activities against C. albicans UKMCC 3001 with 2362.56 mm2/mL. Under TEM, the cell walls and the cytoplasm membrane of S. mutans UKMCC 1019 were disrupted by the antimicrobial substance, causing cell lysis. CONCLUSIONS: Hence, the CCFS from E. faecium DSM 20477 is a potential bacteriocin in future for the treatment of dental caries.

Purification of ß-mannanase derived from Bacillus subtilis ATCC 11774 using ionic liquid as adjuvant in aqueous two-phase system.

The partitioning of ß-mannanase derived from Bacillus subtilis ATCC 11774 in aqueous two-phase system (ATPS) was studied. The ATPS containing different molecular weight of polyethylene glycol (PEG) and types of salt were employed in this study. The PEG/salt composition for the partitioning of ß-mannanase was optimized using response surface methodology. The study demonstrated that ATPS consists of 25% (w/w) of PEG 6000 and 12.52% (w/w) of potassium citrate is the optimum composition for the purification of ß-mannanase with a purification fold (PF) of 2.28 and partition coefficient (K) of 1.14. The study on influences of pH and crude loading showed that ATPS with pH 8.0 and 1.5% (w/w) of crude loading gave highest PF of 3.1. To enhance the partitioning of ß-mannanase, four ionic liquids namely 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4), 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim]BF4), 1-butyl-3-methylimidazolium bromide ([Bmim]Br), 1-ethyl-3-methylimidazolium bromide ([Emim]Br) was added into the system as an adjuvant. The highest recovery yield (89.65%) was obtained with addition of 3% (w/w) of [Bmim]BF4. The SDS-PAGE analysis revealed that the ß-mannanase was successfully recovered in the top phase of ATPS with the molecular size of 36.7kDa. Therefore, ATPS demonstrated a simple and efficient approach for recovery and purification of ß-mannanase from fermentation broth in one single-step strategy.