Catalytic, kinetic and thermodynamic properties of Bacillus pumilus FH9 keratinase conjugated with activated pectin.

Bacillus pumilus FH9 keratinase was covalently coupled to several oxidized polysaccharides. The conjugates were evaluated for the retained activity, kinetic and thermodynamic stability. Among all preparations, the conjugated enzyme with oxidized pectin had the highest recovered activity (71.75%) and the highest thermal stability at 60°C (t1/2=333 min). Compared to the native enzyme, the conjugated preparation exhibited higher optimum temperature, lower activation energy (Ea), lower deactivation constant rate (kd), higher t1/2, and higher decimal reduction time values (D) within the temperature range of 50-80°C. The thermodynamic parameters (ΔH*, ΔG*, ΔS*) of irreversible thermal denaturation for the native and conjugated keratinase were also evaluated. The values of enthalpy of activation (ΔH*), free energy of activation (ΔG*), and free energy of transition state binding (ΔG*E-T) for keratin hydrolysis were lower for the conjugated enzyme. Moreover, there was highly significant impact on improving the values of Vmax/Km, kcat, kcat/Km, and ΔG*E-S for the modified enzyme. Both native and conjugated enzymes were slightly activated by CaCl2 and MgCl2. However, the inhibitory effects of EDTA, HgCl2 and ZnSO4 were more pronounced with the native enzyme.

Isolation and characterization of new poly(3HB)-accumulating star-shaped cell-aggregates-forming thermophilic bacteria.

AIMS: This study aimed at isolating thermophilic bacteria that utilize cheap carbon substrates for the economically feasible production of poly(3-hydroxybutyrate), poly(3HB), at elevated temperatures. METHODS AND RESULTS: Thermophilic bacteria were enriched from an aerobic organic waste treatment plant in Germany, and from hot springs in Egypt. Using the viable colony staining method for hydrophobic cellular inclusions with Nile red in mineral salts medium (MSM) containing different carbon sources, six Gram-negative bacteria were isolated. Under the cultivation conditions used in this study, strains MW9, MW11, MW12, MW13 and MW14 formed stable star-shaped cell-aggregates (SSCAs) during growth; only strain MW10 consisted of free-living rod-shaped cells. The phylogenetic relationships of the strains as derived from 16S rRNA gene sequence comparisons revealed them as members of the Alphaproteobacteria. The 16S rRNA gene sequences of the isolates were very similar (>99% similarity) and exhibited similarities ranging from 93 to 99% with the most closely related species that were Chelatococcus daeguensis, Chelatococcus sambhunathii,Chelatococcus asaccharovorans, Bosea minatitlanensis, Bosea thiooxidans and Methylobacterium lusitanum. Strains MW9, MW10, MW13 and MW14 grew optimally in MSM with glucose, whereas strains MW11 and MW12 preferred glycerol as sole carbon source for growth and poly(3HB) accumulation. The highest cell density and highest poly(3HB) content attained were 4·8g l(-l) (cell dry weight) and 73% (w/w), respectively. Cells of all strains grew at temperatures between 37 and 55°C with the optimum growth at 50°C. CONCLUSIONS: New PHA-accumulating thermophilic bacterial strains were isolated and characterized to produce poly(3HB) from glucose or glycerol in MSM at 50°C. SSCAs formation was reported during growth. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report on the formation of SSCAs by PHA-accumulating bacteria and also by thermophilic bacteria. PHA-producing thermophiles can significantly reduce the costs of fermentative PHA production.

Zobellella denitrificans strain MW1, a newly isolated bacterium suitable for poly(3-hydroxybutyrate) production from glycerol.

AIMS: To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol. METHODS AND RESULTS: Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram-negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98.5% and 96.2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41 degrees C and pH 7.3 and accumulated PHB up to 80.4% (w/w) of cell dry weight. PHB accumulation was growth-associated. Although it was not an absolute requirement, 20 g l(-1) sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol. CONCLUSIONS: A new PHB-accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.