A recombinant cyclodextrin glycosyltransferase cloned from Paenibacillus sp. strain RB01 showed improved catalytic activity in coupling reaction between cyclodextrins and disaccharides

Recombinant cyclodextrin glycosyltransferase (CGTase) was obtained by cloning the PCR gene fragment from thermotolerant Paenibacillus sp. strain RB01 screened from hot spring area in Thailand and cloned into the Escherichia coli expression vector. The nucleotide sequence was analyzed and aligned. Nucleotide sequence of the recombinant CGTase contained an open reading frame of 2139 bp encoding 713 amino acid residues. The recombinant required one-third of culture time and neutral pH to produce CGTase compared to wild type. CGTases from both wild type and transformant were purified in parallel by starch adsorption and DEAE cellulose column. Their biochemical properties such as molecular weight, optimum pH and temperature were quite similar. However, the recombinant enzyme showed improved catalytic activity in the coupling reaction between cyclodextrins (CDs) and some disaccharides. Among several sugars tested with excess βCD, cellobiose was the best substrate followed by leucrose. Very low activity was observed with trehalose, lactose and mellibiose. Sucrose and raffinose showed no activity. The K _m and other kinetic parameters of recombinant enzyme were determined for cellobiose and several cyclodextrin derivatives. Recombinant CGTase showed lower K _m for βCD and its derivatives, with improved activity compared to wild type enzyme.     

Optimization of Lactic Acid Production by Pellet-Form Rhizopus oryzae in 3-L Airlift Bioreactor Using Response Surface Methodology

The influence of two key environmental factors, pH and oxygen transfer coefficient (k _La), was evaluated on the lactic acid production as the main answer and, on the size of cell pellets of the fungal strain Rhizopus oryzae KPS106, as second dependant answer by response surface methodology using a central composite design. The results of the analysis of variance and modeling demonstrated that pH and k _La had a significant effect on lactic acid production by this strain. However, no interaction was observed between these two experimental factors. pH and k _La had no significant influence on the pellet size. Optimal pH and k _La of the fermentation medium for lactic acid production from response surface analysis was 5.85 and of 3.6 h^?1, respectively. The predicted and experimental lactic acid maximal values were 75.4 and 72.0 g/l, respectively, with pellets of an average of 2.54?±?0.41 mm. Five repeated batches in series were conducted with a mean lactic acid production of 77.54 g/l. The productivity was increased from 0.75 in the first batch to 0.99 g/l h in the last fifth batch.     

Investigation of Zr-C,Zr-N,and potential agostic interactions in an organozirconium complex by experimental electron density analysis

The crystal structure and electron density (ED) distribution of an imine coupling product with an open zirconocene, Zr(2,4-C(7)H(11))[(i-Pr)NCHPhCH(2)CMe=CHCMe=CH(2)] (C(7)H(11) = dimethylpentadienyl), have been derived from accurate synchrotron X-ray diffraction measurements. The molecular structure reveals asymmetric coordination of Zr by the pentadienyl (2,4-C(7)H(11)) ligand ( = 2.56(6) A), the butadiene fragment ( = 2.43(5) A), and the amide nitrogen atom (Zr-N = 2.0312(5) A) of the second ligand. The study of the ED and its topological analysis affords new insight into the bonding and electronic structure of the title zirconium complex. The interactions between the metal center and the ligands are represented by a Zr-N bond path and one Zr-C bond path with each of the pentadienyl and butadiene moieties, contrary to the usually depicted global metal-ligand bonding. The butadiene and pentadienyl groups exhibit a polarization of the corresponding pi-like ED in the C-Zr directions, indicating that the whole conjugated systems are nonetheless involved. The 4d atomic orbitals of Zr exhibit unusual populations according to ligand field considerations, which reveal a high degree of sigma-donation from the conjugated pi systems of both ligands. As deduced from numerical integration over the topologically defined atomic basins, the Zr to ligand charge transfer is 1.48 e to the C(17)NH(24) ligand and 0.68 e to dimethylpentadienyl. Topological analysis of a short intramolecular Zr.(C,H) contact provides no indication of the presence of agostic interactions, despite a small Zr-N-C angle of 102.87(4) degrees. Thus, no bond path and BCP (bond critical point) of the ED are found in the Zr.(C,H) region, which would have provided evidence for such direct interactions, nor is there any evidence for charge accumulation between the Zr and H atoms, or for lengthening of the C-H bond involved in the putative interaction. These characteristics, similar to those in other distorted situations, may be common for other electron-deficient d(0) complexes.