Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis

There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST, respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and, ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3 \u2013 5\ub0C increase in the apparent melting temperature (Tm) of the mutants over the native BcEST that has a Tm of 50\ub0C was outperformed by TtEST, a naturally-occurring homolog with a Tm of 65\ub0C. Protein modeling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the \u3b1/\u3b2 fold-hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally-occurring thermostable biocatalyst that outperforms experimentally-evolved homologs that carry out the same hydrolysis.Peer reviewed: YesNRC publication: Ye

Effects of Oxide Additives on the Phase Structures and Electrical Properties of SrBi4Ti4O15 High-Temperature Piezoelectric Ceramics

In this work, SrBi4Ti4O15 (SBT) high-temperature piezoelectric ceramics with the addition of different oxides (Gd2O3, CeO2, MnO2 and Cr2O3) were fabricated by a conventional solid-state reaction route. The effects of oxide additives on the phase structures and electrical properties of the SBT ceramics were investigated. Firstly, X-ray diffraction analysis revealed that all these oxides-modified SBT ceramics prepared presented a single SrBi4Ti4O15 phase with orthorhombic symmetry and space group of Bb21m, the change in cell parameters indicated that these oxide additives had diffused into the crystalline lattice of SBT and formed solid solutions with it. The SBT ceramics with the addition of MnO2 achieved a high relative density of up to 97%. The temperature dependence of dielectric constant showed that the addition of Gd2O3 could increase the TC of SBT. At a low frequency of 100 Hz, those dielectric loss peaks appearing around 500 °C were attributed to the space-charge relaxation as an extrinsic dielectric response. The synergetic doping of CeO2 and Cr2O3 could reduce the space-charge-induced dielectric relaxation of SBT. The piezoelectricity measurement and electro-mechanical resonance analysis found that Cr2O3 can significantly enhance both d33 and kp of SBT, and produce a higher phase-angle maximum at resonance. Such an enhanced piezoelectricity was attributed to the further increased orthorhombic distortion after Ti4+ at B-site was substituted by Cr3+. Among these compositions, Sr0.92Gd0.053Bi4Ti4O15 + 0.2 wt% Cr2O3 (SGBT-Cr) presented the best electrical properties including TC = 555 °C, tan δ = 0.4%, kp = 6.35% and d33 = 28 pC/N, as well as a good thermally-stable piezoelectricity that the value of d33 was decreased by only 3.6% after being annealed at 500 °C for 4 h. Such advantages provided this material with potential applications in the high-stability piezoelectric sensors operated below 500 °C

Monooxygenase-catalyzed Baeyer-Villiger oxidations: CHMO versus CPMO

Cyclopentanone monooxygenase (CPMO) from Comamonas sp. NCIMB 9872 expressed in E. coli was evaluated as a potential new bioreagent for Baeyer-Villiger oxidations of 4-alkoxy- and halo-substituted cyclohexanones (10 examples). The results were compared with those obtained in oxidations catalyzed by an engineered E. coli strain expressing cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. CPMO was found to have modest to good stereoselectivity and broader substrate acceptability than CHMO. The stereoselectivities of the two enzymes were generally opposite. It appears, therefore, that the two engineered strains can be useful and complementary reagents for enantioselective Baeyer-Villiger oxidations of certain prochiral ketones.NRC publication: Ye

Microstructures and Electrical Conduction Behaviors of Gd/Cr Codoped Bi3TiNbO9 Aurivillius Phase Ceramic

In this work, a kind of Gd/Cr codoped Bi3TiNbO9 Aurivillius phase ceramic with the formula of Bi2.8Gd0.2TiNbO9 + 0.2 wt% Cr2O3 (abbreviated as BGTN−0.2Cr) was prepared by a conventional solid-state reaction route. Microstructures and electrical conduction behaviors of the ceramic were investigated. XRD and SEM detection found that the BGTN−0.2Cr ceramic was crystallized in a pure Bi3TiNbO9 phase and composed of plate-like grains. A uniform element distribution involving Bi, Gd, Ti, Nb, Cr, and O was identified in the ceramic by EDS. Because of the frequency dependence of the conductivity between 300 and 650 °C, the electrical conduction mechanisms of the BGTN−0.2Cr ceramic were attributed to the jump of the charge carriers. Based on the correlated barrier hopping (CBH) model, the maximum barrier height WM, dc conduction activation energy Ec, and hopping conduction activation energy Ep were calculated with values of 0.63 eV, 1.09 eV, and 0.73 eV, respectively. Impedance spectrum analysis revealed that the contribution of grains to the conductance increased with rise in temperature; at high temperatures, the conductance behavior of grains deviated from the Debye relaxation model more than that of grain boundaries. Calculation of electrical modulus further suggested that the degree of interaction between charge carriers β tended to grow larger with rising temperature. In view of the approximate relaxation activation energy (~1 eV) calculated from Z″ and M″ peaks, the dielectric relaxation process of the BGTN−0.2Cr ceramic was suggested to be dominated by the thermally activated motion of oxygen vacancies as defect charge carriers. Finally, a high piezoelectricity of d33 = 18 pC/N as well as a high resistivity of ρdc = 1.52 × 105 Ω cm at 600 °C provided the BGTN−0.2Cr ceramic with promising applications in the piezoelectric sensors with operating temperature above 600 °C

Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli-Expressed Cyclopentanone 1,2-Monooxygenase

Cyclopentanone 1,2-monooxygenase, a flavoprotein produced by Pseudomonas sp. strain NCIMB 9872 upon induction by cyclopentanol or cyclopentanone (M. Griffin and P. W. Trudgill, Biochem. J. 129:595-603, 1972), has been utilized as a biocatalyst in Baeyer-Villiger oxidations. To further explore this biocatalytic potential and to discover new genes, we have cloned and sequenced a 16-kb chromosomal locus of strain 9872 that is herein reclassified as belonging to the genus Comamonas. Sequence analysis revealed a cluster of genes and six potential open reading frames designated and grouped in at least four possible transcriptional units as (orf11-orf10-orf9)-(cpnE-cpnD-orf6-cpnC)-(cpnR-cpnB-cpnA)-(orf3-orf4 [partial 3′ end]). The cpnABCDE genes encode enzymes for the five-step conversion of cyclopentanol to glutaric acid catalyzed by cyclopentanol dehydrogenase, cyclopentanone 1,2-monooxygenase, a ring-opening 5-valerolactone hydrolase, 5-hydroxyvalerate dehydrogenase, and 5-oxovalerate dehydrogenase, respectively. Inactivation of cpnB by using a lacZ-Km(r) cassette resulted in a strain that was not capable of growth on cyclopentanol or cyclopentanone as a sole carbon and energy source. The presence of σ(54)-dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-β-thio-d-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts

A flax-retting endopolygalacturonase-encoding gene from Rhizopus oryzae

A polygalacturonase from the filamentous fungus Rhizopus oryzae strain sb (NRRL 29086), previously shown to be effective in the retting of flax fibers, was shown by the analysis of its reaction products on polygalacturonic acid to be an endo-type. By zymogram analysis, the enzyme in the crude culture filtrate appeared as two active species of 37 and 40 kD. The endopolygalacturonase-encoding gene was cloned in Escherichia coli and its translated 383-amino acid sequence found to be identical to that of a presumed exopolygalacturonase found in R. oryzae strain YM9901 and 96% identical to a hypothetical protein (RO3G_04731.1) in the sequenced genome of R. oryzae strain 99-880. Phylogenetic analysis revealed the presence of an unique cluster of Rhizopus polygalacturonase sequences that are separate from other fungal polygalacturonases. Conservation of 12 cysteines appears to be a special feature of this family of Rhizopus polygalacturonase sequences.NRC publication: Ye

Two-layer continuous-process design for the biodesulfurization of diesel oils under bacterial growth conditions

An aqueous\ufffdorganic two-layer partitioning and continuous process was designed to make efficient use of growing cells for the biodesulfurization of diesel oils. This biphasic system was shown to have a significant advantage over batch and fed-batch processes in the maintenance of the biodesulfurization activity for an extended period of time. Specifically, it was demonstrated that in a 2-L bioreactor 1500 ppm total sulfur was removed from 250 ml diesel oil within a 5-day period.NRC publication: Ye