In this study, a fast carbonyl reductases colorimetric screening method for discovering stereoselective carbonyl reductases was established by combining the reverse alcohol oxidation with the azoreductase-catalyzed reduction of azo dye. When azo dye (Orange I , 4-(4-hydroxy-1-naphthylazo) benzenesulfonic acid) and azoreductase (AzoB) were added into the reaction system of alcohol oxidation catalyzed by carbonyl reductase, the produced NAD(P)H served as electron donor for the azoreductase to reduce the azo dye, resulting the color fade. Hence, the carbonyl reductases can be screened by the obvious color change. When chiral alcohol was used as the substrate, the activity and stereoselectivity of carbonyl reductases can be screened at the same time.
Alcohol Oxidoreductases ; chemistry ; Alcohols ; chemistry ; Azo Compounds ; chemistry ; Coloring Agents ; chemistry ; High-Throughput Screening Assays ; NADH, NADPH Oxidoreductases ; chemistry ; NADP ; chemistry ; Oxidation-Reduction ; Stereoisomerism

The monocot mannose-binding lectin can inhibit HIV from infecting the target cells. The total RNA of Zephyranthes grandiflora was extracted and reversely transcribed into cDNA. Degenerate primers were designed based on the conserved regions of other monocot mannose-binding agglutinins by homology alignment. The 694bp full-length cDNA of Zephyranthes grandiflora agglutinin (ZGA) was cloned by RT-PCR, 3' and 5' RACE (rapid amplification of cDNA ends). The start codon and stop codon of ZGA were at 37-39bp and 529-531bp respectively. The NCBI Blast analysis result showed that ZGA gene encoded a protein precursor with signal peptide, mature protein and C-terminal cleavage sequence. The mature ZGA protein contained 106 amino acids residues and its molecular weight was 11.6KD. The percentages of identity of the deduced mature ZGA protein with those of Galanthus nivalis agglutinin, Narcissus hybrid cultivar agglutinin, Lycoris radiate agglutinin and Clivia miniata agglutinin were 71.8%, 81%, 81.8% and 84.5%, respectively. Blocks analysis revealed that ZGA had three functional domains and three mannose-binding boxes (QDNY).
Agglutinins ; genetics ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Liliaceae ; genetics ; Mannose-Binding Lectin ; genetics ; Molecular Sequence Data ; Sequence Analysis, DNA

Horizontal gene transfer(HGT)has long been considered as a principal force for an organism to gain novel genes in genome evolution. Homology search, phylogenetic analysis and nucleotide composition analysis are three major objective approaches to arguably determine the occurrence and directionality of HGT. Here, 21 genes that possess the potential to horizontal transfer were acquired from the whole genome of Magnaporthe grisea according to annotation, among which three can-didate genes(corresponding protein accession numbers are EAA55123, EAA47200 and EAA52136)were selected for further analysis. According to BLAST homology results, we subsequently conducted phylogenetic analysis of the three candidate HGT genes. Moreover, nucleotide composition analysis was conducted to further validate these HGTs. In addition, the functions of the three candidate genes were searched in COG database. Consequently, we conclude that the gene encoding protein EAA55123 is transferred from Clostridium perfringens. Another HGT event is between EAA52136 and a certain metazoan's corresponding gene, but the direction remains uncertain. Yet, EAA47200 is not a transferred gene.