STUDY OF TWO MEDICINAL HERBS LEUCAS ASPERA AND CISTUS LAURIFOLIUS FOR THEIR PROSTAGLANDIN INHIBITORY AND ANTIOXIDANT COMPONENTS

研究科: 千葉大学大学院医学薬学府学位:千大院医薬博甲第薬6

Structural Insight into and Mutational Analysis of Family 11 Xylanases: Implications for Mechanisms of Higher pH Catalytic Adaptation

<div><p>To understand the molecular basis of higher pH catalytic adaptation of family 11 xylanases, we compared the structures of alkaline, neutral, and acidic active xylanases and analyzed mutants of xylanase Xyn11A-LC from alkalophilic <i>Bacillus</i> sp. SN5. It was revealed that alkaline active xylanases have increased charged residue content, an increased ratio of negatively to positively charged residues, and decreased Ser, Thr, and Tyr residue content relative to non-alkaline active counterparts. Between strands β6 and β7, alkaline xylanases substitute an α-helix for a coil or turn found in their non-alkaline counterparts. Compared with non-alkaline xylanases, alkaline active enzymes have an inserted stretch of seven amino acids rich in charged residues, which may be beneficial for xylanase function in alkaline conditions. Positively charged residues on the molecular surface and ionic bonds may play important roles in higher pH catalytic adaptation of family 11 xylanases. By structure comparison, sequence alignment and mutational analysis, six amino acids (Glu16, Trp18, Asn44, Leu46, Arg48, and Ser187, numbering based on Xyn11A-LC) adjacent to the acid/base catalyst were found to be responsible for xylanase function in higher pH conditions. Our results will contribute to understanding the molecular mechanisms of higher pH catalytic adaptation in family 11 xylanases and engineering xylanases to suit industrial applications.</p></div

Additional file 1: Figure S1. of Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis

Schematic diagram of a high-throughput screening of the positive mutant. Table S1. Primers used for random mutagenesis, site-directed mutagenesis and site saturation mutagenesis. (DOC 325 kb

Structural models of six-point mutation sites around the catalytic center in Xyn11A-LC (PDB: 4IXL).

<p>(A) E16Q. (B) L46V/A/G. (C) R48G. (D) S187G. E16, L46, R48 and S187 are shown in green. The corresponding mutation sites are shown in cyan. V46, A46, and G46 are shown in cyan, magenta, and yellow, respectively. Hydrogen bonds and salt bridges are represented in yellow by dashed lines.</p

Solvent-exposed residues, hydrogen bonds content and the number of ionic bonds of structure-determined family 11 mesophilic xylanases.

<p>Solvent-exposed residues, hydrogen bonds content and the number of ionic bonds of structure-determined family 11 mesophilic xylanases.</p

The type of secondary structure and the number of hydrogen bonds between β6 and β7 of family 11 mesophilic xylanases with known structure and pH-dependent activity.

<p>t = 4.667,P = 0.001.</p><p>The type of secondary structure and the number of hydrogen bonds between β6 and β7 of family 11 mesophilic xylanases with known structure and pH-dependent activity.</p

Structure-based sequence alignment of family 11 mesophilic xylanases with known structure and pH-dependent activity.

<p>Coils, arrows, and the symbol T represent helices, strands, and turns, respectively.</p

Effect of pH on the activity of wild-type Xyn11A-LC and mutants.

<p>(A) pH-dependent relative activities of the wild type and mutants E16Q, W18Y, N44D, R48G, and S187G. (B) pH-dependent specific activities of the wild type and mutants E16Q, W18Y, N44D, R48G, and S187G. (C) pH-dependent relative activities of the wild type and mutants L46V/A/G. (D) pH-dependent specific activities of the wild type and mutants L46V/A/G.</p

TALENs-Assisted Multiplex Editing for Accelerated Genome Evolution To Improve Yeast Phenotypes

Genome editing is an important tool for building novel genotypes with a desired phenotype. However, the fundamental challenge is to rapidly generate desired alterations on a genome-wide scale. Here, we report TALENs (transcription activator-like effector nucleases)-assisted multiplex editing (TAME), based on the interaction of designed TALENs with the DNA sequences between the critical TATA and GC boxes, for generating multiple targeted genomic modifications. Through iterative cycles of TAME to induce abundant semirational <i>indels</i> coupled with efficient screening using a reporter, the targeted fluorescent trait can be continuously and rapidly improved by accumulating multiplex beneficial genetic modifications in the evolving yeast genome. To further evaluate its efficiency, we also demonstrate the application of TAME for significantly improving ethanol tolerance of yeast in a short amount of time. Therefore, TAME is a broadly generalizable platform for accelerated genome evolution to rapidly improve yeast phenotypes

Additional file 1: of Genome shuffling of the nonconventional yeast Pichia anomala for improved sugar alcohol production

Fig. S1. The colorimetric assay of sugar alcohols. a The flow chart of the colorimetric method for sugar alcohol screening. b The correlation of the two sugar alcohol-detection methods by linear regression. H and C represent the HPLC and colorimetric methods, respectively. Fig. S2. Comparison of the DNA content among the parent and shuffled strains, as determined by flow cytometry. The DNA content is shown for a haploid control strain S. cerevisiae BY4741, haploid parent strain P. anomala HP, diploid strain P. anomala TIB-x229 and shuffled strains GS2-1, GS2-2 and GS2-3