Title: Characterization of a poly(butylene adipate- co -terephthalate) hydrolase from the mesophilic actinobacteria Rhodococcus fascians Soulenthone P, Tachibana Y, Suzuki M, Mizuno T, Ohta Y, Kasuya KI Ref: Polymer Degradation and Stability, 154:109481, 2021 : PubMed
Poly(butylene adipate- co -terephthalate) (PBAT) possesses excellent film-forming ability and biodegrad- ability. Therefore, it is considered to be a promising mulching film material that eliminates the need for recovery. In the applications that require PBAT degradation in the field after use, it is important to un- derstand the biodegradation mechanism at moderate temperatures. We have previously isolated from the soil the mesophilic actinobacteria Rhodococcus fascians NKCM2511 that biodegraded PBAT under moderate temperature conditions (20-30 C). In this study, to clarify the mechanism of PBAT degradation by the strain NKCM2511, a DNA fragment carrying the gene pbath Rf responsible for the PBAT degradation activity was cloned. The gene encoded a 216-amino-acid-long protein designated as PBATH Rf . Homology modeling revealed that PBATH Rf belongs to the alpha/ betahydrolase fold family, lacking the lid domain covering the active site. PBATH Rf degraded PBAT film at 30 C at the rate of 0.10 +/- 0.03 mg/cm 2 /d and was capable of degrad- ing several other aliphatic polyester films. Liquid chromatography revealed that PBATH Rf preferentially cleaved the ester bond between 1,4-butanediol and adipic acid rather than that between 1,4-butanediol and terephthalic acid (T). This characteristic of PBATH Rf may explain the low degradation rate of the aliphatic - aromatic copolyester PBAT, compared to the rate of degradation of aliphatic polyesters without T. In addition, liquid chromatography showed that PBATH Rf released T, mono(2-hydroxyethyl) terephthalic acid, and bis(2-hydroxybutyl) terephthalate from an amorphous poly(ethylene terephthalate) (PET) film. However, no significant change in the PET film surface after the treatment with PBATH Rf was found by scanning electron microscopy. This is the first report of an enzyme from the mesophilic actinobacteria Rhodococcus fascians that can hydrolyze various polyesters, including PBAT, and catalyze hydrolysis on the surface of an amorphous PET film. This study also provides insight into the biodegradation mechanism of PBAT in the actual field as it describes an enzyme from a naturally occurring organism that acts in the medium temperature range.
Title: Lifestyle improvement or anti-dementia drugs in Alzheimer's disease: A comment on Kapaki et al. The cognitive effects of cholinesterase inhibitor treatment in every-day practice. Curr Med Res Opin 2005; 21(6): 871-5 Niwa F, Mizuno T, Nakagawa M Ref: Curr Med Res Opin, :1, 2019 : PubMed
Title: Mutational analysis of Kex2 recognition sites and a disulfide bond in tannase from Aspergillus oryzae Koseki T, Otsuka M, Mizuno T, Shiono Y Ref: Biochemical & Biophysical Research Communications, 482:1165, 2017 : PubMed
Aspergillus oryzae tannase (AoTanA), which contains two Kex2 recognition sites at positions Arg311 and Arg316, consists of two subunits that are generated by the cleavage of tannase gene product by the Kex2 protease. Based on the crystal structure of feruloyl esterase from Aspergillus oryzae (AoFaeB), which has been classified as a member of the fungal tannase family, the catalytic triad residues of AoTanA are predicted to be Ser195, Asp455, and His501, with the serine and histidine residues brought together by a disulfide bond of the neighboring cysteines, Cys194 and Cys502. In this study, we investigated the functional role of the Kex2 recognition sites and disulfide bond between the neighboring cysteines in AoTanA. We constructed a double variant (R311A/R316A), a seven amino-acid deletion variant of region Lys310-Arg316 (DeltaKR), and two single variants (C194A and C502A). While the R311A/R316A variant exhibited the two bands similar to wild type by SDS-PAGE after treatment with endoglycosidase H, the DeltaKR variant exhibited only one band. R311A/R316A variation had no effect on tannase activity and stability. Meanwhile, the DeltaKR variant exhibited higher activity compared to the wild-type. The activities of the C194A and C502A variants decreased considerably (<0.24% of the wild-type) toward methyl gallate.
The use of biodegradable plastic films made of poly(butylene adipate-co-terephthalate) (PBAT) to improve crop production has been proposed. Because the film after use is expected to be degraded on site, it is important to understand the biodegradation mechanism of PBAT in aerobic and mild temperature conditions. We therefore isolated three PBAT-degrading strains, NKCM3201, NKCM3202, and NKCM3101, from soil environments. Phylogenetic analysis revealed that the strains are closely related to Bacillus pumilus. Strain NKCM3201, which degraded PBAT film at the fastest rate (12.2 mug/day/cm2) and grew well at 30 C to 40 C in aerobic conditions, was selected for further analysis. We cloned the 648-bp coding region of the PBAT hydrolase (PBATHBp) gene, which encodes a 215-amino acid protein containing a signal peptide of 34 residues. Mutation analyses revealed that PBATHBp belongs to the serine hydrolase superfamily, with a catalytic triad composed of Ser77, Asp133, and His156. Homology 3D modeling of PBATHBp using Bacillus subtilis 168 lipase as a template showed that the enzyme belongs to the alpha/beta hydrolase fold family, which lack a lid domain on its surface. PBATHBp hydrolyzed PBAT, poly(butylene succinate-co-adipate) (PBSA), poly(ethylene succinate) (PESu), and polycaprolactone (PCL) films at a degradation rate of 14.3, 3.3 x 10+2, 7.0 x 10+2, and 1.1x 10+2 mug/cm2/day, respectively. Liquid chromatography-mass spectrometry analysis of degradation products from PBAT revealed that PBATHBp hydrolyses ester bonds between butanediol and terephthalate (B-T bonds) at much slower rates than ester bonds between adipate and butanediol. This ester bond preference may explain the very slow PBAT degradation rate compared to PBSA, PESu, and PCL. This is the first report of a PBAT hydrolase from an aerobic mesophilic bacterium, and may contribute to our understanding of PBAT biodegradation under mild temperature conditions.
Title: Biochemical characterization of Aspergillus oryzae native tannase and the recombinant enzyme expressed in Pichia pastoris Mizuno T, Shiono Y, Koseki T Ref: J Biosci Bioeng, 118:392, 2014 : PubMed
In this study, the biochemical properties of the recombinant tannase from Aspegillus oryzae were compared with those of the native enzyme. Extracellular native tannase was purified from a commercial enzyme source. Recombinant tannase highly expressed in Pichia pastoris was prepared as an active extracellular protein. Purified native and recombinant tannases produced smeared bands with apparent molecular masses of 45-80 kDa and 45-75 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, the native enzyme yielded molecular masses of 33 kDa and 30 kDa, whereas the recombinant enzyme yielded molecular masses of 34 kDa and 30 kDa. Purified native and recombinant tannases had an optimum pH of 4.0-5.0 and 5.0, respectively, and were stable up to 40 degrees C. After N-deglycosylation, both enzymes exhibited reduced thermostability. Catalytic efficiencies of both purified enzymes were greater with natural substrates, such as (-)-catechin, (-)-epicatechin, and (-)-epigallocatechin gallates, than those with synthetic substrates, such as methyl, ethyl, and propyl gallates. However, there were no activities against the methyl esters of ferulic, p-coumaric, caffeic, and sinapic acids, which indicate feruloyl esterase activity, or the ethyl, propyl, and butyl esters of 4-hydroxybenzoic acid, which indicate paraben hydrolase activity.
As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.
The gene cluster responsible for ML-236B (compactin) biosynthesis has recently been characterized from P. citrinum No. 41520. Here, we describe how the ML-236B-producing strain was improved using a cosmid-mediated recombination technique. The introduction of the cosmid pML48, which contains seven of the nine ML-236B biosynthetic genes, into P. citrinum No. 41520 resulted in transformants which produced increased amounts of ML-236B. Southern analysis showed that pML48 had been incorporated by a homologous recombination event, and all high producers possessed two copies of each of the seven genes, mlcA- mlcF and mlcR, suggesting that increased dosage of the biosynthetic gene cluster was responsible for the enhanced production of ML-236B. On the other hand, various kinds of mutants with decreased titers of ML-236B were also obtained. Characterization of one such mutant, designated as T48.28, which was more sensitive to ML-236B than the parental strain, suggested that one of the ML-236B biosynthetic genes, mlcD, which encodes a putative HMG-CoA reductase, was involved in conferring resistance to ML-236B.
Title: Cell and tissue transplantation in zebrafish embryos Mizuno T, Shinya M, Takeda H Ref: Methods Mol Biol, 127:15, 1999 : PubMed
