過熱水蒸気中でのポリ（３－ヒドロキシ酪酸-co-３－ヒドロキシヘキサン酸）の分解機構とバイオマテリアルとしての応用のための分解生成物の特性評価

九州工業大学博士学位論文（要旨）学位記番号：生工博甲第299号 学位授与年月日：平成29年9月22

Effects of (R)-3-hydroxyhexanoate units on thermal hydrolysis of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate)s

In order to clarify effects of (R)-3-hydroxyhexanoate (HHx) unit on the hydrothermal degradation of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) (PHBHHx), two PHBHHxs: P(HB-co-6%-HHx) and P(HB-co-11%-HHx), and poly((R)-3-hydroxybutyrate) (PHB) as a reference were treated by superheated steam at 130e190 C. Interestingly, despite having contents of the HHx unit nearby, contrastive degradation property: the contradictory scission behavior of HB-HHx sequence was confirmed. From analysis of chain-end structures, it was confirmed that HHx unit basically suppressed the hydrolysis of HB-HHx sequence and the formation of crotonoyl chain-end groups rapidly increased at higher temperatures, suggesting the shift to thermal degradation from hydrolysis as a main reaction. On thermal degradation, the contrary upper and lower Ea values: 138 ± 1.9 and 121 ± 1.8 kJ mol1 for P(HB-co-6%- HHx) and P(HB-co-11%-HHx), respectively, compared to Ea value: 126 ± 5.3 kJ mol1 of PHB were obtained. The HHx unit basically acts as suppressing factor of the thermal degradation from Td50 and Td50- Tm values; however, the increase in flexibility of polymer sequence must promote the thermal chain cleavage. Thus, the complex hydrothermal degradation behaviors were considered to be combined results of the suppression effects by hydrophobicity and steric hindrance of propyl group in HHx unit and the promotive effects of lower crystallinity and easier steam diffusion into more flexible amorphous region of PHBHHx

Changes in diad sequence distribution by preferential chain scission during the thermal hydrolysis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Polyhydroxyalkanoates (PHAs) are microbial polyesters produced by many types of bacteria as an intracellular energy reserve material under substrate limiting conditions and in the presence of excessive carbon sources.¹ Poly((R)-3-hydroxybutyrate) (PHB), the most commonly used microbial polyester, was the first member of the PHA family to be discovered, and more than 150 other monomer units have been reported to date.2, 3 Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) (PHBHHx) is a copolymer in the PHA family that consists of randomly distributed (R)-3-hydroxybutyrate (HB) and (R)-3-hydroxyhexanoate (HHx) units.⁴ This type of copolymer exhibits improved mechanical properties and processability compared with those of PHB and poly((R)-3-hydroxyvalerate) (PHBV).⁵ PHBHHx copolymers are currently produced on a large scale and have proven to be biocompatible in clinical studies using mouse fibroblasts cells, and rabbit articular cartilage-derived chondrocytes.⁶ PHBHHx is a highly favorable copolymer of the PHB family due to its biodegradability, flexible mechanical properties and good melt processability

Antioxidant and Antibacterial Properties on Different Tissues of Syzygium Aromaticum

This paper focuses on the effect of three drying treatment (microwave-, oven- and air-drying) on the antioxidant and antibacterial properties on different tissues of syzygium aromaticum

MANAGEMENT OF LIBRARIES IN SINGAPORE

Bachelor'sBACHELOR OF SCIENCE (REAL ESTATE

Effects of one amino acid substitutions at the C-terminal region of thermostable L2 lipase from Bacillus sp. L2

The substitutions of the amino acid residue at the predetermined critical point of the Cterminal of L2 lipase may increase its thermostability and lipase activity. N and C-terminal regions in most proteins are often disordered and flexible. However, some protein function was closely related to flexibility as well as play a role in the enzyme reaction. The critical point of the stability of L2 lipase structure was predicted at position 385 (wild type residue Serine) of the L2 sequence based on I-Mutant2.0 software. The effects of substitution of the amino acids at the critical point with Glutamic acid, Isoleucine, and Valine were analyzed with Molecular Dynamics (MD) simulation by using Yet Another Scientific Artificial Reality Application (YASARA) software and it showed that the best predicted mutant L2 lipases had lower RMSD value as compared to L2 lipase. It indicated that the three mutants had higher compactness in the structure consequently enhancing the stability. From RMSF analysis, mutations had reduced the flexibility of the enzyme. The best predicted mutants (S385E, S385I, and S385V) were produced in the experimental lab by site-directed mutagenesis. The mutant L2 lipases (60.4 kDa) were purified to homogeneity by a single chromatography step before proceeding with characterization. There were high lipase activities produced by purified mutant L2 lipases at a temperature range of 60-85 °C with the optimum temperature of 80 ºC, 75 °C and 70 °C for S385E, S385V, and S385I lipases respectively. The optimum temperature for recombinant L2 lipase was at 70 °C. Mutant L2 lipases (S385E and S385V) had higher optimum temperature compared to recombinant L2 lipase. The optimum pH for mutant L2 lipases (S385E and S385V) was found to be at pH 8 and for S385I was at pH 9, whereas the optimum pH for recombinant L2 lipase was at pH 9. S385I lipase was more thermostable as compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 hours preincubation. The stability of S385V lipase in varies organic solvents was higher as compared to recombinant L2 lipase. S385V lipase had relative activities higher than 100% which 111% in DMSO, 105% in acetone, 123% in diethyl ether and 124% in nhexane. Tm values for S385V and S385E lipases were at 85.96 °C and 84.85 °C and the values were higher as compared to recombinant L2 lipase which is only 66.73 °C. This showed the higher thermal stability of S385E and S38V lipases as compared to recombinant L2 lipase. Thus, the substitutions at the predetermined critical point of the Cterminal (Ser385) changed the functionality of the protein structure towards the activity, stability, and flexibility of L2 lipase. The critical point mutation towards the structure of L2 lipase provided a very advantageous strategy for the improvement of enzyme with better function to adapt with harsh environment

Evaluation of degradation products and kinetics of poly(3-hydroxybytyrate-co-3-hydroxyhexanoate) superheated steam hydrolysis for the production of biocompatible oligoesters

In order to apply polyhydroxyalkanoate (PHA) as various biomedical materials, controlled hydrolysis of PHAs was conducted using superheated steam (SHS) for the production of oligoesters. Changes in mass and molecular weight of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) were monitored during the SHS treatment in a range of 130-190 °C. Produced oligoesters were analyzed using 1H and 13C-NMR to determine the chemical structures. Obtained kinetic parameters of degradation and chemical structures of oligoesters revealed that the SHS hydrolysis of PHBHHx preferentially proceeded at HHx*HB sequence and specifically occurred according to the SHS treatment temperatures, resulting in the formation of crotonyl-chain end at temperatures above 170°C