3D プリンターとナノテクノロジーを駆使する環境調和型の新規酵素反応システムによる有用物質の合成
所属：東京工業大学 生命理工学院 生命理工学系 博士後期課程 2 年（助成時）同上 博士後期課程 3 年（現 在）Kotchakorn T.sriwong
Biocatalysis has been a promising and green approach for various industries. However, the limitations of free
enzymes are their instability and unable to be recycled. Therefore, enzyme immobilization has been addressedto solve the problems. Nowadays, the advent of nanotechnology and material science have availed in the successof enzyme immobilization. In the meantime, 3D-printer technologies have significantly been developed due totheir advantages, such as being versatile, convenient, time-saving, and inexpensive. Therefore, this researchutilized nanotechnology and 3D-printertechnology to develop new approachesfor

enzymeimmobilization

andbeneficial compounds production (Fig.
1).

Severalnovel

enzymes

immobilized

in/on

novel

supportexamples,organic-materials,

forwereinorganic nanocrystal, graphene-based
nanomaterials, and the surface of 3Dprinted

reactors.

The

immobilizedenzymes exhibited superior properties tothat of free enzymes. In addition, we
successfully

fabricated

3D-printed

bioreactors and established a continuousflow process to produce an enantiopure
alcohol. Eventually, we intend toproduce beneficial compounds on alargescalefrom

ourdeveloped

continuous-flow biocatalytic system toFig. 1 Applications of nanotechnology and 3D-printer inenzyme immobilization for beneficial compound production

promote green industries.
1. Organic-inorganic nanocrystal formation
This immobilization strategy is simple and effective. We successfully immobilized various novel enzymes such

as Geotrichum candidum acetophenone reductase (GcAPRD), Geotrichum candidum aldehyde dehydrogenase(GcALDH), and Baeyer–Villiger monooxygenase (FBVMO), by this method. We found that all of the enzyme
nanocrystals

exhibitedsuperior

properties to the free enzyme, such as
better stability and recyclability. Inaddition, we synthesized beneficial
compounds by GcAPRD nanocrystal

Fig. 2 Beneficial compounds produced by GcAPRD nanocrystal

and achieved a high reaction yield withexcellent enantioselectivity (Fig. 2).
2. Graphene-based nanomaterials
Graphene oxide (GO) and reduced
grapheneoxide(rGO)

havebeenFig. 3 Beneficial compounds produced by rGO-GcAPRD

attractive carriers for enzyme immobilization because of their unique characteristics, for examples, strong
mechanical strength and large specific area. Thus, we immobilized GcAPRD on GO and rGO via physicaladsorption. It appeared that rGO loaded GcAPRD with a high immobilization yield (maximum ⁓3 mg
protein/mg rGO) and retained high enzyme catalytic activity after immobilization (104%). This approach solved
the trade-off relationship problems between immobilization yield and the retained enzyme activity found ingeneral immobilization methods. Fig. 3 shows examples of beneficial compounds synthesized by rGOGcAPRD.
3. 3D-printed bioreactor of GcAPRDa)b)

We designed bioreactors by computer-aided design(CAD) programs and fabricated them with a 3Dprinter using polypropylene as a material. Then,the surfaces of the 3D-printed reactors werefunctionalized by mussel-inspired polydopamine,
glutaraldehyde, and polyethylenimine for GcAPRD

Fig. 4 a) CAD of the microfluidic bioreactor,
and b) 3D-printed microfluidic bioreactor

immobilization. We found that the immobilized GcAPRD performed sufficient activity and excellent
enantioselectivity after immobilization. Additionally, we successfully established continuous flow processes
utilizing 3D-printed microfluidic bioreactors (Fig. 4) with immobilized GcAPRD to produce an enantiopure
alcohol, and it could operate up to 117-144 h.
要約：酵素は、様々な分野において、環境にやさしい触媒として有用である。しかし、遊離の酵素は不安定な場合もあり、また、再利用できないなどの問題がある。これらの問題を解決するためには、酵素を固定化する必要がある。本研究では、3D プリンターを利用し、また、ナノテクノロジーを駆使することにより、酵素の固定化に成功した。酵素としては、acetophenone reductase(GcAPRD)、aldehyde dehydrogenase (GcALDH)、および、Baeyer–Villiger monooxygenase (FBVMO)を用いた。固定化法としては、有機―無機ハイブリッドナノクリスタルを作成する方法、酸化グラフェン上に固定化する方法、および、3D プリンターで作成したリアクター表面に表面処理を行なった後に酵素を固定化する方法に成功した。さらにフロー系の反応にも成功した。