13 概 要 自然界において結晶性大部分は，種々の微生 物が生産す（CBH）にって分解さ てい。しかしなが，CBH の反応には基質，酵素双方の 性質が関してお，さに反応が固液界面でという特 徴有していことか，定量的な解析が非常に難しい。そ こで本研究では，結晶性の表面積 CBH の最大 吸着量（A max ）か見積，反応の吸着量（A） A max で割った表面密度（r＝A/A max ）に対して，吸着した酵素 分子あたの度（k＝v/A）すことで基質の表 面積相殺した。その結果，基質の性質が の反応度に大きく影響していことが示唆さた。 A. はじめに は，植物細胞壁成分の約 50 占多糖 であ，天然で最豊富に存在す有機物であ。地球で は年間 10 10  10 11 が光合成にって生産さていと言 てい(1)が，実際に利用さていのはごく部であ， その用はとどが繊維（ˆber ）としての利用にとどっ てい。しかしなが，京都議定書に基づく二酸化炭素排出 量の削減，原油価格と穀物価格の高騰といった社会的背景 受けて，系の更な用拡大が望 てお，液体燃料化成品の原料として利用 す試がすでに界で始ってい。自然界において は，そのとどが分解性微生物にっ て分解代謝さていことか，微生物そ微生物が 生産す酵素利用す変換に注目が集 13 Trends in Glycoscience and Glycotechnology Vol. 21 No. 117 (2009) pp. 13–22 doi:10.4052/tigg.21.13  2009 FCCA (Forum: Carbohydrates Coming of Age) MINIREVIEW Kinetic Analysis of Cellobiohydrolase: Quantiˆcation of Enzymatic Reaction at a Solid/Liquid Interface Applying the Concept of Surface Density セロビオヒドロラーゼの反応速度論的解析 固液界面における酵素反応を表面密度という概念を用いて定量化する Igarashi, Kiyohiko*; Wada, Masahisa; and Samejima, Masahiro Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan *Corresponding author FAX: 81–3–5841–5258, E-mail: aquarius＠mail.ecc.u-tokyo.ac.jp (Received on December 25, 2008, accepted on January 9, 2009) Key Words: Cellobiohydrolase, Surface density, Solid/liquid interface, Trichoderma cellulase, Cellulose degradation Abstract In nature, crystalline cellulose is mainly degraded by cellobiohydrolases (CBHs) produced by various microorganisms. Since both substrate and enzymatic fea- tures relate to the reaction and the reaction proceeds at a solid/liquid interface, it is quite di‹cult to perform the quantitative analysis of CBH. In the present study, we es- timate the surface area of crystalline celluloses by the ad- sorption maxima (Amax) of CBH, the speciˆc activity of CBH (k＝v/A) was plotted versus surface density ( r＝ A/Amax) to adjust for the diŠerence of surface area, and quantify the reaction rate of CBH. The results obtained from the approach indicate that the feature of crystalline cellulose greatly aŠects the reaction of CBH. A. Introduction Cellulose is a polysaccharide, accounting for ap- proximately 50z of plant cell walls, and is most abundant organic compound in nature. Although 10–100 Mt of cellulose is photosynthesized every year on earth (1), only a limited amount is used at present, mainly as a ˆber source. However, the reduction of carbon dioxide emis- sions ratiˆed in the Kyoto protocol and the recent sharp rise in the price of oil and crops emphasize the importance of better utilization of cellulosic materials. Consequently, application trials of cellulose as a source of liquid fuels and as a raw material of various chemicals have been start- ed in many countries (2). Since cellulose is mainly degrad- ed and metabolized by cellulolytic microorganisms in na- ture, bio-conversion processes using these microorganisms and/or enzymes produced by them have attracted much