New insights into enzymatic hydrolysis of heterogeneous cellulose by using carbohydrate-binding module 3 containing GFP and carbohydrate-binding module 17 containing CFP

Biotechnology for Biofuels

Shuhong Gao³  Chun You³  Scott Renneckar⁴  Jie Bao²  Yi-Heng Percival Zhang¹ 

[1] Gate Fuels Inc., Blacksburg, VA 24060, USA

[2] School of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

[3] Biological Systems Engineering Department, Virginia Tech, 304-A Seitz Hall, Blacksburg, VA 24061, USA

[4] Sustainable Biomaterials Department, Virginia Tech, 230 Cheatham Hall, Blacksburg, VA 24061, USA

关键词: Mono-cherry fluorescent protein;    Crystalline cellulose;    Carbohydrate-binding module;    Enzymatic hydrolysis of heterogeneous cellulose;    Amorphous cellulose;   

Others  :  793614
DOI  :  10.1186/1754-6834-7-24

 received in 2013-10-21, accepted in 2014-02-07,  发布年份 2014

【 摘 要 】

Background

The in-depth understanding of the enzymatic hydrolysis of cellulose with heterogeneous morphology (that is, crystalline versus amorphous) may help develop better cellulase cocktail mixtures and biomass pretreatment, wherein cost-effective release of soluble sugars from solid cellulosic materials remains the largest obstacle to the economic viability of second generation biorefineries.

Results

In addition to the previously developed non-hydrolytic fusion protein, GC3, containing a green fluorescent protein (GFP) and a family 3 carbohydrate-binding module (CBM3) that can bind both surfaces of amorphous and crystalline celluloses, we developed a new protein probe, CC17, which contained a mono-cherry fluorescent protein (CFP) and a family 17 carbohydrate-binding module (CBM17) that can bind only amorphous cellulose surfaces. Via these two probes, the surface accessibilities of amorphous and crystalline celluloses were determined quantitatively. Our results for the enzymatic hydrolysis of microcrystalline cellulose (Avicel) suggested that: 1) easily accessible amorphous cellulose on the surface of Avicel is preferentially hydrolyzed at the very early period of hydrolysis (that is, several minutes with a cellulose conversion of 2.8%); 2) further hydrolysis of Avicel is a typical layer-by-layer mechanism, that is, amorphous and crystalline cellulose regions were hydrolyzed simultaneously; and 3) most amorphous cellulose within the interior of the Avicel particles cannot be accessed by cellulase.

Conclusions

The crystallinity index (CrI), reflecting a mass-average (three-dimensional) cellulose characteristic, did not represent the key substrate surface (two-dimensional) characteristic related to enzymatic hydrolysis.

【 授权许可】

   
2014 Gao et al.; licensee BioMed Central Ltd.

【 图 表 】

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