Research Progress: Direct Purification and Characterization of the Key Cellulosomal Exoglucanase Cel48S


The global demand of clean fuel is becoming more urgent, especially for the developing nations. The sustainable non-food lignocellulosic biomass is an attractive source for biofuel and biochemical production. However, the high cost, complicated and incongruous processes, and water pollution issues have significantly inhibited the development of lignocellulosic bioproducts.

Cellulosome is an extracellular multiprotein complex produced by cellulolytic bacteria. Clostridium thermocellum(C. thermocellum) is known as the first described cellulosome-producing and cellulolytic bacterium and has potential use in lignocellulose bioconversion via consolidated bioprocessing. Cel48S plays key roles and influences the activity and features of cellulosome to a great extent. Thus, it is of great importance to reveal the enzymatic features of Cel48S. However, Cel48S has not been well performed due to difficulties in purifying either recombinant or native Cel48S proteins.

The research group from Qingdao Institute of Bioenergy and Bioprocess Technology(QIBEBT), Chinese Academy of Sciences(CAS), developed a series of genetic tools for non-model microorganisms like C. thermocellum (J Microbiol Methods; PloS One; Appl Microbiol Biotechnol; Biotechnol Biofuels). They performed directed engineering of cellulosome for the in situ purification of the catalytic domain of Cel48S in one step with high purity and full activity. Substantial biochemical and structural analyses based on the native protein were performed subsequently to reveal the true features of the key cellulosomal enzyme. The results confirmed the high activity and substrate specificity of Cel48S_CD from C. thermocellum. The structure of the native Cel48S_CD protein revealed evidence of conformational changes during substrate binding (Figure 1). Additionally, the study provided a reliable method for in situ purification of cellulosomal and other secretive proteins from C. thermocellum.

The related work was published in Biotechnology for Biofuels. This work was supported by the National Key Technology Research and Development Program of China (grant number 2015BAD15B05), the National Natural Science Foundation of China (grant numbers 31470210, 31570029 and 31670735), and the Shandong Province Key Laboratory Union of Carbohydrate Industry Science and Technology (grant number 2015LKH102).

Figure 1. Schematic representation of the Cel48S engineering in C. thermocellum (a), and the substrate preference (b) and structure analysis of Cel48S (c)