Mingardon Florence, Chanal Angélique, López-Contreras Ana M, Dray Cyril, Bayer Edward A, Fierobe Henri-Pierre
Department of Bioénergétique et Ingénierie de Protéines, UPR9036, BIP-CNRS, IBSM, 31, chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Appl Environ Microbiol. 2007 Jun;73(12):3822-32. doi: 10.1128/AEM.00398-07. Epub 2007 Apr 27.
Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.
人工设计的微型纤维小体包含一种嵌合支架蛋白，该支架蛋白展示了一个可选的纤维素结合模块（CBM）以及来自不同物种的细菌黏附素，这些黏附素与经过工程改造带有互补对接蛋白的酶紧密结合。将解纤维梭菌的纤维小体纤维素酶整合到微型纤维小体中，会形成对结晶纤维素活性增强的人工复合物，这是由于支架蛋白携带的CBM诱导的酶接近度和底物靶向性。在本研究中，将一种细菌对接蛋白附加到由嗜热栖瘤胃杆菌产生的6家族真菌纤维素酶Cel6A上，以便随后与解纤维梭菌的各种纤维小体纤维素酶一起整合到微型纤维小体中。真菌Cel6A与细菌5家族内切葡聚糖酶在嵌合微型支架蛋白上的结合对它们对结晶纤维素的活性没有影响。用带有梭菌对接蛋白的嗜热栖瘤胃杆菌同源内切葡聚糖酶Cel5D替代细菌5家族酶得到了类似的结果。相比之下，与游离酶系统相比，由真菌Cel6A和细菌9家族内切葡聚糖酶组成的酶对在嵌合支架蛋白上的复合作用下受到显著刺激（高达2.6倍）。包含Cel6A和一种持续性细菌纤维素酶的酶对的整合通常诱导较低的刺激水平。与仅由细菌纤维素酶组成的微型纤维小体不同，对结晶纤维素活性的增强似乎仅由接近度或CBM效应导致，而不是两者同时作用。本研究首次证明可以生产出可行的设计微型纤维小体，其包括（i）游离（非纤维小体）酶，（ii）真菌酶与细菌酶的组合，以及（iii）一种天然纤维小体中从未发现的纤维素酶类型（6家族）。
