Specific fusion of β-1,4-endoglucanase and β-1,4-glucosidase enhances cellulolytic activity and helps in channeling of intermediates.
Identification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel β-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a β-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G(4)S)(3)-Gluc1C, demonstrated 3.2- and 2-fold higher molar specific activities for β-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5°C and 9°C, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability.
Synthetic Biology and Biofuel Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
Nidhi
N
Adlakha
Sneha
S
Sawant
Annamma
A
Anil
Arvind
A
Lali
Syed Shams
SS
Yazdani
D002266:Carboxymethylcellulose Sodium / Q000378:metabolism*; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D004795:Enzyme Stability; D044643:Glucan 1,4-beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*; D006868:Hydrolysis; D012275:Oryza / Q000378:metabolism; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation / Q000528:radiation effects; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D011993:Recombinant Fusion Proteins; D002480:Cellulase; D044643:Glucan 1,4-beta-Glucosidase; D002266:Carboxymethylcellulose Sodium