남극 톡토기 유래 저온성 베타 만난아제의 3차원 결정 구조 분석

Abstract

The freezing-intolerant Antarctic springtail, Cryptopygus antarcticus Willem, is the most abundant and widespread terrestrial micro-arthropod in the maritime Antarctic region. The adult reaches approximately 1.2 mm in body length with a live weight of 55 - 78 μg, containing 69 - 85 % water. This organism feeds on fungi, unicellular algae and detritus. Therefore, it is thought that C. antarcticus contains various kinds of cold-active enzymes that hydrolyze carbohydrates. From expressed sequence tags (ESTs) library of C. antarcticus, we identified the β-1,4-D-mannanase gene (CaMan) that belongs to GH 5 family. CaMan is a 382-residue protein with a putative signal peptide and exhibits high specific activity toward locust bean gum at an optimal temperature of 303 K and an optimal pH of 3.5. Its optimal temperature is the lowes among those of the known mannanases and the optimal pH is also the lowest except for those of Sclerotium rolfsii and Aspergillus sulphurous enzymes (pH 2.9 and pH 2.4, respectively). Even at 273 - 278 K, CaMan retains 20 - 40 % of its maximum activity. And it shows typical features of a cold-active enzymes which has a high frequency of polar residues such as Asn, Gln and Ser, and a low frequency of hydrophobic residues as well as a low ratio of Arg/(Arg+Lys) compared to the mesophilic β-mannanases. Here, we report the first structure of a GH5 β-mannanase in complex with M5 (CaMan/M5) and presene weight of 55 - 78 μg, containing 69 - 85 % water. This organism feeds on fungi, unicellular algae and detritus. Therefore, it is thought that C. antarcticus contains various kinds of cold-active enzymes that hydrolyze carbohydrates. From expressed sequence tags (ESTs) library of C. antarcticus, we identified the β-1,4-D-mannanase gene (CaMan) that belongs to GH 5 family. CaMan is a 382-residue protein with a putative signal peptide and exhibits high specific activity toward locust bean gum at an optimal temperature of 303 K and an optimal pH of 3.5. Its optimal temperature is the lowes among those of the known mannanases and the optimal pH is also the lowest except for those of Sclerotium rolfsii and Aspergillus sulphurous enzymes (pH 2.9 and pH 2.4, respectively). Even at 273 - 278 K, CaMan retains 20 - 40 % of its maximum activity. And it shows typical features of a cold-active enzymes which has a high frequency of polar residues such as Asn, Gln and Ser, and a low frequency of hydrophobic residues as well as a low ratio of Arg/(Arg+Lys) compared to the mesophilic β-mannanases. Here, we report the first structure of a GH5 β-mannanase in complex with M5 (CaMan/M5) and presen