Abstract
The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U·mg−1, respectively. The Km and Vmax of AcCel12B for CMC were 25.47 mg·mL−1 and 131.75 U·mg−1, respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future.
Highlights
Cellulose, one of the most important components of plant cell walls in the biosphere, is composed of a linear backbone composed of glucose units linked by β-1,4-D-glucosidic bonds
The results suggested that AcCel12B was thermostable in acidic conditions, and the time course of hydrolysis for insoluble substrates was investigated
The recombinant AcCel12B was active under acidic pH and high temperature conditions. These characteristics correspond with the fact that A. cellulolyticus is a thermophilic and acidophilic bacteria whose optimal growth pH and temperature were shown to be pH 5 and 55 °C, respectively [12]
Summary
One of the most important components of plant cell walls in the biosphere, is composed of a linear backbone composed of glucose units linked by β-1,4-D-glucosidic bonds. Thermophilic and hyperthermophilic enzymes generally present some advantages over mesophilic ones, for example, higher thermal, pH, and chemical stabilities, higher reaction temperatures, and higher catalytic rates. These properties are useful for the enzymatic hydrolysis of lignocellulosic materials [5]. The enzymes from P. furiosus and R. marinus, which both showed a temperature optimum of 100 °C, were extremely thermostable [9,10]. SSO1949 from S. solfataricus showed a half-life of about 8 h at 80 °C and pH 1.8, which could be valuable properties for the large-scale hydrolysis of cellulose under high temperatures and acidic conditions [11]. The results suggested that AcCel12B was thermostable in acidic conditions, and the time course of hydrolysis for insoluble substrates was investigated
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