Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates

<div><p>Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from <i>Saccharophagus degradans</i> was fused to the <i>C</i>-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.</p></div

TLC analysis of hydrolysis products of cellotriose, cellotetraose, cellopentaose, cellohexaose, CMC, PASC, filter paper, Avicel, and <i>p</i>-NPC.

<p>A: Hydrolysis (1 h) products of cellotriose and cellotetraose, B: Hydrolysis (1 h) products of cellopentaose and cellohexaose, C: Hydrolysis (5 h) products of CMC and PASC, D: Hydrolysis (16 h) products of filter paper and Avicel, and E: Hydrolysis (1 h) product of <i>p</i>-NPC. M: Standard marker, where G1 to G6 represent glucose, cellobiose, cellotriose, cellotetraose, cellopentasoe, and cellohexaose. Cello-oligosaccharides, CMC, PASC, and <i>p</i>-NPC were treated with 0.1 nmol of Cel5A_2R2-CBM6 at 55°C. The same reaction was performed using Avicel and filter paper with 1.0 nmol of Cel5A_2R2-CBM6. Reactions were performed in the absence (−) and presence (+) of the enzyme.</p

Optimum temperature and pH for Cel5A_2R2 and Cel5A_2R2-CBM6 fusion protein.

<p>Symbols are as follows: Cel5A_2R2 (Open circles) and Cel5A_2R2-CBM6 (Closed circles). The error bars represent the standard deviation of triplicate measurements.</p

Synergistic interaction of cellobiohydrolase (CbhA) from <i>C. thermocellum</i> with Cel5A_2R2 parent protein and Cel5A_2R2-CBM6 fusion protein.

<p>The error bars represent the standard deviation of triplicate measurements.</p

Cel5A mutations covered in this work mapped onto the model of the Cel5A catalytic domain.

<p>Cellobiose at the reaction cavity is displayed as ball-and-sticks (carbon in yellow and oxygen in red). Mutated residues in each mutant are shown as sticks in different colors: D45G in orange from 1R1, V108G, and L240Q in green from 1R2, D275G in cyan from 1R3, N252D in hot pink from 1R4, D40E in purple-blue from 1R5, T195A in magenta from 2R1, F90L in blue from 2R2, the common mutation V256A in red.</p

Specific enzyme activity of Cel5A_2R2 and Cel5A_2R2-CBM6 on various soluble and insoluble cellulosic substrates.

a<p>Specific activity of Cel5A_2R2-CBM6 was statistically significant from wild type Cel5A and mutant Cel5A_2R2 at two-tailed P value is less than 0.0001.</p>b<p>ND represents “Not Detected”, indicating no enzyme activity.</p