A family of thermostable fungal cellulases created by structure-guided recombination

Abstract

SCHEMA structure-guided recombination of 3 fungal class II cellobiohydrolases (CBH II cellulases) has yielded a collection of highly thermostable CBH II chimeras. Twenty-three of 48 genes sampled from the 6,561 possible chimeric sequences were secreted by the Saccharomyces cerevisiae heterologous host in catalytically active form. Five of these chimeras have half-lives of thermal inactivation at 63 degrees C that are greater than the most stable parent, CBH II enzyme from the thermophilic fungus Humicola insolens, which suggests that this chimera collection contains hundreds of highly stable cellulases. Twenty-five new sequences were designed based on mathematical modeling of the thermostabilities for the first set of chimeras. Ten of these sequences were expressed in active form; all 10 retained more activity than H. insolens CBH II after incubation at 63 degrees C. The total of 15 validated thermostable CBH II enzymes have high sequence diversity, differing from their closest natural homologs at up to 63 amino acid positions. Selected purified thermostable chimeras hydrolyzed phosphoric acid swollen cellulose at temperatures 7 to 15 degrees C higher than the parent enzymes. These chimeras also hydrolyzed as much or more cellulose than the parent CBH II enzymes in long-time cellulose hydrolysis assays and had pH/activity profiles as broad, or broader than, the parent enzymes. Generating this group of diverse, thermostable fungal CBH II chimeras is the first step in building an inventory of stable cellulases from which optimized enzyme mixtures for biomass conversion can be formulated.

Fig. 1.

SDS/PAGE gel of candidate CBH II parent gene yeast expression culture supernatants. Gel Lanes (left to right): 1, H. jecorina; 2, empty vector; 3, H. insolens; 4, C. thermophilum; 5, H. jecorina (duplicate); 6, P. chrysosporium; 7, T. emersonii; 8, empty vector (duplicate); 9, H. jecorina (triplicate). Numbers at bottom of gel represent concentration of reducing sugar (μg/mL) present in reaction after a 2-h 50 °C PASC hydrolysis assay. Subsequent SDS/PAGE comparison with BSA standard allowed estimation of H. insolens expression level of 5–10 mg/L.

Fig. 2.

Illustrations of CBH II chimera library block boundaries. (A) H. insolens CBH II catalytic domain ribbon diagram with blocks distinguished by color. CBH II enzyme is complexed with cellobio-derived isofagomine glycosidase inhibitor (13). (B) Linear representation of H. insolens catalytic domain showing secondary structure elements, disulfide bonds and block divisions denoted by black arrows. (C) Sidechain contact map denoting contacts (side chain heavy atoms within 4.5Å) that can be broken upon recombination. The majority of broken contacts occur between consecutive blocks.

Fig. 3.

Number of broken contacts (E) and number of mutations from closest parent (m) for 23 secreted/active and 15 not secreted/not active sample set chimeras.

Fig. 4.

Specific activity, normalized to pH 5.0, as a function of pH for parent CBH II enzymes and 3 thermostable chimeras. Data presented are averages for 2 replicates. Error bars for HJPlus and H. jeco denote values for 2 independent trials. Sixteen-hour reaction, 300 μg of enzyme per gram of PASC, 50 °C, 12.5 mM sodium citrate/12.5 mM sodium phosphate buffer at pH as shown.

Fig. 5.

Long-time cellulose hydrolysis assay results (micrograms of glucose reducing sugar equivalent per microgram of CBH II enzyme) for parents and thermostable chimeras across a range of temperatures. Error bars indicate standard errors for 3 replicates of HJPlus and H. insolens CBH II enzymes. 40-h reaction, 100 μg of enzyme per gram of PASC, 50 mM sodium acetate, pH 4.8.