Frits Goedegebuur

Cloning and relational analysis of 15 novel fungal endoglucanases from family 12 glycosyl hydrolase

Abstract. Cellulases belong to the large family of glycosyl hydrolases (GHs) and are produced by a variety of bacteria and fungi. These extracellular enzymes act as endoglucanases (EGs), cellobiohydrolases or β-glucosidases. In this paper, we describe molecular screening for EGs from the GH familyxa012. Using three homologous sequence boxes deduced from five previously known members of the family, we analysed 22xa0cellulase-producing fungal strains obtained from a diverse area of the fungal kingdom. Polymerase chain reactions using degenerate primers designed to the homologous protein boxes were used to identify the family 12xa0homologues. Several fungi showed the presence of multiple versions of the gene, while amino acid sequence analysis showed diversity in 15xa0novel members of the family, ranging from 26% to 96% similarity. Our sequence analysis shows that the phylogenetic tree of family 12xa0EGs can be divided into four subfamilies: 12-1 (fungal group I), 12-2 (fungal group II), 12-3 (Streptomyces group in which Rhodothermus marinus fits) and 12-4 (Thermophiles group). Erwinia carotovora may form a new subgroup.

Hypocrea jecorina CEL6A protein engineering.

The complex technology of converting lignocellulose to fuels such as ethanol has advanced rapidly over the past few years, and enzymes are a critical component of this technology. The production of effective enzyme systems at cost structures that facilitate commercial processes has been the focus of research for many years. Towards this end, the H. jecorina cellobiohydrolases, CEL7A and CEL6A, have been the subject of protein engineering at Genencor. Our first rounds of cellobiohydrolase engineering were directed towards improving the thermostability of both of these enzymes and produced variants of CEL7A and CEL6A with apparent melting temperatures above 70°C, placing their stability on par with that of H. jecorina CEL5A (EG2) and CEL3A (BGL1). We have now moved towards improving CEL6A- and CEL7A-specific performance in the context of a complete enzyme system under industrially relevant conditions. Achievement of these goals required development of new screening strategies and tools. We discuss these advances along with some results, focusing mainly on engineering of CEL6A.

Expression of novel β-glucanase Cel12A from Stachybotrys atra in bacterial and fungal hosts.

β-glucanase Cel12A from Stachybotrys atra has been cloned and expressed in Aspergillus niger. The purified enzyme showed high activity of β-1,3-1,4-mixed glucans, was also active on carboxymethylcellulose (CMC), while it did not hydrolyze crystalline cellulose or β-1,3 glucans as laminarin. Cel12A showed a marked substrate preference for β-1,3-1,4 glucans, showing maximum activity on barley β-glucans (27.69 U mg(-1)) while the activity on CMC was much lower (0.51 U mg(-1)). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focussing (IEF), and zymography showed the recombinant enzyme has apparent molecular weight of 24 kDa and a pI of 8.2. Optimal temperature and pH for enzyme activity were 50°C and pH 6.5. Thin layer chromatography analysis showed that major hydrolysis products from barley β-glucan and lichean were 3-O-β-cellotriosyl-D-glucose and 3-O-β-cellobiosyl-D-glucose, while glucose and cellobiose were released in smaller amounts. The amino acid sequence deduced from cel12A revealed that it is a single domain enzyme belonging to the GH12 family, a family that contains several endoglucanases with substrate preference for β-1,3-1,4 glucans. We believe that S. atra Cel12A should be considered as a lichenase-like or nontypical endoglucanase.