Cloning of the Trichoderma reesei cDNA Encoding a Glucuronan Lyase Belonging to a Novel Polysaccharide Lyase Family

Abstract

The filamentous fungus Trichoderma reesei produces glucuronan lyase (TrGL) when it is grown on ␤-(134)-polyglucuronate (cellouronate) as a sole carbon source. The cDNA encoding TrGL was cloned, and the recombinant enzyme was heterologously expressed in Pichia pastoris. The cDNA of TrGL includes a 777-bp open reading frame encoding a 20-amino-acid signal peptide and the 238-amino-acid mature protein. The amino acid sequence showed no similarity to the amino acid sequences of previously described functional proteins, indicating that the enzyme should be classified in a novel polysaccharide lyase (PL) family. Recombinant TrGL catalyzed depolymerization of cellouronate endolytically by ␤-elimination and was highly specific for cellouronate. The enzyme was most active at pH 6.5 and 50°C, and its activity and thermostability increased in the presence of Ca 2؉ , suggesting that its calcium dependence is similar to that of other PLs, such as pectate lyases.

A Polysaccharide Lyase from Stenotrophomonas maltophilia with a Unique, pH-regulated Substrate Specificity

“…The presence of multiple higher order oligmers implies Smlt1473 cleaves its substrates in an endolytic manner with the highest conversion to dimer, the terminal product, observed for the substrate with the highest specific activity (poly-GlcA). The predominance of dimers and tetramers as end products, with small amounts of trimer and pentamer, is consistent with cleavage patterns observed for other polysaccharide lyases that process substrates in an endolytic manner (44,53).…”

“…The highest overall specific activity (848.3 Ϯ 6.3 units/mg at pH 7) was for poly-GlcA (Fig. 5A), which is among the highest reported for poly-GlcAspecific lyases (30,44,45). For poly-ManA the specific activity (68.5 Ϯ 2.9 units/mg at pH 9) was also significant (Fig.…”

“…Lyase Activity-Several polysaccharide lyases are reported to have enhanced activity in response to addition of divalent cations such as calcium or magnesium as well as at elevated NaCl concentrations (31,44,49). In the case of Smlt1473, however, activity was significantly inhibited by the presence of both mono-and divalent cations (Table 3).…”

“…8), similar to the endolytic alginate lyases A1-I, A1-II, and A1-III of Sphingomonas sp. (63), as well as the endolytic poly-GulA-specific lyases of Sinorhizobium meliloti M5N1CS (45) and Trichoderma reesei (44). Each of the aforementioned endolytic lyases produce products that vary in size by one sugar ring, including dimers, trimers, tetramers, and pentamers, which is indicative of a random endolytic mechanism in which the enzyme exhibits no bias for the initial size of the substrate, assuming it is larger than the minimum substrate size (64,67).…”

“…Endolytic enzymes bind to an internal site on the polysaccharide, cleave one glycosidic bond, and detach from the substrate, resulting in the simultaneous accumulation of multiple unsaturated products of various sizes (44,45,63,64). Exolytic enzymes bind to the non-reducing end of the polysaccharide and removes mono-, di-, or trisaccharides from the end until the entire macromolecule has been processed, resulting in the accumulation of predominantly one unsaturated product, with little to no accumulation of differently sized oligosaccharides (46, 64 -66).…”