Bioinformatics and functional selection of GH77 4-α-glucanotransferases for potato starch modification.

4-α-glucanotransferases (4αGTs, EC 2.4.1.25) from glycoside hydrolase family 77 (GH77) catalyze chain elongation of starch amylopectin chains and can be utilized to structurally modify starch to tailor its gelation properties. The potential relationship between the structural design of 4αGTs and functional starch modification is unknown. Here, family GH77 was mined in silico for enzyme candidates based on sub-grouping guided by Conserved Unique Peptide Patterns (CUPP) bioinformatics categorization. From + 12,000 protein sequences a representative set of 27 4αGTs, representing four different domain architectures, different bacterial origins and diverse CUPP groups, was selected for heterologous expression and further study. Most of the enzymes catalyzed starch modification, but their efficacies varied substantially. Five of the 4αGTs were characterized in detail, and their action was compared to that of the industrial benchmark enzyme, Tt4αGT (CUPP 77_1.2), from Thermus thermophilus. Reaction optima of the five 4αGTs ranged from ∼40-60 °C and pH 7.3-9.0. Several were stable for a minimum 4 h at 70 °C. Domain architecture type A proteins, consisting only of a catalytic domain, had high thermal stability and high starch modification ability. All five novel 4αGTs (and Tt4αGT) induced enhanced gelling of potato starch. One, At4αGT from Azospirillum thermophilum (CUPP 77_2.4), displayed distinct starch modifying abilities, whereas T24αGT from Thermus sp. 2.9 (CUPP 77_1.2) modified the starch similarly to Tt4αGT, but slightly more effectively. T24αGT and At4αGT are thus interesting candidates for industrial starch modification. A model is proposed to explain the link between the 4αGT induced molecular modifications and macroscopic starch gelation.

New insights to diversity and enzyme-substrate interactions of fungal glucuronoyl esterases.

Glucuronoyl esterases (GEs) (EC 3.1.1.117) catalyze the cleavage of ester-linked lignin-carbohydrate complexes that has high impact on the plant cell wall integrity. The GEs are among the very few known types of hydrolytic enzymes that act at the interface of lignin, or which may potentially interact with lignin itself. In this review, we provide the latest update of the current knowledge on GEs with a special focus on the fungal variants. In addition, we have established the phylogenetic relationship between all GEs and this reveals that the fungal enzymes largely fall into one major branch, together with only a minor subset of bacterial enzymes. About 22% of the fungal proteins carry an additional domain, which is almost exclusively a CBM1 binding domain. We address how GEs may interact with the lignin-side of their substrate by molecular docking experiments based on the known structure of the Cerrena unicolor GE (CuGE). The docking studies indicate that there are no direct interactions between the enzyme and the lignin polymer, that the lignin-moiety is facing away from the protein surface and that an elongated carbon-chain between the ester-linkage and the first phenyl of lignin is preferable. Much basic research on these enzymes has been done over the past 15 years, but the next big step forward for these enzymes is connected to application and how these enzymes can facilitate the use of lignocellulose as a renewable resource. KEY POINTS: Fungal GEs are closely related and are sometimes linked to a binding module Molecular docking suggests good accommodation of lignin-like substructures GEs could be among the first expressed enzymes during fungal growth on biomass.

Enzymatic potato starch modification and structure-function analysis of six diverse GH77 4-alpha-glucanotransferases.

4-α-glucanotransferase (EC 2.4.1.25) mediated glucan transfer in starch provides opportunities for production of clean label starch ingredients with unique gelling properties. 4-α-glucanotransferases can be found in glycoside hydrolase (GH) family GH13, GH57, and in the monospecific glycoside hydrolase family 77 (GH77). Here, pH-temperature optima, steady-state kinetics, potato starch modifying properties and structural folds are reported for six phylogenetically distinct GH77 members, representing four different domain architectures including a novel multi-domain 4-α-glucanotransferase from Lactococcus lactis. Four of the enzymes exhibited starch modifying activity leading to a gradual decrease of the amylose content, elongation of amylopectin chains, and enabled formation of firm starch gels. Unexpectedly, these diverse enzymes catalyzed similar changes in chain length distributions. However, the amylose depletion and amylopectin elongation rates spanned more than two orders of magnitude between the enzyme showing very different specific activities. Tt4αGT from Thermus thermophilus had highest temperature optimum (73 °C) and superior potato starch modifying efficacy compared to the other five enzymes.