Impact of Starch Binding Domain Fusion on Activities and Starch Product Structure of 4-α-Glucanotransferase.

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBDSt1 and SBDSt2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBDGA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBDGA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2-2.4-fold on amylose and decreased 3-9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3-1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBDGA-TuαGT and-SBDSt2-TuαGT showed Kd of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBDSt1-TuαGT had 3-5-fold lower affinity. SBDSt2 contributed more than SBDSt1 to activity, substrate binding, and the stability of TuαGT fusions.

Importance of Inactivation Methodology in Enzymatic Processing of Raw Potato Starch: NaOCl as Efficient α-Amylase Inactivation Agent.

Efficient inactivation of microbial α-amylases (EC 3.2.1.1) can be a challenge in starch systems as the presence of starch has been shown to enhance the stability of the enzymes. In this study, commonly used inactivation methods, including multistep washing and pH adjustment, were assessed for their efficiency in inactivating different α-amylases in presence of raw potato starch. Furthermore, an effective approach for irreversible α-amylase inactivation using sodium hypochlorite (NaOCl) is demonstrated. Regarding inactivation by extreme pH, the activity of five different α-amylases was either eliminated or significantly reduced at pH 1.5 and 12. However, treatment at extreme pH for 5 min, followed by incubation at pH 6.5, resulted in hydrolysis yields of 42-816% relative to controls that had not been subjected to extreme pH. "Inactivation" by multistep washing with water, ethanol, and acetone followed by gelatinization as preparation for analysis gave significant starch hydrolysis compared to samples inactivated with NaOCl before the wash. This indicates that the further starch degradation observed in samples subjected to washing only took place during the subsequent gelatinization. The current study demonstrates the importance of inactivation methodology in α-amylase-mediated raw starch depolymerization and provides a method for efficient α-amylase inactivation in starch systems.

A biochemical comparison of fungal GH6 cellobiohydrolases.

Cellobiohydrolases (CBHs) from glycoside hydrolase family 6 (GH6) make up an important part of the secretome in many cellulolytic fungi. They are also of technical interest, particularly because they are part of the enzyme cocktails that are used for the industrial breakdown of lignocellulosic biomass. Nevertheless, functional studies of GH6 CBHs are scarce and focused on a few model enzymes. To elucidate functional breadth among GH6 CBHs, we conducted a comparative biochemical study of seven GH6 CBHs originating from fungi living in different habitats, in addition to one enzyme variant. The enzyme sequences were investigated by phylogenetic analyses to ensure that they were not closely related phylogenetically. The selected enzymes were all heterologously expressed in Aspergillus oryzae, purified and thoroughly characterized biochemically. This approach allowed direct comparisons of functional data, and the results revealed substantial variability. For example, the adsorption capacity on cellulose spanned two orders of magnitude and kinetic parameters, derived from two independent steady-state methods also varied significantly. While the different functional parameters covered wide ranges, they were not independent since they changed in parallel between two poles. One pole was characterized by strong substrate interactions, high adsorption capacity and low turnover number while the other showed weak substrate interactions, poor adsorption and high turnover. The investigated enzymes essentially defined a continuum between these two opposites, and this scaling of functional parameters raises interesting questions regarding functional plasticity and evolution of GH6 CBHs.

A practical approach to steady-state kinetic analysis of cellulases acting on their natural insoluble substrate.

Measurement of steady-state rates (vSS) is straightforward in standard enzymology with soluble substrate, and it has been instrumental for comparative biochemical analyses within this area. For insoluble substrate, however, experimental values of vss remain controversial, and this has strongly limited the amount and quality of comparative analyses for cellulases and other enzymes that act on the surface of an insoluble substrate. In the current work, we have measured progress curves over a wide range of conditions for two cellulases, TrCel6A and TrCel7A from Trichoderma reesei, acting on their natural, insoluble substrate, cellulose. Based on this, we consider practical compromises for the determination of experimental vSS values, and propose a basic protocol that provides representative reaction rates and is experimentally simple so that larger groups of enzymes and conditions can be readily assayed with standard laboratory equipment. We surmise that the suggested experimental approach can be useful in comparative biochemical studies of cellulases; an area that remains poorly developed.

Direct kinetic comparison of the two cellobiohydrolases Cel6A and Cel7A from Hypocrea jecorina.

Cellulose degrading fungi such as Hypocrea jecorina secrete several cellulases including the two cellobiohydrolases (CBHs) Cel6A and Cel7A. The two CBHs differ in catalytic mechanism, attack different ends, belong to different families, but are both processive multi-domain enzymes that are essential in the hydrolysis of cellulose. Here we present a direct kinetic comparison of these two enzymes acting on insoluble cellulose. We used both continuous- and end-point assays under either enzyme- or substrate excess, and found distinct kinetic differences between the two CBHs. Cel6A was catalytically superior with a maximal rate over four times higher than Cel7A. Conversely, the ability of Cel6A to attack diverse structures on the cellulose surface was inferior to Cel7A. This latter difference was pronounced as the density of attack sites for Cel7A was almost an order of magnitude higher compared to Cel6A. We conclude that Cel6A is a fast but selective enzyme and that Cel7A is slower, but promiscuous. One consequence of this is that Cel6A is more effective when substrate is plentiful, while Cel7A excels when substrate is limiting. These diverse kinetic properties of Cel6A and Cel7A might elucidate why both cellobiohydrolases are prominent in cellulolytic degrading fungi.

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.

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.

Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii.

Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data are available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far. Here, we report a new crystal structure and biochemical characterization of a thermostable endoglucanase from the thermophilic ascomycete Rasamsonia emersonii, ReCel7B. The enzyme was compared with the homologous endoglucanase from the mesophilic model ascomycete Trichoderma reesei (TrCel7B), which unlike ReCel7B possesses an additional carbohydrate-binding module (CBM). With a temperature optimum of 80 °C, ReCel7B displayed a number of differences in activity and ability to synergize with cellobiohydrolases compared to TrCel7B. We improved both binding and kinetics in a chimeric variant of ReCel7B and a CBM, while we observe the opposite effect when the CBM was removed in TrCel7B. The crystal structure of ReCel7B was determined at 2.48 Å resolution, with Rwork and Rfree factors of 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7B has increased rigidity in a number of peripheral loops compared to TrCel7B and fewer aromatics in the substrate-binding cleft. An increased number of glycosylations were identified in ReCel7B, and we propose a stabilizing mechanism for one of the glycans. Global structure-function interpretations of ReCel7B highlight the differences in temperature stability, turnover, binding, and cellulose accessibility in GH7 endoglucanases. DATABASE: Structural data are available in RCSB Protein Data Bank database under the accession number 6SU8. ENZYMES: ReCel7B, endoglucanase (EC3.2.1.4) from Rasamsonia emersonii; ReCel7A, cellobiohydrolase (EC3.2.1.176) from Rasamsonia emersonii; TrCel7B, endoglucanase (EC3.2.1.4) from Trichoderma reesei; TrCel7A, cellobiohydrolase (EC3.2.1.176) from Trichoderma reesei.

Functional analysis of chimeric TrCel6A enzymes with different carbohydrate binding modules.

The glycoside hydrolase (GH) family 6 is an important group of enzymes that constitute an essential part of industrial enzyme cocktails used to convert lignocellulose into fermentable sugars. In nature, enzymes from this family often have a carbohydrate binding module (CBM) from the CBM family 1. These modules are known to promote adsorption to the cellulose surface and influence enzymatic activity. Here, we have investigated the functional diversity of CBMs found within the GH6 family. This was done by constructing five chimeric enzymes based on the model enzyme, TrCel6A, from the soft-rot fungus Trichoderma reesei. The natural CBM of this enzyme was exchanged with CBMs from other GH6 enzymes originating from different cellulose degrading fungi. The chimeric enzymes were expressed in the same host and investigated in adsorption and quasi-steady-state kinetic experiments. Our results quantified functional differences of these phylogenetically distant binding modules. Thus, the partitioning coefficient for substrate binding varied 4-fold, while the maximal turnover (kcat) showed a 2-fold difference. The wild-type enzyme showed the highest cellulose affinity on all tested substrates and the highest catalytic turnover. The CBM from Serendipita indica strongly promoted the enzyme's ability to form productive complexes with sites on the substrate surface but showed lower turnover of the complex. We conclude that the CBM plays an important role for the functional differences between GH6 wild-type enzymes.