Band 3, Heft 1

In brewing, amylases are key enzymes in hydrolyzing barley starch to sugars, which are utilized in fermentation to produce ethanol. Starch fermentation depends on sugars produced by amylases and starch molecular structure, both of which vary with barley grain size. Grain size is a major industrial specification for selecting barley for brewing. An in-depth study is given of how enzyme activity and starch structure vary with grain size, the impact of these factors on fermentable sugar production, and the underlying mechanisms. Micro-malting and mashing experiments were based on commercial methodologies. Starch molecular structural parameters were obtained using size-exclusion chromatography, and fitted using biosynthesis-based models. Correlation analysis using the resulting parameters showed larger grain sizes contained fewer long amylopectin chains, higher amylase activities and soluble protein level. Medium grain sizes released most sugars during mashing, because of higher starch utilization from the action of amylases, and shorter amylose chains. As starch is the substrate for amylase-driven fermentable sugars production, measuring its structure should be a prime indication for mashing performance, and should be used as an industry specification when selecting barley grains for brewing.

Amylosucrase (ASase; EC 2.4.1.4), a versatile enzyme, exhibits three characteristic activities: hydrolysis, isomerization, and transglycosylation. In this study, a novel ASase derived from Deinococcus wulumuquiensis (DWAS) was identified and expressed in Escherichia coli . The optimal reaction temperature and pH for the sucrose hydrolysis activity of DWAS were determined to be 45 °C and 9.0, respectively. DWAS displays relatively high thermostability compared with other ASases, as demonstrated by half-life of 96.7 and 4.7 min at 50 °C and 55 °C, respectively. DWAS fused with 6×His was successfully purified to apparent homogeneity with a molecular mass of approximately 72 kDa by Ni-NTA affinity chromatography and confirmed by SDS-PAGE. DWAS transglycosylation activity can be used to modify isovitexin, a representative flavone C-glucoside contained in buckwheat sprouts to increase its limited bioavailability, which is due to its low absorption rate and unstable structure in the human body. Using isovitexin as a substrate, the major transglycosylation product of DWAS was found to be isovitexin monoglucoside. The comparison of transglycosylation reaction products of DWAS with those of other ASases derived from Deinococcus species revealed that the low sequence homology of loop 8 in ASases may affect the acceptor specificity of ASases and result in a distinctive acceptor specificity of DWAS.

Glycoside phosphorylases in subfamily GH13_18 of the carbohydrate-active enzyme database CAZy catalyse the reversible phosphorolysis of α-glycosidic bonds. They contribute to a more energy-efficient metabolism in vivo , and can be applied for the synthesis of valuable glucosides, sugars or sugar phosphates in vitro . Continuing our efforts to uncover new phosphorylase specificities, we identified an enzyme from the myxobacterium Corallococcus coralloides DSM 2259 that does not feature the signature sequence patterns of previously characterised phosphorylases. The enzyme was recombinantly expressed and subjected to substrate screening. Although it was confirmed that the Corallococcus phosphorylase does not have the same substrate specificity as other phoshorylases from subfamily GH13_18, its true natural substrate remains a mystery for now. Myxobacteria have been widely investigated as producers of numerous bioactive secondary metabolites for decades, but little research has been conducted on myxobacterial proteins. The present study exemplifies the untapped metabolic activities and functional diversity that these fascinating organisms may have left to show.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered copper enzymes that cleave recalcitrant polysaccharides by oxidation. The structure of an Aspergillus oryzae LPMO from the starch degrading family AA13 ( Ao AA13) has previously been determined from an orthorhombic crystal grown in the presence of copper, which is photoreduced in the structure. Here we describe how crystals reliably grown in presence of Zn can be Cu-loaded post crystallization. A partly photoreduced structure was obtained by severely limiting the X-ray dose, showing that this LPMO is much more prone to photoreduction than others. A serial synchrotron crystallography structure was also obtained, showing that this technique may be promising for further studies, to reduce even further photoreduction. We additionally present a triclinic structure of Ao AA13, which has less occluded ligand binding site than the orthorhombic one. The availability of the triclinic crystals prompted new ligand binding studies, which lead us to the conclusion that small starch analogues do not bind to Ao AA13 to an appreciable extent. A number of disordered conformations of the metal binding histidine brace have been encountered in this and other studies, and we have previously hypothesized that this disorder may be a consequence of loss of copper. We performed molecular dynamics in the absence of active site metal, and showed that the dynamics in solution differ somewhat from the disorder observed in the crystal, though the extent is equally dramatic.