Statistical investigation of the bioprocess conditions of alkali combined twin-screw extrusion pretreatment to enhance fractionation and enzymatic hydrolysis of bulgur bran.

BACKGROUND: Bulgur bran (BB) is a potential source for the production of value-added products such as fermentable sugars and xylooligosaccharides (XOs). In this study, alkali combined twin-screw extrusion pretreatment was performed and statistically optimized to enhance fractionation and enzymatic hydrolysis of BB. The pretreatment conditions (barrel temperature, screw speed and alkali impregnation) were optimized by Box-Behnken design (BBD) to obtain the highest hemicellulose separation from BB. The obtained fractions were analyzed for the production of fermentable sugars and XOs. RESULTS: The results revealed that twin-screw extrusion of BB performed at 67 °C barrel temperature and 250 rpm screw speed after alkali impregnation at 0.02 g alkali g-1 biomass concentration provided 40.4% higher hemicellulose separation yield compared to the untreated BB. Alkali combined twin-screw extrusion pretreatment increased the enzymatic hydrolysis yield of BB fourfold, whereas a 13-fold increase was achieved after the separation of hemicellulose from pretreated BB. Xylose (X1)-free xylobiose (X2) was the main product after xylanase hydrolysis of hemicellulose fraction. SEM images confirmed the morphological modifications in BB, which were in agreement with the enhanced fractionation performance and the higher enzymatic hydrolysis yield. CONCLUSION: The results of this study suggested that pretreatment by alkali combined twin-screw extrusion followed by alkali extraction could be a reliable and effective process for fractionation of BB and production of fermentable sugars and XOs. © 2022 Society of Chemical Industry.

Evolving regulatory policies regarding food enzymes produced by recombinant microorganisms.

Bio-based industries rely extensively on the use of enzymatic biocatalysts. The global market for industrial enzymes, of which approximately half is used for food applications, is estimated at $5.5 billion. Most enzymes used in food production worldwide are produced by recombinant DNA techniques. Production and use of food enzymes are regulated by three main bodies: the Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Committee on Food Additives; the European Food Safety Authority; and the U.S. Food and Drug Administration. Regulation in the U.S. follows a largely product-oriented approach while the EU emphasizes production processes. Both systems have, or are developing, lists of approved enzymes to facilitate trade while protecting consumer health and welfare. This paper compares regulatory policies, and presents the growing food industry in Turkey as a case study of a national system responding to the food enzyme production and regulatory landscape.

Structure, recombinant expression and mutagenesis studies of the catalase with oxidase activity from Scytalidium thermophilum.

Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon-optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9 Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis-hydroxychlorin γ-spirolactone, which is rotated 180° with respect to small-subunit catalases. The haem-binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d-type haem to a b-type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide.

Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from Scytalidium thermophilum.

Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8 A resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P2(1) and contained one tetramer per asymmetric unit.

Purification, characterization, and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum.

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 +/- 0.2 and 10.1 +/- 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.