Elucidation of the noncovalent interactions driving enzyme activity guides branching enzyme engineering for α-glucan modification

Branching enzymes (BEs) confer to α-glucans, the primary energy-storage reservoir in nature, a variety of features, like slow digestion. The full catalytic cycle of BEs can be divided in six steps, namely two covalent catalytic steps involving glycosylation and transglycosylation, and four noncatalytic steps involving substrate binding and transfers (SBTs). Despite the ever-growing wealth of biochemical and structural information on BEs, clear mechanistic insights into SBTs from an industrial-performance perspective are still missing. Here, we report a Rhodothermus profundi BE (RpBE) endowed with twice as much enzymatic activity as the Rhodothermus obamensis BE currently used in industry. Furthermore, we focus on the SBTs for RpBE by means of large-scale computations supported by experiment. Engineering of the crucial positions responsible for the initial substrate-binding step improves enzymatic activity significantly, while offering a possibility to customize product types. In addition, we show that the high-efficiency substrate-transfer steps preceding glycosylation and transglycosylation are the main reason for the remarkable enzymatic activity of RpBE, suggestive of engineering directions for the BE family

Antioxidant peptides from freshwater clam extract using

enzymatic hydrolysi

Effects of three extraction methods on the structural and functional properties of insoluble dietary fibers from mycoprotein

Enzymatic, acidic, and alkaline treatments were applied to extract insoluble dietary fiber (IDF) from mycoprotein. The structural properties of IDFs were elucidated using scanning electron microscopy, molecular weight analysis, Fourier transform infrared spectroscopy, and monosaccharide composition analysis. The results indicated that all three treatment methods led to IDFs with higher water holding capacity and oil holding capacity compared to untreated mycoprotein. Moreover, the alkali and enzymatic treated IDFs displayed more loose and porous structures, resulting in higher water holding capacity, oil holding capacity, and emulsifying activity compared to citric acid treated IDFs. The findings of this study can help for better understanding of the application of mycoprotein IDFs and optimizing the IDFs extraction from filamentous fungi

Purification and Characterization of a Novel Alginate Lyase from the Marine Bacterium Bacillus sp. Alg07

Alginate oligosaccharides with different bioactivities can be prepared through the specific degradation of alginate by alginate lyases. Therefore, alginate lyases that can be used to degrade alginate under mild conditions have recently attracted public attention. Although various types of alginate lyases have been discovered and characterized, few can be used in industrial production. In this study, AlgA, a novel alginate lyase with high specific activity, was purified from the marine bacterium Bacillus sp. Alg07. AlgA had a molecular weight of approximately 60 kDa, an optimal temperature of 40 °C, and an optimal pH of 7.5. The activity of AlgA was dependent on sodium chloride and could be considerably enhanced by Mg2+ or Ca2+. Under optimal conditions, the activity of AlgA reached up to 8306.7 U/mg, which is the highest activity recorded for alginate lyases. Moreover, the enzyme was stable over a broad pH range (5.0–10.0), and its activity negligibly changed after 24 h of incubation at 40 °C. AlgA exhibited high activity and affinity toward poly-β-d-mannuronate (polyM). These characteristics suggested that AlgA is an endolytic polyM-specific alginate lyase (EC 4.2.2.3). The products of alginate and polyM degradation by AlgA were purified and identified through fast protein liquid chromatography and electrospray ionization mass spectrometry, which revealed that AlgA mainly produced disaccharides, trisaccharides, and tetrasaccharide from alginate and disaccharides and trisaccharides from polyM. Therefore, the novel lysate AlgA has potential applications in the production of mannuronic oligosaccharides and poly-α-l-guluronate blocks from alginate

Supercritical Fluid CO2 Extraction and Microcapsule Preparation of Lycium barbarum Residue Oil Rich in Zeaxanthin Dipalmitate

The scope of this investigation aimed at obtaining and stabilizing bioactive products derived from Lycium barbarum seeds and peels, which were the byproducts in the processing of fruit juice. Zeaxanthin dipalmitate is a major carotenoid, comprising approximately 80% of the total carotenoid content in the seeds and peels. The method of obtainment was supercritical fluid CO2 extraction, studying different parameters that affect the oil yield and content of zeaxanthin dipalmitate. The optimized protocol to enact successful supercritical fluid CO2 extraction included optimum extraction pressure of 250 bar, temperature at 60 °C over a time span of 2.0 h, and a CO2 flow of 30 g/min, together with the use of a cosolvent (2% ethanol). The yields of oil and zeaxanthin dipalmitate under these optimal conditions were 17 g/100 g and 0.08 g/100 g, respectively. The unsaturated fatty acids were primarily linoleic acid (C18:2), oleic acid (C18:1), and γ-linolenic acid (C18:3), with their contents being as high as 91.85 ± 0.27% of the total fatty acids. The extract was a red-colored oil that was consequently microencapsulated through spray-drying with octenylsuccinate starch, gum arabic, and maltodextrin (13.5:7.5:3, w/w) as wall materials to circumvent lipid disintegration during storage and add to fruit juice in a dissolved form. The mass ratio of core material and wall material was 4:1. These materials exhibited the highest microencapsulation efficiency (92.83 ± 0.13%), with a moisture content of 1.98 ± 0.05% and solubility of 66.22 ± 0.24%. The peroxide content level within the microencapsulated zeaxanthin dipalmitate-rich oil remained at one part per eight in comparison to the unencapsulated oil, following fast-tracked oxidation at 60 °C for 6 weeks. This indicated the potential oxidation stability properties of microcapsule powders. Consequently, this microencapsulated powder has good prospects for development, and can be utilized for a vast spectrum of consumer health and beauty products

The development of low‐calorie sugar and functional jujube food using biological transformation and fermentation coupling technology

Jujube juice has been used as ingredient in a range of foods and dietary supplements. In this study, an enzyme transformation and fermentation coupling technology was applied to increase the nutritional value of concentrated/extracted Jinsi jujube juice. Two enzymes, D‐glucose isomerase (GI) and D‐allulose 3‐epimerase (DAE), were employed to convert the glucose and fructose to a low‐calorie sweeter D‐allulose with a concentration of 110 g/L in jujube juice. Furthermore, the mixed cultures of Pediococcus pentosaceus PC‐5 and Lactobacillus plantarum M were employed to increase the content of nutrition components related to bioactivities and flavor volatiles in jujube juice. Accordingly, this fermentation accumulated 100 mg/L gamma‐aminobutyric acid (GABA), which has neurotransmission, hypotension, diuretic, and tranquilizer effects, and increased the content of branched‐chain amino acids (BCAAs) and many free amino acids (Asp, Glu, Gly, and Ala) at different level. The fermentation not only maintained the concentration of native functional components such as cyclic adenosine monophosphate (cAMP) and minerals, but also increased the content of iron (Fe2+) and zinc (Zn2+), which have blood and eyesight tonic function. The value‐added jujube juice might serve as a low‐calorie and probiotic functional beverage and show high application potential in food industry