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.

Alginase lyase is an important enzyme for the preparation of alginate oligosaccharides (AOS), that possess special biological activities and is widely used in various fields, such as medicine, food, and chemical industry. In this study, a novel bifunctional alginate lyase (AlgH) belonging to the PL7 family was screened and characterized. The AlgH exhibited the highest activity at 45 °C and pH 10.0, and was an alkaline enzyme that was stable at pH 6.0–10.0. The enzyme showed no significant dependence on metal ions, and exhibited unchanged activity at high concentration of NaCl. To determine the function of non-catalytic domains in the multi-domain enzyme, the recombinant AlgH-I containing only the catalysis domain and AlgH-II containing the catalysis domain and the carbohydrate binding module (CBM) domain were constructed and characterized. The results showed that the activity and thermostability of the reconstructed enzymes were significantly improved by deletion of the F5/8 type C domain. On the other hand, the substrate specificity and the mode of action of the reconstructed enzymes showed no change. Alginate could be completely degraded by the full-length and modified enzymes, and the main end-products were alginate disaccharide, trisaccharide, and tetrasaccharide. Due to the thermo and pH-stability, salt-tolerance, and bifunctionality, the modified alginate lyase was a robust enzyme which could be applied in industrial production of AOS.

Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

Asymmetric aldol-type C–C bond formation with ketones used as electrophilic receptor remains a challenging reaction for aldolases as biocatalysts. To date, only one kind of dihydroxyacetone phosphate (DHAP)-dependent aldolases has been discovered and applied to synthesize branched-chain sugars directly using DHAP and dihydroxyacetone (DHA) as substrate. However, the unstable and high-cost properties of DHAP limit large-scale application. Therefore, biosynthesis of branched-chain sugar from low-cost and abundant carbon sources is essential. The detailed catalytic property of l-rhamnulose-1-phosphate aldolase (RhaD) and l-fuculose-1-phosphate aldolase (FucA) from Escherichia coli in catalyzing the aldol reactions with DHA as electrophilic receptors was characterized. Furthermore, we calculated the Bürgi–Dunitz trajectory using molecular dynamics simulations, thereby revealing the original sources of the catalytic efficiency of RhaD and FucA. A multi-enzyme reaction system composed of formolase, DHA kinase, RhaD, fructose-1-phosphatase, and polyphosphate kinase was constructed to in vitro produce dendroketose, a branched-chain sugar, from one-carbon formaldehyde. The conversion rate reached 86% through employing a one-pot, two-stage reaction process. Moreover, we constructed two artificial pathways in Corynebacterium glutamicum to obtain this product in vivo starting from glucose or glycerol. Fermentation with glycerol as feedstock produced 6.4 g/L dendroketose with a yield of 0.45 mol/mol glycerol, representing 90% of the maximum theoretical value. Additionally, the dendroketose production reached 36.3 g/L with a yield of 0.46 mol/mol glucose when glucose served as the sole carbon resource. The detailed enzyme kinetics data of the two DHAP-dependent aldolases with DHA as electrophilic receptors were presented in this study. In addition, insights into this catalytic property were given via in silico simulations. Moreover, the cost-effective synthesis of dendroketose starting from one-, three-, and six-carbon resources was achieved through in vivo and in vitro metabolic engineering strategies. This rare branched-chain ketohexose may serve as precursor to prepare 4-hydroxymethylfurfural and branched-chain alkanes using chemical method.

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.