Abstract
BackgroundAstaxanthin, a naturally occurring carotenoid pigment molecule, displays strong antioxidant, anti-cancer, and immunity-enhancing properties, and is often utilized in food, biomedical, cosmetic, and other industries. Free astaxanthin has better solubility than astaxanthin esters (Ast-E), and is a useful auxiliary ingredient in health foods and medicines. Our goal was to establish an improved enzymatic method for preparation of free astaxanthin from natural sources (e.g., the microalga Haematococcus pluvialis), to expand the potential applications of free astaxanthin.ResultsThe alkaline lipase gene proalip and its propeptide were cloned and successfully fusion-expressed in Pichia pastoris X-33. The recombinant lipase was termed Lipase-YH. Through optimization of culture conditions (medium formulation, pH, added methanol concentration), cell growth (OD600) and secreted enzyme activity respectively reached to 280 and 2050 U/mL in a 50-L autofermentor. Activity of Lipase-YH enzyme powder was about 40,000 U/g. Hydrolysis of Ast-E (extracted from H. pluvialis) by Lipase-YH occurred in aqueous phase, and reaction conditions were optimized based on emulsification method and enzyme/substrate ratio. The highest enzymatic reaction rate was observed for substrate concentration 200 μg/mL, with maximal free astaxanthin yield (80%) at 1 h, and maximal Ast-E hydrolysis rate 96%, as confirmed by TLC, HPLC, and mass spectroscopy.ConclusionA novel, efficient enzymatic process was developed for production of free astaxanthin through hydrolysis of Ast-E. Lipase activity was enhanced, and production cost was greatly reduced. The unique structure of free astaxanthin allows linkage to various functional compounds, which will facilitate development of novel pharmaceutical and food products in future studies.
Highlights
Astaxanthin, a naturally occurring carotenoid pigment molecule, displays strong antioxidant, anti-cancer, and immunity-enhancing properties, and is often utilized in food, biomedical, cosmetic, and other industries
Addition of certain groups to hydroxyl groups of free astaxanthin can increase solubility, which is useful for preparation of derivatives for oral administration; e.g., derivatives based on astaxanthin disuccinic acid disodium salt have been applied for treatment of various neurological disorders [13]
Enzyme activity enhancement by added propeptide fusion expression Amino acid (a.a.) sequence of P. cyclopium var. albus lipase (ALIP) was obtained from GenBank (Seq ID # AAF82375.1), and the signal sequence was predicted by SignalP-4.0 Server program
Summary
Astaxanthin, a naturally occurring carotenoid pigment molecule, displays strong antioxidant, anti-cancer, and immunity-enhancing properties, and is often utilized in food, biomedical, cosmetic, and other industries. Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) is the principal carotenoid pigment in algae, yeasts, plants, crustaceans, and certain fish (notably salmon) [1,2,3]. It has molecular formula C40H52O4 and molecular weight 596.86. Huang et al Biotechnol Biofuels (2018) 11:181 Both free astaxanthin and Ast-E are strongly hydrophobic. Addition of certain groups to hydroxyl groups of free astaxanthin can increase solubility, which is useful for preparation of derivatives for oral administration; e.g., derivatives based on astaxanthin disuccinic acid disodium salt have been applied for treatment of various neurological disorders [13]. Derivatives based on free astaxanthin have been successfully used in nutraceutical and medicinal preparations for treatment of degenerative diseases [14]
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