Identification of a GDSL lipase from Streptomyces bacillaris and its application in the preparation of free astaxanthin.

Astaxanthin shows multiple biological activities, but it is usually linked to different fatty acids and exists in the form of esters. The complexity of astaxanthin esters limits their application in the preparation of sophisticated drugs. Herein, a novel lipase from Streptomyces bacillaris that could hydrolyze astaxanthin esters, named OUC-Sb-lip12, was expressed in Bacillus subtilis. The active site of OUC-Sb-lip12 is probably composed of a dyad of Ser48 and His254, instead of a typical catalytic triad. The lipase was identified to be a GDSL hydrolase, and it showed highest activity at 45 °C and pH 9.0 (glycine-NaOH buffer). OUC-Sb-lip12 showed a good stability at its optimum temperature or a higher temperature, retaining 88.4% and 80.6% of its activity after incubating for 36 h at 45 °C and 55 °C, respectively. OUC-Sb-lip12 could effectively hydrolyze astaxanthin esters in Haematococcus pluvialis oil, generating free astaxanthin. Under the optimum conditions, 96.29% astaxanthin esters were hydrolyzed in 12 h. In addition, B.subtilis is a GRAS model strain and it could efficiently secrete lipase in 9 h, making the lipase potential for scale production of free astaxanthin, which could be further used in the preparation of specific astaxanthin esters with specific functions.

Enhancement of linoleic acid content stimulates astaxanthin esterification in Coelastrum sp.

Most natural astaxanthin is fatty acid-esterified in microalgae to prevent oxidation. However, the factors influencing astaxanthin esterification (AE) are poorly understood. In this study, obstacles to AE in Coelastrum sp. HA-1 were investigated. Only half of the astaxanthin molecules in HA-1 were esterified, but AE was stimulated with exogenous linoleic acid (LA) and ethanol treatment. Astaxanthin esters and total astaxanthin (TA) with exogenous LA were elevated to 3.82-fold and 2.18-fold of control levels, respectively. Treatment with 3% (v/v) ethanol enhanced transcription of the Δ12 fatty acid desaturase gene, which caused more oleic acid (OA) to be converted to LA. Furthermore, the contents of astaxanthin esters and TA were 2.42-fold and 1.61-fold control levels, respectively. These findings confirmed that AE was upregulated by increasing LA content. Thus, a large concentration of OA alone does not increase astaxanthin accumulation in HA-1, and a certain amount of LA was necessary for AE.

Efficient heterologous expression of an alkaline lipase and its application in hydrolytic production of free astaxanthin.

BACKGROUND: 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. 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. RESULTS: The 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. CONCLUSION: A 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.