| Astaxanthin is a kind of xanthophyll carotenoids,which exists in microorganisms and marine animals.It has strong antioxidant capacity due to its special molecular structure.Astaxanthin is widely used in food,feed,nutritional health products and medicines.The powerful physiological effects of astaxanthin have attracted the attention of researchers,their research mainly focuses on the yield and production methods of astaxanthin.At present,the cultivation technology of Haematococcus pluvialis is mainly adopted to produce astaxanthin,but the growth conditions of Haematococcus pluvialis are harsh and the cultivation period is long in the process,and the contradiction between the accumulation of astaxanthin and the accumulation of algal cell biomass leads to a certain limitation on the efficient production of natural astaxanthin by Haematococcus pluvialis.Therefore,to get rid of the difficulties of cultivation and shorten the cultivation time is still a problem that needs to be studied in astaxanthin production process.In this paper,a new approach was developed.The protein β-carotene hydroxylase(SpCRTR)encoded by β-carotene hydroxylase gene(SpcrtR)from Spirulina platensis was secreted and expressed in Saccharomyces cerevisiae.Spirulina platensis and recombinant Saccharomyces cerevisiae were fermented by mixed fermentation technology.SpCRTR used astaxanthin in Spirulina platensis to metabolize the precursor canthaxanthin,and finally catalytic conversion of canthaxanthin to astaxanthin.compared with Haematococcus pluvialis culture,this culture route not only shortened the culture time,but also got rid of the restriction of the conditions in the culture process.First,SpcrtR gene was cloned and its encoded protein was analyzed by bioinformatics.SpCRTR catalytic function was verified by expression of SpcrtR gene in prokaryotic cells.Secondly,transforming SpcrtR into wild-type Saccharomyces cerevisiae to realize SpCRTR secretory expression to obtain recombinant Saccharomyces cerevisiae strain.Thirdly,Spirulina platensis and recombinant yeast strains were cultured by fermentation technology.The accumulation of astaxanthin in the fermentation broth was analyzed qualitatively and quantitatively by HPLC.Astaxanthin in fermentation broth was extracted and purified by organic solvent extraction and silica gel column chromatography.Finally,the purified astaxanthin was applied to H2O2-damaged macrophages Raw 264.7 by cell engineering technology.The antioxidant activity of the purified astaxanthin to macrophages raw 264.7 was proved by detecting antioxidant enzymes.The expression of caspase-3 and caspase-9 mRNA was detected by morphological observation,mitochondrial membrane potential change and molecular means to verify the activity of astaxanthin to resist apoptosis.The main conclusions of this study are as follows:(1)The β-carotene hydroxylase gene SpcrtR was cloned from Spirulina platensis for the first time.The total length of SpcrtR is 906 bp,which encodes a 301 amino acid residue with a molecular weight of 28 kDa.the molecular formula is C1652H2435N425O399S8 and the isoelectric point(pI)is 8.90.Bioinformatics analysis showed that there were six transmembrane domains in SpCRTR.The amino acids encoded by SpcrtR were highly homologous with the amino acids encoded by SpcrtR from different species and their structures were analyzed.There were two highly conserved histidine domains in SpCRTR:"HXXXXH"和"HXXHH".In addition,SpcrtR was closely related to the gene of algae and far related to the amino acid in plants and bacteria.(2)The expression vector pGEX-6p-1-SpcrtR was successfully constructed and co-transformed into prokaryotic host with pACCAR16△crtX.The expression of SpCRTR was preliminarily determined by SDS-PAGE and Western Bloting analysis.Carotenoids in Escherichia coli were extracted and analyzed by HPLC,the results showed that SpCRTR catalyzed(3-carotene to produce zeaxanthin,which further indicated that SpCRTR had the catalytic function.(3)The expression vector pYES2/NT-A-S-SpcrtR was successfully constructed in the eukaryotic expression system of Saccharomyces cerevisiae.The expression of SpCRTR in Saccharomyces cerevisiae was confirmed by SDS-PAGE and Western Blotting.The recombinant Saccharomyces cerevisiae S7 strain was screened,and the astaxanthin was transformed from nonexistence to existence after fermentation of Spirulina platensis and recombinant Saccharomyces cerevisiae S7,The results showed that the content of astaxanthin was the highest after 18 h fermentation with 4%Spirulina platensis.The concentration of canthaxanthin before fermentation was 0.24 ± 0.07 μg/mL,and no canthaxanthin was found in the fermented solution.the astaxanthin content was 0.25 ± 0.02μg/mL.Secondly,the concentration of astaxanthin was 7 ± 0.28 mg/mL after being purified by organic solution extraction and silica gel chromatography column.(4)After that oxidative damage model of macrophage 264.7 H2O2 was established,when astaxanthin concentration was 40 μM,MDA activity was 61.78%lower than that of damaged macrophages 264.7(P<0.01),When the concentration of astaxanthin was 20 μM,the activity of SOD was significantly recovered and increased by 31.44%(P<0.01),which indicated that the purified astaxanthin had antioxidant function.Astaxanthin can prevent cell morphology damage and mitochondrial membrane potential changes,and astaxanthin may inhibit caspase-3 and caspase-9 mRNA expression through mitochondrial apoptosis pathway,thus protecting and preventing cell apoptosis. |
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