| Schizochytrium sp.is a heterotrophic single-cell microalgae,which can accumulate large amounts of lipid with strong security and high purity.Schizochytrium sp.was regarded as the best algal for industrial production of docosahexaenic acid(DHA).The growth process of Schizochytrium sp.was divided into three stages: cell growth,lipid accumulation and lipid turnover.Fatty acid shifts would be accelerated during the lipid turnover stage,which also resulted a phenomenon of DHA enrichment.The production of fatty acid was influenced by carbon sources.Thus,analysis of fatty acids migration during the fermentation under different carbon sources is meaningful to the quality of single cell oil from Schizochytrium sp.In this study,compositions of microbial oil extracted from Schizochytrium sp.S31 were investigated thoroughly.Lipidomics analysis was adopted to illustrate the migration of fatty acids.Also,the primary research of lipase involved in the process of fatty acids migration was conducted.Firstly,a series of methods were adopted to analyze the compositions of the algal oil extracted at lipid accumulation stage.The oil was separated into neutral lipids(74.83%),polar lipids(2.69%)and glycolipid(22.68%).Triacylglycerol was the major glyceride(93.81%).A small amounts of diacylglycerol(2.93%)and monacylglycerol(2.86%)were also detected.Fatty acid analysis proved that palmitic acid and DHA were the major fatty acids in all lipid classes.DHA distributed in the neutral lipids was higher than its distribution in polar lipids or glycolipid.Futhermore,more than eighty species of triacylglycerols were identified by using UPLC-Q-ToF-MS.22:6-22:6-16:0 and 18:1-22:6-14:0 were at relatively higher contents.Secondly,effects of different carbon sources on fatty acids migration were investigated.During the lipid turnover stage,the biomass and lipid content would both decrease without the influence of carbon sources.However,phenomenon of lipid turnover was more obvious when using glycerol as carbon source.After lipid turnover,the content of palmitic acid would decreased to 28.23% from 41.18%.But the DHA content would be enriched to 50.43% from 31.20%.Triacyglycerol changes indicated 14:0-14:0-16:0,14:0-16:0-16:0 and 16:0-16:0-16:0 were the majors,which would be accumulated during the cell growth stage.Their preferential hydrolysis promoted the enrichment of 16:0-22:6-22:6,16:0-22:6-22:5,22:6-22:6-22:6 and 22:6-22:6-22:5.Meanwhile,four triacylglycerols were identified as biomarkers by lipidomic analysis,which contributing most to the discrimination of the two fermentation styles.Finally,the lipase participated in the migration of fatty acids were determined by means of bioinformatics.The STGL1 with a size of 2885 bp was obtained,which encoding Stgl1 p with 961 amino acids.The similarities of Stgl1 p to reported Tgl4 p in Saccharomyces cerevisiae was 34%.Higher resemblance was shown in the range of 326-569 in amino acid sequence.Both of them contain the consensus sequence(GSSAG)characteristic for lipase.Furthermore,the STGL1 gene was successfully cloned into expression vector pPIC9 K,and transformed into Pichia pastoris GS115.Cultures of recombined strain performed active enzyme activity in the supernatant after induction for 48 h.Stgl1 p showed a hydrolysis capacity for triacylglycerol of olive oil by rhodamine based plate and HPLC-ELSD detection. |
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