| Nowadays,there is waste in the use of all kinds of resources on the earth.Therefore,finding alternative renewable energy materials is a problem that many scientists are committed to solve.Microalgae widely exist in temperate and tropical soil,fresh water and ocean;they are small,autotrophic and easy to cultivate;at present,there are a lot of researches on cellulase production by microorganisms in academic circles,but few researches on cellulase production by microalgae and its application in cellulose degradation.The microalgae in this experiment,a single celled green alga attached to the stem of Roystonea regia,can secrete cellulose.This research provides a new plant source for the research of cellulase and a new idea for the comprehensive utilization of agricultural waste.In this study,the key factors of producing cellulase from microalgae were obtained by response surface optimization method,and various enzyme proteins were analyzed under light and dark conditions;the optimized enzyme solution was used to treat banana straw,and the cellulose structure characteristics were analyzed;then the whole genome of the strain was sequenced and assembled,and the key gene regulating cellulase was obtained.The main results are as follows.(1)The microalgal strain A3-8 that was previously isolated from the stem of Roystonea regia was discovered in this research to have the capacity to digest cellulose.After purification by single cell inoculation,an algael strain named A3-8-1 was obtained,and confirmed to be Heveochlorella roystonensis by molecular and morphological methods.The characteristics of cellulase production were tested,and the qualitative analysis of cellulase and other enzyme proteins under light and dark conditions was carried out.The results showed that the cellulase index of A3-8-1cultured in light was higher than that cultured in dark,and the EAI values of light and dark were similar,which were 2.77(P < 0.05)and 2.62(P < 0.05),respectively.In the qualitative analysis of other enzymes and proteins,lipase was produced in light culture and dark culture,respectively.And xylanase was also produced.Amylase was produced in dark culture;protease,urease and esterase were not produced in light and dark culture.(2)Four kinds of enzyme-producing media were used to determine the cellulase producing ability of the strain A3-8-1.The results showed that the activities of endocellulase,exocellulase,β-glucosidase and filter paper enzyme in PM3 medium were the highest with 13.43 U / ml,17.36 U / ml,14.18 U / ml and 11.04 U / ml,respectively.(3)The PM3 cellulase producing medium was used as the basic medium of A3-8-1,and the subsequent cellulase producing conditions were optimized by response surface methodology.Plackett Burman design was used to evaluate the effects of 8factors including 2x Filner’s beijernicks solution,1M potassium phosphate stock,trace mineral solution,sucrose,CMC,light,p H and temperature on the cellulase activity of the strain A3-8-1.The response surface methodology was used to optimize the p H value of 5.75 and temperature of 27.5 ℃.(4)The structure and characteristics of banana straw cellulose were analyzed by TGA,DTA,XRD,FTIR,SEM and EDX.The comprehensive results showed that: after treating,the hydrogen bonds and weak intermolecular interactions of cellulose were destroyed,and the content of crystalline cellulose was reduced;the lignin bound to cellulose was significantly reduced;the structure of cellulose was seriously damaged,and a large number of massive corrosion products and honeycomb like products were produced;the C and O of cellulose were significantly reduced,indicating that the cellulose was highly degraded after enzyme treatment.(5)The whole genome of A3-8-1 was sequenced by the combination of the third generation sequencing Pac Bio and the second generation sequencing Illumina Hiseq.After assembling and analyzing the sequencing data,the full length of the genome of A3-8-1 was 70.9 mega bases,including 139 scaffolds from nuclear genome,1 from chloroplast genome,and 1 from mitochondrion genome,and the longest fragment was2.7 mega bases.After further elongation using both Pac Bio and Illumina sequences,the nuclear genome was assembled into 47 pseudomolecules,in which Ps Mol3 contains both 5’ and 3’ telomeric sequence and is a intact chromosome.A total of 29 telomeric sequence was identified in the genome,indicating that algal strain A3-8-1 contains at least 15 chromosomes.(6)During annotation,a cellulose gene Hr Cellulase-1 was identified in the genome.It has length of 3900 bp,including14 exons,and the c DNA is 2502 bp,encoding 833 amino acid residues.There is a signal peptide of 23 amino acids at the N terminal,suggesting that it is secreted outside of the cell.Phylogenetic analysis indicated that Hr Cellulase-1 is an endoglucanase. |
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