| China is rich in straw reserves,but the comprehensive utilization efficiency of straw is not high,and a considerable part of it is burned as fuel,which not only wastes a huge amount of biomass resources,but also causes environmental pollution.The use of biological method to degrade straw is not only environmentally friendly and pollution-free,but also can produce a lot of ancillary products with economic benefits.The main component of corn straw is lignocellulose.Lignin and hemicellulose in lignocellulose are closely intertwined,wrap cellulose and combine in the way of covalent bond to form a three-dimensional network of complex structure.as a result,microorganisms or enzymes can not directly contact cellulose and hemicellulose,and the degradation effect is greatly reduced.The straw is first pretreated by microorganisms or enzymes to degrade lignin in the straw and release cellulose and hemicellulose.After subsequent biodegradation,cellulose and hemicellulose will eventually be transformed into glucose and xylose,which will greatly improve the utilization effect of corn straw.In order to degrade lignin in straw,the expression vectors of laccase,lignin peroxidase and manganese peroxidase(p YES2-P?LC-r DNA,p YES2-P?Li C-r DNA,p YES2-P?MC-r DNA)were constructed.The constructed expression vectors were transferred into Saccharomyces cerevisiae by r DNA integration.The high expression of laccase,lignin peroxidase and manganese peroxidase in Saccharomyces cerevisiae was realized.The copy number of transformants was determined by microdrop digital PCR technique,and the relationship between copy number and enzyme activity was studied.Through the degradation of alkali lignin by laccase,lignin peroxidase and manganese peroxidase,the effect and mechanism of lignin degradation enzyme system on lignin were explored.Finally,the conditions of lignin degradation of Saccharomyces cerevisiae were optimized by response surface methodology.The results are as follows:1.The expression plasmid of laccase(p YES2-P?LC-r DNA)was constructed and transformed into Saccharomyces cerevisiae.10 transformants with different copy numbers of 1,3,4,5,6,7,8,9,11 and 12 were obtained by microdrop digital PCR.According to the investigation of its enzyme production ability,it was found that when the Lac copy number was less than 7,the enzyme activity increased with the increase of copy number.When the number of copies was 8,the highest enzyme activity was425U/L,which was 4.04 times higher than that of 1 copy.When the copy number was more than 8,the enzyme activity decreased with the increase of copy number,and the expression plasmid of lignin peroxidase(p YES2-P?Li C-r DNA)was constructed and transformed into Saccharomyces cerevisiae.12 transformants with different copy numbers of 1,2,4,5,6,7,8,9,10,11,12 and 13 were obtained by microdrop digital PCR.According to the investigation of its enzyme production ability,it was found that when the Li P copy number was less than 7,the enzyme activity increased with the increase of copy number.When the number of copies was 7,the highest enzyme activity was 367U/L,which was 3.46 times higher than that of 1 copy.When the copy number was more than 7,the enzyme activity decreased with the increase of copy number.When the copy number was more than 10:00,the enzyme activity tended to be stable,and the expression plasmid of manganese peroxidase(p YES2-P?MC-r DNA)was constructed and transformed into Saccharomyces cerevisiae.10 transformants with 3,4,5,7,8,9,10,11,13 and 14 copies were obtained by microdrop digital PCR.According to the investigation of its enzyme production ability,it was found that when the Mn P copy number was less than 9,the enzyme activity increased with the increase of copy number.When the copy number was 9,the highest enzyme activity was 204U/L.When the copy number was more than 9,the enzyme activity decreased with the increase of copy number.2.When laccase,lignin peroxidase and manganese peroxidase were used to treat alkali lignin alone and in different combinations,the degradation rates of the three enzymes were 22.15%,15.44% and 12.21%,respectively,and the effect of laccase was the best.the degradation rate of laccase was significantly higher than that of the combination without laccase,which proved that laccase played a major role in the action of the three enzymes.In particular,the combined action of the three enzymes had the best effect on the degradation of alkali lignin,and the degradation rate was 28.98%.Afterdegradation,holes appeared on the surface of alkali lignin,the structure was destroyed,and the specific surface area increased.Lignin degradation enzymes mainly destroy the apparent structure and key chemical bonds of lignin,and most of the degradation products are aromatic small molecular compounds.Lignin-degrading enzymes can lead to the ring-opening reaction of macromolecular benzene ring structure and groups in alkali lignin and the cleavage of chemical bonds.3.The fermentation conditions of lignin degradation enzyme Saccharomyces cerevisiae for lignin degradation by Saccharomyces cerevisiae were optimized.the results showed that when the initial p H value of culture medium was 6.86,fermentation temperature was 29.4? and stirring speed was 147 rpm,the degradation rate of corn straw lignin was53.7%,It is 18% higher than that before optimization.After the detection of electron microscope,specific surface area and Fourier transform infrared spectrum,the smooth surface of degraded corn straw was eroded,holes appeared,the specific surface area increased by about 3 times compared with that before degradation,and the chemical bonds of lignin such as conjugated carbonyl group were greatly reduced.it is fully proved that lignin in corn straw is degraded,and the lignin degradation enzyme Saccharomyces cerevisiae has excellent ability to degrade lignin in corn straw.In this study,laccase,lignin peroxidase and manganese peroxidase were expressed in Saccharomyces cerevisiae for the first time,and the engineering bacteria obtained had high ability to degrade lignin in corn stalk.It can effectively increase the content of cellulose and hemicellulose in the treated corn straw,which lays the foundation for the efficient degradation of treated corn straw by cellulase and hemicellulase. |
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