Heterologous Expression Characteristics Of Lignin Peroxidase H2 From Phanerochaete Chrysosporium

Lignin peroxidases (LiP) secreted by white rot fungi are capable of degrading a variety of persistent environmental pollutants. Direct use of the enzymes can achieve much faster degradation of pollutants and would represent a new direction of research and development. However, the enzyme fermentation has been confined by the natural characteristics of white rot fungi, thereby limiting their practical application. Heterologous expression of LiP offers a way to solve the problem. A few of attempts to heterologous express the LiP gene in Escherichia coli, and some other hosts have been reported, while further research is necessary to make the expression successful. The present study targets at cloning the LiPH2 gene, encoding the lignin peroxidase, into both Pichia pastoris and Escherichia coli, and expressing the enzyme protein by the recombinant strains. Properties of these expressions were charactered.Both the original gene and the synthetic gene of LiPH2 were used as target genes in this research. The original gene was obtained by performing RT-PCR on the total RNA extracted from Phanerochaete chrysosporium. The synthetic gene was obtained by chemical synthesis with the gene sequence being optimized.The two genes were all successfully inserted into different yeast expression vectors. The recombinant vectors containing different secretion signals and selectable markers were then transformed into the P. pastoris genomes. Fourteen different recombinant strains were constructed. As a result, only two of them were identified to express the LiPH2. Factors that affect the expression were explored and the results are summarized as: Either theα-factor signal peptide or native secretion signal of LiPH2 was adequate for both the secretion and expression of the protein; Using vectors of pPIC9K and pPIC3.5K led to no expression of LiPH2 in GS115 and SMD1168 while using vectors of pPICZαA and pPICZA were more successful; The disruption of PEP4 gene in P. pastoris had an adverse effect on the in vitro expression of LiPH2; C-terminal addition of amino acids could promote the expression of the synthetic gene. The recombinant protein was in an inactive form. The molecular weight of the recombinant protein was larger than that of the native protein, which suggested that over glycosylation may be responsible for the inactivation of the enzyme protein. The tag addition may also have its influence on it.The original gene was also transfermed into E. coli. Four different recombinant strains were constructed. As a result, three of them were identified to express the soluble recombinant LiPH2. The yield of soluble recombinant LiPH2 expressed by E. coli was higher than that secreted by P. pastoris. The N-terminal addition of fusion tags could promote the soluble protein expression in E. coli cells. The native secretion signal of LiPH2 led the recombinant LiPH2 extracellular, though the yield is quite low. The recombinant protein was in an inactive form which might due to the N-terminal addition of amino acids.The results above built a good foundation for further research.