Heterologous expression of athermostable manganese peroxidase from Dichomitus squalens in Phanerochaete chrysosporium.
Heterologous expression of athermostable manganese peroxidase from Dichomitus squalens in Phanerochaete chrysosporium.
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黄孢原平革菌中角鲨烯的不耐热锰过氧化物酶的异源表达。
DOI：
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发表时间：
2001
影响因子：
3.9
通讯作者：
M. Gold
中科院分区：
文献类型：
--
作者：
Dongmei Li;H. Youngs;M. Gold
角叉丝菌（Dichomitus squalens）属于白腐真菌，其表达锰过氧化物酶（MnP）和漆酶，但不表达木质素过氧化物酶（LiP）。为进一步研究D.角鲨烷，我们异源表达的酶在充分研究的担子菌，黄孢原毛革菌。将黄孢原毛平革菌的甘油醛-3-磷酸脱氢酶（gpd）启动子与D.角鲨烷，翻译起始位点上游5 bp，并置于含有Ural基因作为选择标记的载体中。纯化的重组蛋白（rDsMnP）在动力学和光谱特性上与来自D.角鲨烯和黄孢原毛平革菌（PcMnP）。测定了rDsMNP的N-末端氨基酸序列，并与预测的序列相同。在rDsMNP和PcMNP中，前肽的切割遵循保守的氨基酸基序（A-A-P-S/T）。而D.角鲨烯的热稳定性比其黄孢原毛平革菌同系物高得多，在55摄氏度下的半衰期长15至40倍。如先前对于PcMnP所证明的，添加外源性MnII和CdII赋予rDsMnP额外的热稳定性。然而，与PcMnP不同，ZnII还赋予rDsMnP在55 ° C下的一些额外的热稳定性。注意到在65 ° C下对rDsMnP的热稳定性的金属特异性影响的一些差异。
Dichomitus squalens belongs to a group of white-rot fungi which express manganese peroxidase (MnP) and laccase but do not express lignin peroxidase (LiP). To facilitate structure/function studies of MnP from D. squalens, we heterologously expressed the enzyme in the well-studied basidiomycete, Phanerochaete chrysosporium. The glyceraldehyde-3-phosphate-dehydrogenase (gpd) promoter of P. chrysosporium was fused to the coding region of the mnp2 gene of D. squalens, 5 bp upstream of the translation start site, and placed in a vector containing the ural gene as a selectable marker. Purified recombinant protein (rDsMnP) was similar in kinetic and spectral characteristics to both the wild-type MnPs from D. squalens and P. chrysosporium (PcMnP). The N-terminal amino acid sequence of the rDsMnP was determined and was identical to the predicted sequence. Cleavage of the propeptide followed a conserved amino acid motif (A-A-P-S/T) in both rDsMnP and PcMnP. However, the protein from D. squalens was considerably more thermostable than its P. chrysosporium homolog with half-lives 15- to 40-fold longer at 55 degrees C. As previously demonstrated for PcMnP, addition of exogenous MnII and CdII conferred additional thermal stability to rDsMnP. However, unlike PcMnP, ZnII also confers some additional thermal stability to rDsMnP at 55 degrees C. Some differences in the metal-specific effects on thermal stability of rDsMnP at 65 degrees C were noted.
裂褶菌乳清苷-5-单磷酸脱羧酶URA1基因的序列分析。
影响因子：
3.5
作者：
Froeliger,EH;Ullrich,RC;Novotny,CP
通讯作者：
Novotny,CP
白腐担子菌 Dichomitus squalens 漆酶的纯化和表征。
DOI：
10.1006/abbi.1998.0625
发表时间：
1998
期刊：
Archives of biochemistry and biophysics.
影响因子：
--
作者：
Perie,FH;Reddy,GV;Blackburn,NJ;Gold,MH
通讯作者：
Gold,MH
影响因子：
2.9
作者：
Gelpke, MDS;Moënne-Loccoz, P;Gold, MH
通讯作者：
Gold, MH