Selinheimo Emilia, Saloheimo Markku, Ahola Elina, Westerholm-Parvinen Ann, Kalkkinen Nisse, Buchert Johanna, Kruus Kristiina
VTT Technical Research Centre of Finland, Espoo, Finland.
FEBS J. 2006 Sep;273(18):4322-35. doi: 10.1111/j.1742-4658.2006.05429.x.
A homology search of the genome database of the filamentous fungus Trichoderma reesei identified a new T. reesei tyrosinase gene tyr2, encoding a protein with a putative signal sequence. The gene was overexpressed in the native host under the strong cbh1 promoter, and the tyrosinase enzyme was secreted into the culture supernatant. This is the first report on a secreted fungal tyrosinase. Expression of TYR2 in T. reesei resulted in good yields, corresponding to approximately 0.3 and 1 g.L(-1) tyrosinase in shake flask cultures and laboratory-scale batch fermentation, respectively. T. reesei TYR2 was purified with a three-step purification procedure, consisting of desalting by gel filtration, cation exchange chromatography and size exclusion chromatography. The purified TYR2 protein had a significantly lower molecular mass (43.2 kDa) than that calculated from the putative amino acid sequence (61.151 kDa). According to N-terminal and C-terminal structural analyses by fragmentation, chromatography, MS and peptide sequencing, the mature protein is processed from the C-terminus by a cleavage of a peptide fragment of about 20 kDa. The T. reesei TYR2 polypeptide chain was found to be glycosylated at its only potential N-glycosylation site, with a glycan consisting of two N-acetylglucosamines and five mannoses. Also, low amounts of shorter glycan forms were detected at this site. T. reesei TYR2 showed the highest activity and stability within a neutral and alkaline pH range, having an optimum at pH 9. T. reesei tyrosinase retained its activity well at 30 degrees C, whereas at higher temperatures the enzyme started to lose its activity relatively quickly. T. reesei TYR2 was active on both l-tyrosine and l-dopa, and it showed broad substrate specificity.
对丝状真菌里氏木霉的基因组数据库进行同源性搜索，鉴定出一个新的里氏木霉酪氨酸酶基因tyr2，其编码一种带有假定信号序列的蛋白质。该基因在强cbh1启动子控制下在天然宿主中过表达，酪氨酸酶被分泌到培养上清液中。这是关于分泌型真菌酪氨酸酶的首次报道。在里氏木霉中表达TYR2可获得高产率，在摇瓶培养和实验室规模的分批发酵中，酪氨酸酶产量分别约为0.3和1 g·L⁻¹。里氏木霉TYR2通过三步纯化程序进行纯化，包括凝胶过滤脱盐、阳离子交换色谱和尺寸排阻色谱。纯化后的TYR2蛋白分子量（43.2 kDa）明显低于根据假定氨基酸序列计算出的分子量（61.151 kDa）。根据通过片段化、色谱、质谱和肽测序进行的N端和C端结构分析，成熟蛋白是通过约20 kDa肽片段的C端切割加工而成。发现里氏木霉TYR2多肽链在其唯一的潜在N-糖基化位点进行了糖基化，聚糖由两个N-乙酰葡糖胺和五个甘露糖组成。此外，在该位点还检测到少量较短的聚糖形式。里氏木霉TYR2在中性和碱性pH范围内表现出最高活性和稳定性，最适pH为9。里氏木霉酪氨酸酶在30℃时能很好地保持其活性，而在较高温度下酶开始相对较快地失去活性。里氏木霉TYR2对L-酪氨酸和L-多巴均有活性，并且表现出广泛的底物特异性。
