【作者】 洪宇植；

【导师】 肖亚中；

【作者基本信息】 安徽大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 将漆酶保守序列引物扩增法与长距离反向PCR（LD-IPCR）技术相结合,建立了真菌漆酶基因的新型克隆体系,从栓菌AH28-2（Trametes sp.AH28-2）中克隆得到三个漆酶同工酶基因（lacA、lacB和lacC）和一个漆酶假基因lcD。这些同工酶基因与其它担子菌漆酶在氨基酸水平上呈现出很高的相似性。lacA、lacB和lacC的5′-非编码区均存在多个可能的顺式作用元件一致序列,包括TATA box、CAAT box、MRE、XRE和CreA结合位点,这三个基因5′-非编码区的平均G+C含量分别为59%、60%和52%。lacA和lacB的TATAbox位于起始密码子上游约-100bp处,与其不同,lacC的TATA box位于-172bp。Cu²⁺的存在对于Trametes sp.AH28-2漆酶同工酶基因的表达是必需的,并且Cu²⁺浓度的不同对同工酶的诱导效应不同,低浓度的Cu²⁺（<0.5mmol/L）能够诱导产生LacA和一新型漆酶,而高浓度的Cu²⁺（1-2mmol/L）只诱导合成LacA。侧链基团不同的芳香化合物能够诱导特定漆酶同工酶的表达,邻甲苯胺诱导产生单一的漆酶组分（LacA）,而3,5-二羟基甲苯主要诱导产生同工酶LacB。由3,5-二羟基甲苯诱导,在培养后期LacA与LacB的比率下降。葡萄糖对于Trametes sp.AH28-2漆酶的表达具有抑制效应,以纤维二糖代替葡萄糖引起发酵液漆酶活力急剧增长。定量RT-PCR结果表明,漆酶mRNA转录本的累积伴随着发酵液中相应漆酶活力的增加。 将漆酶同工酶lacA的cDNA克隆到pPIC9K载体上,以α-因子前导序列为分泌信号,在Pichiapastoris GS115细胞中进行异源活性表达。在含有0.3mmol/LCu²⁺、0.2%（w/v）丙氨酸的BMM培养基（pH6.0）中,20℃条件下最有利于重组漆酶的合成,最适甲醇添加量为0.5%（v/v）,摇瓶发酵获得的最高漆酶产量为4.0mg/L（1360U/mg）。重组漆酶LacA对底物ABTS的表观Km为24.6μmol/L,与天然LacA的Km（25.0μmol/L）基本相近,两种漆酶的含糖量（8.5%）相同。然而,与天然酶相比,重组LacA与底物ABTS或愈创木酚反应的最适pH有向酸性漂移的趋势。重组LacA与天然LacA的最适稳定pH分别为5.5和8.5,并且,更多还原

【Abstract】 The PCR amplification using the degenerate primers, based on the Cu-binding conserved sequences of laccase, and the long distance-inverse PCR technique were used to clone novel fungal laccase genes. Three novel laccase isozyme genes (lacA, lacB and lacC) as well as a laccase pseudo-gene (lcD) have been identified from basidiomycete Trametes sp. AH28-2. These genes display a high similarity with other basidiomycete laccases at the amino acid level. An inferred TATA box and several putative CAAT, MRE, XRE and CreA consensus sequences were identified in the 5’-noncoding regions of lacA, lacB and lacC. For the three promoters of lacA - lacC, the average G+C contents are 59%, 60% and 52% respectively. Different from the TATA boxes of lacA and lacB at about -100 upstream of the initial translation codon, the TATA box of lacC is located at -172. On the other hand, copper ion is necessary for the synthesis of all laccase isozymes in Trametes sp. AH28-2. Moreover, copper at different concentrations has an effect on the expression of different isozyme components, with lower Cu~2+ concentration (< 0.5 mmol/L) inducing LacA and a novel laccase while the latter will disappear when increasing Cu~2+ concentration up to 1-2 mmol/L. More interestingly, different aromatic compounds can induce the production of distinct laccase isozymes, with o-toluidine inducing the expression of LacA while 3,5-dihydrotoluene mainly stimulating the production of LacB. Upon induction by 3,5-dihydrotoluene, the ratio of LacA to LacB decreased in the later phase of induction. It was also observed the glucose-repression effect on the laccase expression in Tramete sp. AH28-2, with the laccase activity in cultures increasing rapidly when the glucose was displaced with cellobiose. Furthermore, quantitative RT-PCR showed that the accumulation of laccase mRNA transcripts is accompanied with the increase of corresponding enzyme activity in cultures.The lacA cDNA from Trametes sp. AH28-2 was cloned into the expression vector pPIC9K, using the leader sequence of a-factor as the secretion signal, and heterologously expressed in Pichia pastoris strain GS115. The highest laccase production of 4.0 mg/L (1360 U/mg) was obtained in shaken-flask "fermentation at 20°C in BMM medium containing 0.3 mmol/L Cu2+ and 0.2% (w/v) alanine, with the addition amount of 0.5% (v/v) methanol being most suitable for laccase synthesis. The apparent Km for ABTS (24.6 umol/L) and the carbohydrate content of the recombinant LacA (8.5%) are approximately identical to that of native LacA. However, the optimal pHs of recombinant LacA to substrates ABTS and guaiacol have a trend of shift to acidity in comparison with native LacA. In addition, the pHs optimum for enzyme stability are respectively 5.5 and 8.5 for the recombinant and native laccases, and recombinant LacA is more stable than the latter in pH2.2-6.0. Thermal stability was also investigated. Furthermore, mutagenesis of a transformant was performed by low-energy nitrogen ion implantation and one mutant (MAI) of excellent laccase producer was isolated with laccase production of 7.7 mg/L (1085 U/mg) in cultures being 92.5% more than the control (4.0 mg/L). Compared to the non-mutant recombinant LacA, five amino acid residues have been detected to mutate in the peptide sequence of the mutant laccase (MAI-LacA) when deduced from the corresponding cDNA sequence where seven nucleotides have been transformed. Several characters are identical for the two recombinant laccases while the catalytic ability of them has a slight variation. The apparent Km of MAI-LacA (28.8 umol/L) has an increase of 17.7% and the relative activity has a decrease of 20.2% in comparison with the non-mutant recombinant LacA. Moreover, the thermal stability of MAI-LacA is superior to the latter.更多还原