漆酶对酚类化合物生物催化氧化动力学研究——以儿茶酚和表儿茶素为模式底物

以白腐菌漆酶为试材,儿茶酚和表儿茶素为模式底物进行了酶促氧化反应动力学参数的测定。结果表明,以儿茶酚和表儿茶素为底物,漆酶催化氧化的产物最大吸收波长分别为388和408nm,作用最适pH值分别为5.5和5.75;最适反应温度均为55℃。漆酶对2种底物的催化氧化反应均符合米氏方程规律。在25℃反应条件下测得儿茶酚和表儿茶素2种底物的Km值分别为0.279和0.145mol/L;Vmax分别为0.114和1.139A/(U.min)。漆酶对表儿茶素的催化活性大大高于对儿茶酚的,作用于其混合物时需以儿茶酚的酶用量为准,即底物浓度0.5mol/L时加酶0.04U/mL。     

木质素降解相关酶类测定标准方法研究

在比较研究和具体实践的基础上,提出了漆酶、锰过氧化物酶和木素过氧化物酶活力测定的标准方法,详细介绍了各标准方法的具体操作步骤及其在实际研究中的分析应用.     

一株产漆酶细菌的分离、鉴定及对造纸黑液处理的初步研究

利用铜离子作为筛选剂,从凉水国家级自然保护区的土样中分离纯化出一株具有高漆酶活性的细菌.通过形态学观察、生理生化特性测定及16S rDNA序列分析,鉴定其为枯草芽孢杆菌(Bacillus subtilis),命名为B.subtilis WD23.根据GenBank上公布的Baccillus subtilis cotA ...     

偏肿革裥菌漆酶基因克隆及启动子序列分析

以优化后的漆酶培养基为基础,通过RT-PCR和RACE技术相结合,从偏肿革裥菌 Lenzites gibbosa 菌株中获得编码漆酶基因的cDNA及Genomic DNA的全长序列,Genomic DNA大小为2 165 bp.通过比较该漆酶基因的cDNA和Genomic DNA的全长序列,发现该基因包含11个外显子和10个内含子.cDNA序列的全长为1 873 bp,其中包含一个完整的ORF,长度为1 563 bp,编码520个氨基酸.序列在氨基酸水平上与彩绒革盖菌 Trametes versicolor 的相似性评价最高,相似性达83%.通过SEFA-PCR的方法,扩增得到漆酶基因起始密码子上游长986 bp的启动子序列.分析表明,该启动子区域上除分布有TATA-box、CAAT-box以及AP2等基本的转录起始元件外,还存在有多个潜在的顺式作用元件序列位点,包括7个MRE元件、2个STRE元件、1个HSEs元件、7个氮因子结合位点等.这些结果表明,不同的外源诱导物可以调节偏肿革裥菌漆酶基因的表达.     

漆酶不同施用方法对土壤DDT污染修复的研究

该文从投加量、分次投加和灭菌等方面,研究了漆酶不同施用方法对其修复土壤DDT污染效果的影响.研究结果表明,土壤中DDT各组分(P,P'-DDE除外)及DDT总量(DDTs)的降解率均随着漆酶投加量的增加而显著提高,在投加量为每克土加酶6U时DDTs的降解率达到50.63%:漆酶两次投加对土壤中DDT各组分及总量的降解率均显著高于一次投加,DDTs的降解率提高11.4%;漆酶处理对于非灭菌土壤和灭菌土壤中DDT总量的降解率几乎相当,但各组分则有显著差异,其中P,P'-DDE、O,P'-DDT和P,P'-DDD在非灭菌土壤中的降解率低于灭菌土壤,而P,P'-DDT则相反.     

北方梭囊孔菌漆酶基因DNA片段的克隆与序列分析

根据报道的10几种真菌漆酶氨基酸序列的保守区域设计了一对简并引物,以北方梭囊孔菌总DNA为模板,通过PCR扩增到一个1 201 bp的基因片段.将片段序列在NCBI的BLAST软件上进行同源性搜寻,显示99个序列,全部为漆酶基因及其类似基因,因而认为克隆到的片段就是北方梭囊孔菌漆酶基因片段.经过与同源性最高的粗毛革孔菌（laccase lcc1）同一区段编码区序列进行ClustalW比较,其中有6个间隔区,确定为内含子区.去除内含子后,推导的氨基酸序列为286个残基.经与其他几种真菌漆酶的氨基酸序列进行ClustalW比对,同源性均在51%以上.      

不同白腐真菌复配对底物碳和氮的利用规律的研究

通过单一白腐真菌和不同白腐真菌复配对底物碳、氮消耗的比较,得出随着碳、氮的消耗,无论是单一菌还是复合菌,酶活峰值均发生在对数消耗阶段;并且缓慢消耗阶段越长,产酶量越低;在产酶最高的青顶拟多孔菌的参与下,对底物碳氮的利用显著,同时提高产酶量,缩短达到酶活峰值所用时间;在不产酶菌株的参与下,延长对底物碳氮的吸收时间和达到酶活峰值所用时间,对产酶量的影响较小。     

Preserving boiled eggs with a sterilization system employing microbial laccase and wood vinegar

A combination of a microbial laccase and wood vinegar was used to treat the shell surface of boiled eggs. The laccase enhanced the sterilization effect of phenolic compounds contained in the wood vinegar and resulted in prevention of microbial infection of the boiled eggs after storage for 21 days at 40°C and 75% relative humidity. Significant phenoxy radicals, formed by the catalytic reaction of the laccase, could be detected in the shell of the boiled eggs.     

Laccase Gene LAC1 of Colletotrichum lagenarium Is Not Essential for Melanin Biosynthesis and Pathogenicity

Laccase has been shown to oxidize 1,8-dihydroxynaphthalene (1,8-DHN) in the final step of melanin biosynthesis in several fungi. In this study, a laccase gene (LAC1) was cloned from Colletotrichum lagenarium that synthesizes 1,8-DHN melanin, and characterized. To clone the LAC1 sequences, genomic DNA was subject to polymerase chain reactions (PCR) with degenerate oligonucleotide primers that were designed on the basis of amino acid sequences conserved among characterized laccases from other ascomycetes Botrytis cinerea, Neurospora crassa, Aspergillus nidulans, and Cryphonectria parasiticus. The LAC1 gene contained an open reading frame composed of 589 codons and three introns of 51, 49, and 57 nucleotides. The deduced amino acid sequence of Lac1p had high similarity to that of laccase from N. crassa and significant homology with those of multicopper blue proteins. Under melanin-induced culture of this fungus, laccase activity significantly increased and LAC1 expression was also detected. However, the lac1Δ mutants retained laccase activity and had no significant phenotypic differences in melanin production or pathogenicity from the wild-type strain. Received 23 February 2001/ Accepted in revised form 29 March 2001     

白腐真菌F9产漆酶发酵条件的优化

采用液体摇瓶培养方法,探讨了碳源、氮源、pH值、培养温度等各种因素对白腐真菌分泌漆酶能力的影响,采用正交试验对主要的影响因素进行了优化。优化的培养条件为:马铃薯200 g/L,葡萄糖20 g/L,酵母膏5 g/L,MgSO41.5 g/L,VB10.1 g/L,ZnSO40.05 g/L,MnSO40.05g/L,KH2PO43.0 g/L,pH 6,120 r/min,28℃发酵到10 d左右粗酶液酶活达到109.1μ/mL。