木质素过氧化物酶基因5′端上游调控序列的分析

黄孢原毛平革菌（Ｐｈａｎｅｒｏｃｈａｅｔｅ ｃｈｒｙｓｏｓｐｏｒｉｕｍ）能产生降解木质的胞外木质过氧化物酶（ＬＩＰ）和锰过氧化物酶（ＭｎＰ）同工酶。为研究ＬＩＰ基因的转录调控机理,对ＬＩＰ基因（ＧＬＧ３和ＧＬＧ６）的５′端上游序列进行亚克隆,获得６个亚克隆ＤＮＡ片段,然后应用凝胶迁移率变动分析技术筛选能与菌体蛋白质专一性结合的ＤＮＡ片段。结果表明：ＬＩＰ基因ＧＬＧ６的５′端上游有一个约６７０ｂｐ的     

一氧化氮在启动黄孢原毛平革菌木质素降解酶合成中的作用

[目的]通过了解一氧化氮在启动黄孢原毛平革菌合成木质素过氧化物酶(LiP)中的作用及其作用机制,弄清白腐菌启动次生代谢的调控机制.[方法]以黄孢原毛平革菌原种pc530及突变种pcR5305为研究对象,弄清在不同营养条件下NO含量的动态变化及其与合成LiP之间的关系,再通过添加外源NO供体SNP、NO淬灭剂cPTIO对两菌株合成LiP的影响分析,揭示NO在白腐菌启动合成LiP中的作用和作用机理.[结果]两菌株均能在两种不同营养条件下产生NO,但NO的产生量与菌株及其营养状况有关,富营养使pc530产生NO量低且严重延后,而pcR5305产生NO并不需要营养饥饿激发且产生量显著高于pc530.除了LiP峰值出现时间迟于NO峰值时间外,NO含量与LiP合成呈正相关性;外施SNP对合成LiP有促进作用,但对pcR5305的促进作用没有pc530明显;15 mmol/L cPTIO使黄孢原毛平革菌合成LiP均大为降低,但并没有完全抑制产生LiP.[结论]黄孢原毛平革菌可能通过产生NO启动LiP的合成,但NO并不直接参与或影响合成LiP,NO更可能是作为一种上游的信号分子起作用.除了NO外,可能还有其它与NO有相互促进作用的信号分子也参与了LiP合成的调控.与pc530具有不同的产生NO的机制可能就是pcR5305抗营养阻遏合成木质素降解酶的机理.     

木质素过氧化物酶基因5'端上游调控序列的分析

黄孢原毛平革菌（Ｐｈａｎｅｒｏｃｈａｅｔｅｃｈｒｙｓｏｓｐｏｒｉｕｍ）能产生降解木质素的胞外木质素过氧化物酶（ＬＩＰ） 和锰过氧化物酶（ ＭｎＰ）同工酶。为研究ＬＩＰ基因的转录调控机理, 对ＬＩＰ基因（ ＧＬＧ３ 和ＧＬＧ６） 的５′端上游序列进行亚克隆, 获得６ 个亚克隆ＤＮＡ 片段, 然后应用凝胶迁移率变动分析技术筛选能与菌体蛋白质专一性结合的ＤＮＡ片段。结果表明： ＬＩＰ基因ＧＬＧ６ 的５′端上游有一个约６７０ ｂｐ 的ＤＮＡ 片段能与总蛋白质组分专一性结合, 其核苷酸序列分析表明该片段可能含有蛋白质结合的序列特征。研究结果初步显示, 黄孢原毛平革菌可能存在有与ＬＩＰ基因上游某些顺式调控元件相互作用的蛋白质, 调控着ＬＩＰ基因的转录表达。     

黄孢原毛平革菌木质素过氧化物酶基因在甲醇毕赤酵母中的表达

将编码黄孢原毛平革菌木质素过氧化物酶(lip)的cDNA克隆到酵母整合型质粒pMETA上,电转化Ade缺陷型甲醇毕赤酵母(Pichiamethanolica)PMAD16,通过MD平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDSPAGE分析和木质素过氧化物酶活力测定等方法鉴定,表明带自身信号肽的黄孢原毛平革菌木质素过氧化物酶基因(lip)在甲醇毕赤酵母中得到表达。优化其发酵培养条件,以藜芦醇为底物进行酶活测定,其酶活可达932U/L。相应发酵指数为12.94U/h·L。比出发菌株提高了24.18%。     

黄孢原毛平革菌生产锰过氧化物酶的发酵条件研究

目的 :研究黄孢原毛平革菌产锰过氧化物酶的发酵条件。方法 :对培养条件进行优化 ,采用正交设计法对培养基组分进行优化。结果与结论 :优化培养条件为 :接种量 1 6× 10 6 个孢子 L ,pH 4 4～ 4 8,温度 36℃～ 4 0℃ ,转数 12 0r/min。优化培养基参数为 :葡萄糖 5g L ,酒石酸铵 1 3mmol L ,吐温 - 80 1 2g L ,Mn2 + 0 9mmol L。     

黄孢原毛平革菌合成木素过氧化物酶的营养调控

本文研究了营养条件对黄孢原毛平革茵(Phanerochale chrysosporium)ME-116合成木素过氧化物酶及其同工酶组分的影响.在最适培养条件下获得1500U/L的酶活.高效液相色谱分离的5个同工酶组分中以P_2组分含量最高.低碳高氮培养基最适于酶的合成.降低氮和KH_2PO_4含量致使各组分含量下降,而改变MgSO_4和CaCl_2浓度对P_2组分无影响.表面活性剂吐温80主要通过提高细胞膜透性而增加酶的合成.黎芦醇对5种同工酶组分的合成均有诱导作用.培养基中各营养因子对木素过氧化物酶的合成存在着复杂的交互作用.     

培养于天然冷杉木片的黄孢原毛平革菌木质素过氧化物酶基因表达的RTPCR分析

利用RTPCR方法分析了生长于冷杉木片上的黄孢原毛平革菌木质素过氧化物酶基因lipＡ２（GLG3）、lipＣ1(GLG2)、lipＣ2(GLG5)、lipＤ２(GLG1)、lipE（LP0811）的表达。结果发现在不同的培养时间里仅有特定的基因表达,在第２周时仅有lipＡ2(GLG3)基因表达,在第４周时未检测到任何基因的表达,在第６周时lipＤ2(GLG1)和lipＣ1(GLG2)基因表达,在第８周时仅有lipＡ2(GLG3)基因表达。这些结果说明,在冷杉木片上培养的黄孢原毛平革菌的lip基因表达具有明显的时间特异性,并且与限定培养基中得到的结果明显不同。     

培养于天然冷杉木片的黄孢原毛平革菌木质素过氧化物酶基因表达的RT-PCR分析

利用RT-PCR方法分析了生长于冷杉木片上的黄孢原毛平革菌木质素过氧化物酶基因lipA2(GLG3)、lipC1(GLG2)、lipC2(GLG5)、lipD2(GLG1)、lipE(LPO811)的表达。结果发现在不同的培养时间里仅有特定的基因表达,在第2周时仅有lipA2(GLG3)基因表达,在第4周时未检测到任何基因的表达,在第6周时lipD2(GLG1)和lipC1(GLG2)基因表达,在第8周时仅有lipA2(GLG3)基因表达。这些结果说明,在冷杉木片上培养的黄孢原毛平革菌的lip基因表达具有明显的时间特异性,并且与限定培养基中得到的结果明显不同。     

黄孢原毛平革菌木素降解酶系的研究进展

黄孢原毛平革菌木素降解酶系主要由木素过氧化物酶、锰过氧化物酶和乙二醛氧化酶组成。由于该酶系特殊的降解机制,除了木质素,它能降解许多种类的有机污染物,因此在环保方面有巨大的应用前景。本文主要综述了国内外对该酶系的研究进展。     

黄孢原毛平革菌基因启动子的分离与鉴定

利用启动子探针型载体pSUPV8直接在大肠杆菌(Escherichia coli)中分离黄孢原毛平革菌(Phanerochaete chrysosporium)基因启动子片段,获得6个潮霉素抗性(Hyg-r)重组子。对重组子CH2、CH6进行序列分析,结果发现它们都存在真核生物基因启动子的保守序列;用原生质体转化法将其转化黄孢原毛平革菌,仅pCH6获得了潮霉素抗性转化子;PCR和斑点杂交分析表明,pCH6已成功导入黄孢原毛平革菌,并启动潮霉素抗性基因的表达。     

Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Aspergillus niger

The lignin peroxidase (isoenzyme H8) of Phanerochaete chrysosporium was expressed in Aspergillus niger under the control of plant nopaline synthase (NOS) promoter and terminator. H8 mRNA was produced in this heterologous system. Western blot analysis showed that the recombinant protein was of size similar to the native H8. The extracellular lignin peroxidase activity in these primary constructs was positive, though weak (1.12 nKat mg^–1).     

Solid-state fermentation of natural birch lignin by Phanerochaete chrysosporium

A strain of white rot fungus, Phanerochaete chrysosporium Burds. ME446, has been characterized with respect to the extent and rate of Betula nigrificans lignin and non-lignin conversion by solid-substrate fermentation for different culture conditions. Moisture content, inoculum density, nitrogen supplementation and autoclaving of birch solids significantly affected lignin conversion rates and yields in 20 day fermentations. Oxygen favoured lignin over non-lignin conversion at partial pressures of 1.0 atm. Oxygen pressures of 2.0 atm severely inhibited both lignin and non-lignin conversions. Carbon dioxide partial pressures of 0.25, 0.5 and 1.0 atm at oxygen pressures of 1.0 atm increasingly inhibited both lignin and non-lignin conversion rates and yields. The results of these studies demonstrate the effects of major process variables and suggest a need to control the gas environment for process optimization.     

Stimulation of ligninolytic enzyme production in Phanerochaete chrysosporium by polyoxyalkanes

AIMS: Poly(ethylene glycol) (PEG) and some substances similar to PEG in chemical structure were tested as stimulators of ligninolytic enzyme production in shaken culture of Phanerochaete chrysosporium. METHODS AND RESULTS: The substances that caused high enzymatic activity were linear polymers [poly(ethylene glycol), poly(propylene glycol), poly(butylene glycol) and poly(vinyl alcohol)] and cyclic polymers (crown ether). They can have terminal groups other than -OH [PEG (di)methyl ether, PEG sulphate, PEG derivative with the amino group and xanthate]. The maximum lignin peroxidase activities were compared with the surface pressure caused by the stimulator. Addition of polymers composed of charged monomer units did not increase the enzymatic activity and the fungi did not grow at all on addition of polymers having a fixed positive charge. CONCLUSIONS: Lignin peroxidase activity was increased after the addition of polymers with uncharged monomer units. It was higher and its maximum was reached in a shorter time on addition of polymers with higher molecular weights. SIGNIFICANCE AND IMPACT OF STUDY: Beside Tweens there are several polymers that stimulate ligninolytic enzyme production in shaken culture of P. chrysosporium. Their characteristics are: similarity to PEG in chemical structure, having uncharged monomer units and high molecular weight.     

Polymerisation of lignins by ligninases from Phanerochaete chrysosporium

Abstract Four major hemoproteins were purified by isoelectric focusing from an extracellular crude enzyme preparation, produced by the white rot fungus Phanerochaete chrysosporium under carbon-limited conditions. Both the crude enzyme and the purified proteins oxidised milled wood lignin, HCl-dioxane-extracted straw lignin and alkali straw lignin in the presence of hydrogen peroxide. The oxidation resulted mainly in further polymerisation of the lignins and was enhanced by addition of veratryl alcohol to the reaction mixture. Alkali straw lignin was also polymerised by horseradish peroxidase, although veratryl alcohol had no influence on this reaction.     

粉刺侧孢霉产木质素过氧化物酶酶活性与pH值的关系

研究了粉刺侧孢霉 (Phanerochaetechrysosporium)在以微晶纤维素为C源时 ,木素过氧化酶与 pH值的关系 .试验采用 37～ 39°C下液体振荡培养 ,藜芦醇为产酶诱导剂 ,测试不同起始 pH值所得木素过氧化物酶活性 .结果表明 ,起始 pH值 6.0所得酶活最高达 0 .1 2 6U·ml- 1 ,说明以微晶纤维素代替葡萄糖为C源 ,其最佳 pH值相对较高 .菌丝球直径与酶活呈正相关 .