【作者】 韦元雅；

【导师】 陈五岭；

【作者基本信息】 西北大学 ， 微生物学， 2009， 硕士

【摘要】 木质素是地球上廉价、丰富的可再生资源,其生物降解是自然界碳素循环关键的一步,但由于它是一种高度复杂的、不定型的芳香环聚合物,很难被降解,因此给全球造成了严重的环境污染。由于传统的物理和化学方法并不能从根本上解决这一问题,生物技术便成为环境界和生物界愈发关注的新生力量。如何利用微生物产生的木质素降解酶系来分解和转化木质素成了新的研究课题,并引发了一轮又一轮的研究热潮。目前人们对木质素酶的研究多集中于白腐菌,并以黄孢原毛平革菌（P.chrysosporium）为模式菌。黄孢原毛平革菌（P.chrysosporium）合成的木质素降解酶系中起主要作用的是胞外过氧化物酶系,包括木质素过氧化物酶（Lignin Peroxidase,LiP）和锰过氧化物酶（ManganesePeroxidase,MnP）,其中MnP在工业和农业中具有广泛的应用前景。但由于从自然界筛选出的黄孢原毛平革菌其产MnP较低,无法直接应用于生产实践,故对黄孢原毛平革菌进行改良,使其获得更高的酶活是广泛应用的前提。同时,迄今为止,大多关于黄孢原毛平革菌发酵的研究多为液体发酵,成本较高且对环境条件有一定要求,无法有效应用在大规模生产中。因此,对黄孢原毛平革菌的固体发酵条件进行研究更具有重要的现实意义。本研究从长有褐色或者乳白色锈斑的树皮上分离出4株可以降解染料的菌株,结合乳酸钠法测酶活,筛选出1株产酶能力较高的菌株W3,其MnP活力为8.59Wg,经形态鉴定为黄孢原毛平革菌。由正交试验确定出该菌原生质体制备和再生的最佳条件:酶浓度为1.5mg/mL,酶解时间为2h,酶解温度为30℃。其中所采用的酶液是将蜗牛酶（S）（5mg/mL）、纤维素酶（C）（4mg/mL）用pH5.0的0.4 mol/L NH₄Cl-0.3%β-巯基乙醇0.2mol/L磷酸缓冲液溶解,按S:C=5:4配比而成。随后用波长为632.8nm,输出功率为12mw,光斑直径为1.5mm的He-Ne激光在照射距离30cm处对制备出的原生质体进行激光诱变。通过对突变株的酯酶同功酶谱、产酶能力及遗传稳定性进行分析,选育出产酶能力较强、遗传稳定性良好的7#菌株作为工业发酵的新出发菌株,其MnP活力为14.28U/g。通过单因素试验确定固体发酵培养基优化方案为:添加浓度为0.015mmol/g的Mn²⁺和浓度为0.1%的Tween-20;通过正交试验确定菌体的固体发酵条件为:菌种驯化时间5d,接种量0.5%,发酵温度37℃,发酵基质含水率85%。在该条件下对黄孢原毛平革菌进行固体发酵,其MnP活力达到20.71U/g。实验证明,亲本菌株经过激光诱变和固体发酵条件优化,其MnP活力提高了141.1%。更多还原

【Abstract】 Lignin is a kind of inexpensive and affluent renewable resource on the earth.The biodegradation of lignin is the pivotal step in the circulation of carbon in nature.However, due to its extremely complicated and indefinite structure,such polymer is hard to be degraded. Consequently,serious environment pollution has been resulted in around the world. Traditional ways,both physical and chemical,can not resolve this problem effectively and thoroughly.In such conditions,biotechnology is an optimum choice for scientists.How to decompose and convert lignin into unharmful matter with catabolic enzymes produced by microorganism has become a new topic in scientific field,which strongly attracted scientists’ eyes.In recent years,most studies related to lignin’s catabolic enzymes have been focused on white-rot fungi and Phanerochaete chrysosporium has been regarded as mode fungus.The catabolic enzymes produced by Phanerochaete chrysosporium include lignin peroxidase（Lip） and manganese peroxidase（MnP） and MnP has a broad prospect in application,both in industry and agriculture.Yet,the MnP produced by natural strains exhibit low activity,which has been restrict its practical use in production.In such a case,finding a new way to improve strains’ MnP activity is in urgent demand and it’s the base of widely application.What’s more, so far,most studies on fermentation of Phanerochaete chrysosporium have been concentrated on fluid fermentation,which is highly cost and has strict requirements of circumstances.Thus, making studies on Phanerochaete chrysosporium’s solid fermentation is of practical importance.Four strains which could effectively degrade dye were separated from bark with brown or ivory white locus ferrugineus.One strain with the strongest MnP activity was selected by measuring enzyme activity with natrium lacticum,which is 8.59U/g.According to morphous characteristics,it was regarded as Phanerochaete chrysosporium.Orthogonal experiment was carried out to determine the optimum conditions of Phanerochaete chrysosporium protoplast production and regeneration.The results showed that the strains should be treated with 1.5mg/mL enzyme,at 30℃,for 2 hours.The enzyme was a mixture of glusulase and cellulase. They were dissolved with 0.4 mol/L amchlor-0.3%β-ME and 0.2mol/L phosphate buffer, according to S:C=5:4.Then,He-Ne laser（λ=632.8nm,output power was 12mw,the diameter of light spot was 1.5nm） irradiated protoplasts for different time in a distance of 30cm.7# strain with 14.28U/g MnP activity and better genetic stability was selected after analysis of esterase isozymogram,MnP activity and genetic stability.The solid fermentation medium should be added with 0.015mmol/g Mn²⁺ and 0.1%Tween-20 and the fermentation conditions were as follows:domestication time of strains was 5d,inoculation rate was 0.5%,temperature was 37℃and water ratio was 85%.Under such circumstances,the MnP activity of Phanerochaete chrysosporium hit 20.71U/g.The study showed that after laser mutagenesis and optimization of solid fermentation conditions,strains’ MnP activity could be raised by 141.1%.更多还原