摘要
木质素是植物材料中仅次于纤维素的最丰富的组份。木质素的降解、转化和利用关系到制浆造纸工业清洁化生产及污染环境的生物修复等领域。对木质素高效降解的菌株主要有真菌、细菌和放线菌,其中白腐真菌是研究最多的菌种,其木质素降解过程的实质是氧化过程,酚氧化酶是其中的关键酶,它们对于天然木质素的再利用有不可低估的作用。白腐真菌是自然界中最有效的木质素降解微生物之一,其分解转化木质素主要依赖生长代谢过程中产生的木质素降解酶系完成,但目前仍存在所用的白腐真菌种类不多且产酶能力和产酶量都有限等问题。细菌和放线菌降解木质素的研究还不多见。开发研究新的降解木质素的真菌、细菌或放线菌,对丰富菌种资源、开发高产木质素降解酶的菌株具有重要意义。本文的研究内容如下:
1.本文通过对从玉米秸杆表面筛选得来的产木质素降解酶的细菌菌株B7、B8、B9和真菌菌株F2、F3、F4、F5、F7、F8、F10、F12进行进一步评价,得到产木质素降解酶能力强的菌株B7和F4。菌种鉴定命名B7菌株为Enterobacter cloacae B7。F4菌株命名为Fusarium annulatum strain F4。
2.通过单因子及正交优化试验确定了F4菌株产木质素降解酶的最佳碳源为淀粉,用量为2.0g/l,最佳氮源为酒石酸铵,用量为1.0g/l,最佳pH为5。此条件下得出的漆酶活力达3827.1U/l。木质素过氧化物酶活力达8602.2U/l。B7菌株产木质素降解酶的最佳碳源为麦芽糖,用量为2.0g/l,最佳氮源为酒石酸铵,用量为1.2g/l。最佳pH为4.5,此条件下的漆酶活力达210.6U/l。木质素过氧化物酶活力达2150.6U/l。进行诱导剂对B7菌株产木质素降解酶的影响显示,诱导剂对B7菌株产木质素降解酶有诱导作用,其中二甲苯胺诱导效果较好,在O.O1mM条件下漆酶活力可达2222.2U/l。木质素过氧化物酶活力达8602.2U/l。
3.对木质素降解酶的酶学性质的研究发现,漆酶的作用最适pH为3.5,低浓度的铜离子对漆酶活力有促进作用,浓度过高则抑制。漆酶具有较好的温度耐受性,在5～65℃间酶活力都具有较高酶活力,酶活力无明显变化。木质素过氧化物酶的最适pH为2.5,低浓度的铁离子对漆酶活力有促进作用,浓度过高则抑制。
Lignin is a class of substances constitute with polymer of aromatic alcohols,found in wood-based organizations,a major role is through formation of intertwined with Net to hardening cell wall.Lignin is mainly located between the cellulose fibers,as a role of resistance.In woody plants,the lignin 25%,is the world's second most abundant organic compounds after cellulose.With the rise of awareness the problem of resources crisis,such as environmental pollution,and the renewable of the natural polymer,human pay more and more attention to biodegradable nature.Waste and the resources renewable,are the major issues of contemporary economic and social development,as well as the new requirements to the contemporary science and technology.
Lignin degradation,conversion and use are related to clean production of today's pulp and paper industry,and the Sound processing of toxic phenolic compounds and bioremediation of environmental pollution,lignin-degrading strains are mainly fungi,bacteria and actinomycetes,white-rot fungi are the most studied species,the lignin degradation process are the real oxidation process,phenol oxidase is one of the key enzyme.Their role in natural lignin re-use should not be underestimated.
In nature,White-rot fungi are the most effective lignin degradation microbial,the decomposition of lignin rely largely on the process of lignin degradation enzymes achieve during the its growth and metabolism,since the late 80's of 20th century,many aspects of the study against the white-rot fungi in the degradation of pollutants,production of lignin-degrading enzyme characteristics,optimization of fermentation conditions and biological characteristics of the molecular level are carry on both at home and abroad,but just a narrow range of white-rot fungi are used,enzyme production capacity and a amount of enzyme production are limited.The research of bacteria and Actinomycete bacteria on lignin degradation are rare,it is significance to research and development of new bacteria or actinomycetes to degrade lignin,and can rich strains resources.
In this paper,bacteria strains B7,B8,B9 and fungal strains F2,F3,F4,F5,F7,F8,F10, F12 were screened form a corn straw surface,all have the Capacity of produce lignin-degrading enzyme.after screened I choise B7 and F4 strains for advanced reseach,and optimized the lignin-degrading enzyme production conditions of F4 and B7 strains with Orthogonal test.inhance the production of lignin-degrading enzyme and studied the physical and chemical properties of lignin-degrading enzyme.
1.optimized the condition for Ligninolytic enzymes production
Established at the experimental conditions obtained:the best culture conditions of F4 strain producing ligninolytic enzymes as follow:carbon source for starch 2g/l,ammonium nitrogen for the tartaric acid 1g/l,the best pH was 4.5.Under these conditions the laccase activity reached up to 3827.1u / L,Lignin peroxidase activity of 8602.2U/l.The best culture conditions of B7 strain producing lignin-degrading enzyme producing as follow:carbon source is maltose 2g/l.tartaric acid ammonium nitrogen for the 1.2g/l.Optimum pH of 4.5. Under these conditions the laccase activity reached up to 210.6U/l.,Lignin peroxidase activity of 8602.2U/l.The induced For B7 strain produce lignin-degrading enzyme have show that Inducer have an impact on lignin-degrading enzyme production.xylidine have a best induced,under the conditions at 0.01mM xylidine,the laccase activity can be of 2222.2U/l. Lignin peroxidase activity of 8602.2U/l.
2.Enzymatic properties
Crude enzyme obtained from the fermentation liquid,obtained purified laccase and lignin peroxidase after purified.The optimal pH is 4 of laccase from enzyme study,low concentrations of copper ions have a promotion on the laccase activity,high concentration inhibitory the laccase activity.Laccase has good temperature tolerance,between the 5-65℃,there no significant changes in laccase activity.The optimum pH is 2.5 of Lignin peroxidase,low concentrations of iron ions have an promotion on the Lignin peroxidase activity,high concentration inhibitory the Lignin peroxidase activity.
3.ligninolytic enzymes production capacity compared of F4 and B7 strains
The ability of F4 strain producing ligninolytic enzymes is high then B7,and easy culture, F4 strain con production a mount of lignin-degrading enzyme even without inducer.but B7 strain producing ligninolytic enzymes has a shorter incubation time also has certain advantages,shorten the fermentation time in industrial production ligninolytic enzymes,improve the fermentation efficiency.
引文
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