摘要
本文通过丁香醛联氮和愈创木酚显色的方法,从47株丝孢菌中首次筛选获得28株产漆酶菌株,其中12株具有高产漆酶能力。对其中Monodictys asperospera(ZGZII_(04)093-1),Monodictys desquamata(ZGZII_(03)003)及Berkleasmium inflatum(ZGZII_(04)014)三菌株进行产漆酶发酵条件探索。M.asperospera(ZGZII_(04)093-1)的发酵条件为:250 mL三角瓶中装入100 mL改进的Kirk产酶培养基,包含葡萄糖2.5 g/L,酒石酸铵1 g/L,接种6菌饼,于pH 6.0,28℃,120 r/min振荡发酵培养264 h后酶活达到峰值,为618.67 U/mL。M.desquamata(ZGZII_(03)003)的发酵条件为:250 mL三角瓶中装入50 mL改进的Kirk产酶培养基,包含葡萄糖10g/L,酒石酸铵0.2 g/L,接种4菌饼,于pH 6.0,28℃,120 r/min振荡发酵培养288h,漆酶活性达到最高值,为573.33 U/mL。B.inflatum(ZGZII_(04)014)的发酵条件为:250 mL三角瓶中装入50 mL改进的Kirk产酶培养基,包含葡萄糖10 g/L,硝酸铵1 g/L,接种5菌饼,于pH 6.0,28℃,120 r/min振荡发酵培养336 h,漆酶活性达到最高值,为921.33 U/mL。
进一步对菌株M.asperospera(ZGZII_(04)093-1)所产漆酶进行分离纯化及酶学性质研究,结果表明:粗酶液经硫酸铵盐析、DEAE-纤维素层析及丙烯葡聚糖凝胶S-300层析纯化,纯化倍数为8.1,回收率为5.7%。漆酶分子量约为77 kDa,最适反应温度为55℃,最适反应pH 6.0,以丁香醛连氮为底物时K_m为0.163 mmol/L,V_(max)为0.194 mmol/(L·min),含糖量为18.14%,Cu~(2+)对漆酶有明显抑制作用。
In this paper,using syringaldazine and O-methoxyphenol as chromogenic substrates,twelve new wood-degrading strains of hyphomycete were screened from forty-seven hyphomycetes including three strains with high level of laccase production.Optimization of fermentation conditions was studied respectively.The results showed that the best condition of M.asperospera (ZGZII_(04)093-1) laccase production was 100ml modified Kirk laccase-producing culture media in 250ml flask with 6 fungal disks containing the following elements:2.5 g/L glucose,1 g/L ammonium tartrate,adjusted to pH 6.0 at 28℃.After the incubation of 120 r/min for the duration of 264 h,the maximum units of laccase was 618.67 U/mL.The best condition of M.desquamata (ZGZII_(03)003) laccase production was 50ml modified Kirk laccase-producing culture media in 250ml flask with 4 fungal disks containing the following elements:10 g/L glucose,0.2 g/L ammonium tartrate,adjusted to pH 6.0 at 28℃.After the incubation of 120 r/min for the duration of 288 h,the maximum units of laccase was 573.33 U/mL.The best condition of B.inflatum (ZGZII_(04)014) laccase production was 50ml modified Kirk laccase-producing culture media in 250ml flask with 5 fungal disks containing the following elements:10 g/L glucose,1g/L ammonium tartrate,adjusted to pH 6.0 at 28℃.After the incubation of 120 r/min for the duration of 336h,the maximum units of laccase was 921.33 U/mL.
The laccase of M.asperospera(ZGZII_(04)093-1) was purified by ammonium sulfate precipitation,DEAE-cellulose and sephacryl S-300.Purification of about 8.1 fold was achieved with an overall yield of 5.7%.Its molecular weight was estimated to be about 77 kDa.The optimum temperature and pH of the laccase activity were 55℃and 6.0,respectively.Kinetic studies of the laccase showed that the K_m and the V_(max) for using syringaldazine as substrate was 0.163 mmol/L and 0.194 mmol/(L·min),respectively.The carbohydrate content was 18.14%.In addition,it was found that laccase activity was significantly inhibited by Cu~(2+).
引文
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