A Study on Ligninolytic Enzyme-producing Fungi
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摘要:
目的 获得高产木质素降解酶（LMLs，包括漆酶Lac、锰过氧化物酶MnP、木质素过氧化物酶LiP）的菌株，为木腐菌菌种资源的合理开发和应用提供一定理论依据。 方法 对9株具有产LMLs能力的木腐菌进行产酶筛选，并对筛选出的菌株的生物学特性进行研究。 结果 结果表明：在9株木腐菌中，产LMLs能力较高的菌株分别为LS136（Lac：0.18 U·L-1、LiP：109.68 U·L-1、MnP：140.38 U·L-1）、LJ485（Lac：136.99 U·L-1、LiP：9.53 U·L-1、MnP：72.05 U·L-1）和LJ496（Lac：163.39 U·L-1、LiP：9.50 U·L-1、MnP：74.36 U·L-1）。生物学特性研究结果显示3种菌株最适生长温度均为25℃左右，适宜的生长pH值在4~8。此外，3种菌株对0~8 mmol·L-1的重金属Cr3+、Pb2+以及0~4 mmol·L-1的Cu2+均有较强的耐受性。当Cr3+为1~4 mmol·L-1时，对LJ485和LJ496的生长具有一定的促进作用。 结论 菌株LS136、LJ485和LJ496分别属于韧革菌属Stereum sp.彩绒革盖菌Trametes versicolor和小薄孔菌Antrodiella sp.，具有较强的产LMLs能力和一定的重金属耐受性，为降解木质素的菌株开发提供了菌种资源参考。 Abstract:Objective To search for wood-rotting fungi that are highly effective in producing ligninolytic enzymes(LMLs), such asLac, MnP, and LiP. Methods Nine strains of wood-rotting fungi with known ability to produce LMLs were screened and their biological characteristics studied. Results The strains with high capacity of generating LMLs and their yields on each of the LMLs were found to be:LS136 (yielding Lac at 0.18 U·L-1, LiP at 109.68 U·L-1, and MnP at 140.38 U·L-1), LJ485 (yielding Lac at 136.99 U·L-1, LiP at 9.53 U·L-1, and MnP at 72.05 U·L-1), and LJ496 (yielding Lac at 163.39 U·L-1, LiP at 9.50 U·L-1, and MnP at 74.36 U·L-1). The optimum growth temperature for the 3 fungal strains was approximately 25℃, and pH ranged 4 to 8. The strains were all highly tolerant to Cr3+ or Pb2+ at concentrations of 0-8 mmoL·L-1 and Cu2+ at 0-4 mmoL·L-1. At the concentrations between 1-4 mmoL·L-1, Cr3+ promoted the growth of LJ485 and LJ496. Conclusion The identified LS136 was thought to be a Stereum sp., LJ485 identified as Trametes versicolor, and LJ496 considered an Antrodiella sp.. They all demonstrated varying capacities of producing LMLs with a tolerance to heavy metals. The information would be useful for further investigation on the utilization of these lignin degrading fungi. -
Key words:
- wood-rotting fungus /
- ligninolytic enzymes /
- biological characteristics
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表 1 9株木腐菌在发酵培养基中的木质素降解酶活性变化
Table 1. Ligninolytic enzyme activities in fermentation media of 9 strains of ligninolytic fungi
[单位/(U·L-1)] 木质素降解酶
Ligninolytic enzymes菌株
Strain0 d 3 d 6 d 9 d 12 d 15 d 18 d 21 d 漆酶Lac LS77 - 0.79±0.35a 8.84±3.40ab 10.78±1.16ab 11.92±10.38ab 20.10±15.68ab 57.96±10.92c 23.61±4.69b LS136 - 0.12±0.17a - - 0.18±0.15a - 0.06±0.05a 0.15±0.12a LS180 - 4.78±6.76a 0.28±0.19a 0.44±0.16a 2.47±2.32a 0.47±0.37a 0.01±0.01a 0.17±0.17a LJ367 - 0.06±0.08a - - 0.11±0.16a - 0.01±0.01a - LJ417 - 0.57±0.66a 0.12±0.17a 0.24±0.24a 4.30±4.09a 5.52±3.51a 60.40±16.25b 127.38±1.26c LJ460 - 0.06±0.09a - - 0.47±0.13a 0.20±0.29a - - LJ483 - 3.23±2.33a 20.76±22.74a 3.68±2.85a 3.66±5.17a 1.85±2.62a - 0.19±0.00a LJ485 - 10.65±3.13a 65.24±11.01b 105.17±12.95c 136.99±11.93d 120.69±11.24cd 133.36±1.29cd 127.52±2.23cd LJ496 - 6.06±2.13a 47.08±7.24b 138.43±14.51c 161.28±0.75d 163.39±2.96d 130.53±5.17c 126.73±3.82c 木质素过氧化物酶LiP LS77 - 7.28±5.76a 2.47±2.51a 1.79±1.29a 7.67±1.69a 2.15±0.22a 6.67±0.00a - LS136 - - 5.09±1.96a 9.09±5.11a 55.91±25.20ab 109.68±54.61b 17.37±8.03a - LS180 - 2.80±3.95a 2.29±3.24a 2.22±3.14a 4.59±3.78a 10.00±6.89a 3.98±0.54a - LJ367 - 21.72±30.72a 2.33±1.69a 1.25±1.17a 7.31±2.04a 4.84±2.15a 4.19±0.18a - LJ417 - 36.31±28.15b 1.79±1.31a 2.11±1.06a 12.11±5.10ab 4.19±1.75a 3.84±1.91a - LJ460 - 1.68±2.38a 1.97±1.77a 15.30±6.15b 5.10±1.99a 4.05±2.42a 5.00±3.60a - LJ483 - 1.54±1.16a 2.07±1.48a 1.29±1.82a 13.55±0.00c 6.99±0.00b 5.16±0.00b - LJ485 - 0.22±0.30a 3.69±0.98a 9.53±10.19a 4.19±0.32a 5.96±2.38a 4.80±1.49a - LJ496 - 11.29±9.01a 2.29±1.53a 3.15±4.46a 9.50±4.45a 9.28±1.14a 6.02±0.99a - 锰过氧化物酶MnP LS77 - 63.15±1.46b 24.92±1.63a 19.74±5.79a 27.33±12.48a 23.79±6.75a 14.36±4.04a 22.00±4.12a LS136 - 45.00±24.56a 117.00±22.17b 113.46±34.38b 121.00±62.38b 140.38±34.08b - 3.95±1.67a LS180 - 30.38±1.15b 21.15±5.77ab 22.87±7.99ab 14.92±1.85a 14.54±4.54a 20.00±6.31ab 21.54±3.23ab LJ367 - 41.13±58.16a 19.79±6.23a 25.69±3.77a 26.77±16.72a 23.54±0.15a 20.72±1.34a 22.87±0.76a LJ417 - 51.90±47.73a 26.26±1.13a 21.79±3.23a 31.95±7.23a 26.62±3.83a 18.67±1.27a 27.31±4.96a LJ460 - 18.69±3.31ab 31±0.44cd 36.87±6.46d 23.85±1.78bc 18.21±2.18ab 10.77±9.58a 19.08±0.00ab LJ483 - 17.46±4.38a 30.15±5.23b - 42.62±0.00c 27.08±0.00b 14.15±0.00a 18.92±0.00a LJ485 - 15.08±0.00a 49.44±4.46ab 72.05±31.43b 41.49±7.27ab 29.49±4.66a 22.82±3.47a 30.92±8.38a LJ496 - 36.62±28.80a 28.51±2.59a 38.54±1.95a 56.77±17.24ab 74.36±12.11b 32.36±6.40a 25.38±8.61a 注：“-”表示未检测到酶活。表中不同小写字母表示酶活在P < 0.05水平存在显著差异。表 3~6同。
Note：“-”means undetected.Different letters indicate the same type of wood rot fungi in the fermentation culture medium, and there are significant differences at P < 0.05 levels at different times.The same as Table 3-6.
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表 2 不同温度对木质素降解菌生长速度的影响
Table 2. Effect of temperature on growth of ligninolytic fungi
[单位/(mm·d-1)] 菌株
Strain5℃ 15℃ 25℃ 35℃ 45℃ 55℃ LS136 1.13±0.00b 6.31±0.09c 11.25±0.00d 0.63±0.00a 0.63±0.00a 0.63±0.00a LJ485 0.83±0.00a 4.83±0.24b 15.00±0.00d 5.33±0.24c 0.83±0.00a 0.83±0.00a LJ496 0.83±0.00a 5.33±0.00c 15.00±0.00d 1.29±0.04b 0.83±0.00a 0.83±0.00a
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表 3 不同pH值对木质素降解菌生长速度的影响
Table 3. Effect of pH on growth of ligninolytic fungi
[单位/(mm·d-1)] 菌株
Strain3 4 5 6 7 8 9 LS136 1.50±0.00b 6.06±0.06c 8.56±0.19e 11.13±0.13f 11.06±0.06f 6.59±0.03d 0.64±0.00a LJ485 0.83±0.00a 8.88±1.10c 12.25±0.08de 13.75±1.25e 10.04±0.79cd 5.79±0.21b 0.83±0.00a LJ496 0.83±0.00a 12.67±0.00c 15.00±0.00d 14.67±0.33d 15.00±0.00d 14.17±0.83d 5.42±0.42b
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表 4 不同浓度Cu2+对木质素降解菌生长速度的影响
Table 4. Effect of Cu2+ concentration on growth of ligninolytic fungi
[单位/(mm·d-1)] 菌株
Strain1 mmol·L-1 2 mmol·L-1 4 mmol·L-1 8 mmol·L-1 16 mmol·L-1 LS136 11.25±0.00c 11.13±0.00c 8.94±0.06b 0.97±0.34a 0.63±0.00a LJ485 15.00±0.00d 13.25±0.08c 10.58±0.25b 0.83±0.00a 0.83±0.00a LJ496 15.00±0.00c 15.00±0.00c 12.25±0.83b 0.83±0.00a 0.83±0.00a
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表 5 不同浓度Cr3+对木质素降解菌生长速度的影响
Table 5. Effect of Cr3+ concentration on growth of ligninolytic fungi
[单位/(mm·d-1)] 菌株
Strain0.25 mmol·L-1 0.50 mmol·L-1 1.00 mmol·L-1 2.00 mmol·L-1 4.00 mmol·L-1 8.00 mmol·L-1 16.00 mmol·L-1 LS136 11.15±0.21d 11.30±0.00d 11.20±0.14d 11.30±0.00d 10.30±0.00c 5.60±0.00b 3.05±0.07a LJ485 - - 10.90±1.30b 11.25±0.07b 13.50±0.28c 10.20±0.71b 2.50±0.28a LJ496 15.00±0.00d 15.00±0.00d 13.70±0.00c 13.6±0.85c 15.00±0.00d 12.05±0.35b 3.20±0.00a
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表 6 不同浓度Pb2+对木质素降解菌生长速度的影响
Table 6. Effect of Pb2+ concentration on growth of ligninolytic fungi
[单位/(mm·d-1)] 菌株Strain 0.5 mmol·L-1 1.0 mmol·L-1 2.0 mmol·L-1 4.0 mmol·L-1 8.0 mmol·L-1 16.0 mmol·L-1 LS136 11.30±0.00c 11.30±0.00c 11.30±0.00c 11.30±0.00c 10.05±0.07b 2.55±0.64a LJ485 - 14.60±0.57d 11.00±0.28bc 11.55±0.35c 10.10±0.57b 6.00±0.28a LJ496 15.00±0.00d 15.00±0.00d 15.00±0.00d 13.40±0.14c 9.50±0.71b 0.80±0.00a
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