裂褶菌产酶条件优化及对秸秆的降解
Optimization of enzyme production and straw degradation by Schizophyllum commune
收稿日期: 2023-03-21 接受日期: 2023-04-21
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Received: 2023-03-21 Accepted: 2023-04-21
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对一株保存的裂褶菌Schizophyllum commune Fr.的产酶能力进行测定,在苯胺蓝和愈创木酚平板上出现褪色反应和红棕色氧化圈,表明该菌株具有较强产过氧化物酶和漆酶能力。在单因素试验和均匀试验的基础上,通过人工神经网络算法优化液体发酵的产酶条件,优化后漆酶酶活达到29.385 U/mL。应用玉米秸秆栽培裂褶菌,发现降解木质素的酶类主要为漆酶,在菌丝满袋时漆酶酶活达到最大48.333 U/mL,具有较强降解木质素能力,在工业、农业等方面具有较广阔的开发应用前景。
关键词:
The enzyme production capacity of a strain of Schizophyllum commune was measured. Fading reaction and red brown oxidation ring of S. commune appeared on aniline blue and guaiacol plate indicated that the strain had strong ability to produce peroxidase and laccase. On the basis of single factor test and uniform test, artificial neural network algorithm was used to optimize the enzyme production conditions of liquid fermentation. The result showed that the optimized laccase activity reached 29.385 U/mL. The laccase activity reached the maximum of 48.333 U/mL in bagful time of cultivation using cornstalks as substrate. The lignin degrading enzymes were mainly laccase, which had broad development and application prospects in industry and agriculture.
Keywords:
本文引用格式
鲁丽颖, 卢士欣, 申东晨, 刘思远, 刘峥, 徐恒, 董爱荣.
LU Liying, LU Shixin, SHEN Dongchen, LIU Siyuan, LIU Zheng, XU Heng, DONG Airong.
我国地域辽阔,农业资源丰富,随着农作物产量的增加,秸秆等农作物废弃物也不断增加,据统计,我国秸秆产出量约为全球总量的20%-30%,也是秸秆年产量最多的国家(李文龙等2021;张芳芳等2021)。但目前大部分秸秆没有得到合理开发利用,不仅造成资源浪费还伴随着环境污染等问题(Chen et al. 2017)。秸秆中的纤维素、半纤维素和木质素以共价键的形式存在,形成一个稳定、天然的高分子组合物屏障,不易被降解(Ten & Teunissen 2001;Chen et al. 2011;刘云鹏2017)。应用物理方法、化学方法和生物方法等处理方式可以有效降解木质素,其中利用细菌、真菌及放线菌等微生物对秸秆进行处理具有多种优势(Fujimoto et al. 2005;姜洁2010;Li et al. 2020;Mei et al. 2020)。有研究表明真菌尤其是白腐菌的降解能力优于放线菌和细菌,选择有较强木质素降解能力的菌株及合适的产酶条件能够加快农作物废弃物的降解,减少污染。
裂褶菌Schizophyllum commune Fr.隶属于担子菌门Basidiomycota、蘑菇纲Agaricomycetes、蘑菇目Agaricales、裂褶菌科Schizophyllaceae (戴玉成等2022),是一种常见食用菌(Wu et al. 2019),一般生长于春夏秋季节雨后的针阔叶枯木和腐木上,但偶尔也生长在活立木上(戴玉成2005, 2012),野生种分布广泛,除南极外其余各地均有分布(Le et al. 2007;岳诚等2019),亚、非地区的人们多食用枯枝上生长的野生裂褶菌(Ohm et al. 2010)。裂褶菌是典型的白腐菌,生长过程中可产生漆酶、过氧化物酶及纤维素酶等多种酶类(吴柯军2017),并通过这些酶使植物细胞中的纤维素、半纤维素和木质素转化为糖类物质,供自身生长,同时子实体着生的木质基部被酶腐烂为海绵团块状并褪色为淡色(安琪等2023;白长胜等2023),且在纯培养体系中,白腐菌类可将木质素降解为CO2和H2O (Reid 1995;李慧蓉2005)。本研究以从东北林业大学林业示范中心采集分离到的一株裂褶菌为研究对象,通过单因素试验确定最佳碳源、氮源的种类及初始pH和培养时间,通过均匀试验对产酶培养基配方进行优化,结合人工神经网络算法寻找高产酶发酵条件,最后对栽培过程中不同栽培阶段的酶活变化进行研究,为裂褶菌的开发利用和玉米秸秆降解机制的研究提供参考。
1 材料与方法
1.1 材料
1.1.1 试验菌株
菌株采集于东北林业大学林业示范中心的水曲柳枯枝上,经分离纯化后转接到PDA斜面试管中,于4 ℃冰箱冷藏保存。
1.1.2 培养基
PDA培养基:削皮土豆200 g,葡萄糖20 g,琼脂17 g,蒸馏水1 000 mL;PDA-苯胺蓝培养基:在1 L PDA培养基中加入0.1 g苯胺蓝溶解备用;PDA-愈创木酚培养基:在1 L PDA培养基中加入1 mL愈创木酚混匀备用;基础产酶培养基:葡萄糖10 g,蛋白胨5 g,牛肉膏3 g,KH2PO4 1.5 g,NaCl 1 g,MgSO4 1 g,VB1 10 mg。
将上述各培养基放于高压灭菌锅,121 ℃灭菌20 min后使用。
栽培料培养基:玉米秸秆78%,玉米芯10%,麦麸10%,石灰1%,蔗糖1%,装袋后121 ℃高温灭菌2 h冷却至室温后备用。
1.2 方法
1.2.1 菌株的分离、纯化
将采集的样品用75%乙醇消毒30 s,无菌水冲洗3-5次,取裂褶菌子实体样品的组织接种于PDA平板中,放置于25 ℃恒温培养箱培养。接种后每天观察菌丝生长情况并记录,当菌落生长到1 cm左右时,挑取无污染菌落边缘的菌丝转移至新的PDA平板和斜面试管中进行纯化培养和菌种保存工作。
1.2.2 产酶能力测定
苯胺蓝和愈创木酚是用于检测产酶能力的2种物质,漆酶能够与愈创木酚反应产生红棕色氧化圈,而过氧化物酶能使苯胺蓝发生褪色反应。因此可以通过显色现象和脱色现象判断菌株是否能产生漆酶(laccase)、木质素过氧化物酶(lignin peroxidase)和锰过氧化物酶(manganese peroxidase)。用5 mm打孔器在超净工作台内对活化的菌株打孔,挑取一块菌饼分别接种于PDA-愈创木酚和PDA-苯胺蓝培养基中,25 ℃恒温培养,观察是否产生红棕色显色反应和脱色反应。
1.2.3 裂褶菌产酶条件优化
(1) 碳源对产酶的影响:将基础发酵培养基中的碳源分别以相同含量的葡萄糖、蔗糖、乳糖、麦芽糖和玉米粉替代,每个处理重复3次。
(2) 氮源对产酶的影响:将基础发酵培养基中的氮源分别以相同含量的蛋白胨、麦麸、氯化铵、硝酸钾和硝酸钠代替基础发酵培养基中的蛋白胨,每个处理重复3次。
(3) pH对菌丝产酶的影响:将基础产酶培养基的pH分别调至4、5、6、7和8,每个处理重复3次。
(4) 培养时间对产酶的影响:在接种后10 d内,每天取发酵液,5 000 r/min离心15 min后保存备用。
1.2.4 均匀试验及人工神经网络建模优化产酶培养基
1.2.5 酶活测定
在活化的平板上用5 mm打孔器取菌饼,接种于装有100 mL液体培养基的锥形瓶内,25 ℃、150 r/min振荡培养7 d,培养结束后过滤菌丝收集滤液,5 000 r/min离心15 min,取上清液-20 ℃冷冻保存用于酶活检测。
LAC活性测定:总反应体系共4 mL,包括0.1 mol/L pH 4.5的醋酸-醋酸钠缓冲液3.3 mL,0.5 mmol/L ABTS溶液0.5 mL,粗酶液200 μL,在30 ℃水浴反应3 min,测定420 nm吸光度下3 min内的吸光度变化,以沸水灭活10 min的酶液为空白。
MnP活性测定:总反应体系共4 mL,包括50 mol/L pH 2.5的琥珀酸-琥珀酸钠缓冲液2.5 mL,1.6 mol/L MnSO4溶液0.4 mL,粗酶液1 mL,于37 ℃水浴,加入100 μL H2O2启动反应,测定3 min内240 nm下的吸光度变化,以沸水灭活10 min的酶液为空白。
LiP活性测定:总反应体系共4 mL,包括250 mol/L pH 3.0的酒石酸-酒石酸钠缓冲液2.5 mL,10 mol/L 藜芦醇0.4 mL,粗酶液1 mL,30 ℃水浴,加入100 μL H2O2启动反应,在310 nm处测定3 min内的吸光度变化,以沸水灭活10 min的酶液为空白。
1.2.6 菌袋及粗酶液制备
将各栽培料种类按比例称出,加水至手握有水渗出但不滴落为宜,装入菌袋121 ℃高温灭菌2 h,冷却至室温后放入超净工作台接种。接种后的菌袋放入干净环境中黑暗培养,出菇阶段给予散射光,每天喷水保持湿度。
在各栽培阶段取菌袋栽培料表层下2 cm处栽培料,保存于-20 ℃冰箱备用。取3 g栽培料,加入蒸馏水,28 ℃水浴锅提取4 h,过滤保留滤液,将残渣重复提取2-3次,合并滤液即为粗酶液。
2 结果与分析
2.1 裂褶菌产酶能力测定
图1
图1
裂褶菌在PDA-愈创木酚平板上的显色反应(A)和在PDA-苯胺蓝平板上的脱色反应(B)
Fig. 1
Chromogenic reaction of Schizophyllum commune on PDA-guaiacol plate (A) and decolorization reaction on PDA-aniline blue plate (B).
2.2 裂褶菌产酶条件优化
2.2.1 培养时间对菌丝产酶的影响
酶类物质与白腐真菌的生长相关,产酶量越大对木质素的利用率越高。在发酵过程中,MnP和LiP均未被检测出,但LAC一直具有活性,说明裂褶菌丝生长主要依靠LAC降解物质来供应。随着培养时间的增加,LAC活力呈现先上升后下降的趋势,在培养的第7天,酶活达到最大为33.89 U/mL;第7天后酶活开始下降,原因可能是培养基中营养物质耗尽,菌丝产酶能力下降(图2)。
图2
图2
培养时间和pH对菌丝产漆酶的影响
Fig. 2
Effects of culture time and pH on mycelium laccase production.
2.2.2 pH对菌丝产酶的影响
pH对裂褶菌产漆酶的影响见图2。随着pH的增加,酶活先上升后下降,在pH 5和6时,产漆酶较高,在pH为6时达到最高13.27 U/mL。
2.2.3 碳源对菌丝产酶的影响
裂褶菌菌丝在分别添加以10 g/L的葡萄糖、蔗糖、乳糖、麦芽糖和玉米粉为碳源的培养基中均可产生漆酶,但种类不同产酶量也不同。碳源对漆酶的影响见图3,当以葡萄糖、蔗糖、乳糖和麦芽糖这类单糖为碳源时,菌丝生长分泌的漆酶量较少;以玉米粉为碳源时,产酶量达到峰值为8.09 U/mL。玉米粉的营养物质丰富,且价格便宜,因此选择玉米粉作为产酶培养基的碳源。
图3
图3
碳源和氮源对菌丝产漆酶的影响
Fig. 3
Effects of carbon and nitrogen sources on laccase production in mycelium.
2.2.4 氮源对菌丝产酶的影响
在添加5 g/L的蛋白胨、麦麸、氯化铵、硝酸钾、硝酸钠为氮源时,菌丝产漆酶差距较大。裂褶菌在以蛋白胨、氯化铵、硝酸钾、硝酸钠类无机氮源的培养基中产酶量均低于以麦麸为有机氮源时的产酶量,在麦麸为氮源的培养基中,菌丝产酶量最大为7.84 U/mL。因此宜选用麦麸作为产漆酶培养基的氮源(图3)。
2.2.5 均匀试验结合人工神经网络建模优化
在培养时间7 d和初始pH 6条件下,通过6因素5水平的均匀试验得到实测值,结合人工神经网络算法估计得到仿真值,实测值和仿真值见表1,对比仿真值和真实值,漆酶酶活的误差较小,该模型可信度较高。
表1 均匀试验及结果
Table 1
试验号 Test No. | 玉米粉 Corn flour (g/L) | 麦麸 Wheat bran (g/L) | 牛肉膏 Beef paste (g/L) | 磷酸二氢钾 KH2PO4 (g/L) | 氯化钠 NaCl (g/L) | 硫酸镁 MgSO4 (g/L) | 漆酶酶活 Laccase enzyme activity (U/mL) |
---|---|---|---|---|---|---|---|
实测值1 Actual measured value 1 | 6 | 3 | 3 | 2.5 | 0.75 | 1.5 | 11.707 |
仿真值1 Simulation value 1 | 6 | 3 | 3 | 2.5 | 0.75 | 1.5 | 11.807 |
实测值2 Actual measured value 2 | 8 | 7 | 1 | 2.5 | 1 | 1.25 | 15.307 |
仿真值2 Simulation value 2 | 8 | 7 | 1 | 2.5 | 1 | 1.25 | 15.845 |
实测值3 Actual measured value 3 | 10 | 1 | 4 | 2 | 1.5 | 1 | 17.193 |
仿真值3 Simulation value 3 | 10 | 1 | 4 | 2 | 1.5 | 1 | 17.306 |
实测值4 Actual measured value 4 | 12 | 5 | 1 | 2 | 0.5 | 0.75 | 17.260 |
仿真值4 Simulation value 4 | 12 | 5 | 1 | 2 | 0.5 | 0.75 | 17.835 |
实测值5 Actual measured value 5 | 14 | 9 | 4 | 1.5 | 0.75 | 0.5 | 28.723 |
仿真值5 Simulation value 5 | 14 | 9 | 4 | 1.5 | 0.75 | 0.5 | 28.385 |
实测值6 Actual measured value 6 | 6 | 1 | 2 | 1.5 | 1.25 | 1.5 | 11.393 |
仿真值6 Simulation value 6 | 6 | 1 | 2 | 1.5 | 1.25 | 1.5 | 11.161 |
实测值7 Actual measured value 7 | 8 | 5 | 5 | 1 | 1.5 | 1.25 | 15.743 |
仿真值7 Simulation value 7 | 8 | 5 | 5 | 1 | 1.5 | 1.25 | 15.409 |
实测值8 Actual measured value 8 | 10 | 9 | 2 | 1 | 0.5 | 1 | 20.590 |
仿真值8 Simulation value 8 | 10 | 9 | 2 | 1 | 0.5 | 1 | 20.776 |
实测值9 Actual measured value 9 | 12 | 3 | 5 | 0.5 | 1 | 0.75 | 18.697 |
仿真值9 Simulation value 9 | 12 | 3 | 5 | 0.5 | 1 | 0.75 | 18.350 |
实测值10 Actual measured value 10 | 14 | 7 | 3 | 0.5 | 1.25 | 0.5 | 22.603 |
仿真值10 Simulation value 10 | 14 | 7 | 3 | 0.5 | 1.25 | 0.5 | 22.692 |
2.2.6 优化后裂褶菌菌丝产漆酶情况
根据人工神经网络模型对培养基的各成分进行预测,经验证试验可知真实值和预测值相差较小,经优化的液体发酵培养基发酵,漆酶酶活达到29.385 U/mL (表2),高于优化前培养基的产酶量。
表2 人工神经网络寻优结果
Table 2
试验号 Test No. | 玉米粉 Corn flour (g/L) | 麦麸 Wheat bran (g/L) | 牛肉膏 Beef paste (g/L) | 磷酸二氢钾 KH2PO4 (g/L) | 氯化钠 NaCl (g/L) | 硫酸镁 MgSO4 (g/L) | 漆酶酶活 Laccase enzyme activity (U/mL) |
---|---|---|---|---|---|---|---|
仿真值 Simulation value | 14 | 7.2 | 4 | 1.3 | 0.48 | 0.57 | 29.394 |
实测值 Actual measured value | 14 | 7.2 | 4 | 1.3 | 0.48 | 0.57 | 29.385 |
2.3 不同栽培阶段裂褶菌酶活变化
图4
图4
不同栽培阶段酶活变化情况
Fig. 4
Changes of enzyme activity in different cultivation stages.
3 讨论
本研究对采集的样品子实体经过分离、纯化、形态学及DNA鉴定后确定该菌株为裂褶菌。裂褶菌属于典型木腐菌,主要生长于树木枝干上,能够分泌降解木质素的相关酶,经过产酶能力测试,发现其可产生漆酶和过氧化物酶,在秸秆降解方面具有重要价值。裂褶菌产酶的相关报道主要集中在液体发酵过程中各发酵条件对其产酶的影响,有试验表明裂褶菌产羟甲基纤维素酶(CMC)、滤纸酶(FPA)和微晶纤维素酶(AVI)的高峰期各异,且各培养条件对产酶的影响也不相同(曾亚岚等2012);李梦洁等(2011)对固体和液体培养基产酶规律进行探讨,发现固体发酵产酶效率是液体发酵的6.83倍,产酶能力更高。所以可以利用高产酶的菌种对秸秆进行发酵,在降解秸秆的同时收获子实体,产生附加值。
本研究发现该菌种在液体发酵过程中产漆酶能力较强,通过单因素试验及均匀试验对裂褶菌产漆酶能力进行探究,并结合人工网络算法对其产LAC液体培养基进行优化,进而得到高产酶的培养基。有研究发现裂褶菌产漆酶最适pH为4.5 (赵文娟等 2018),与本研究最适pH 5.0接近。应用高产LAC的液体菌种进行玉米秸秆降解试验,在栽培过程中漆酶酶活高于锰过氧化物酶,起主导作用,后期2种酶活均下降,原因可能是由于培养料中糖类物质不足无法满足菌丝生长要求。郭晓威等(2017)筛选出一株高效降解秸秆能力的菌种,在秸秆固态发酵过程中产生漆酶和锰过氧化物酶,与本研究结果相同,但该菌株锰过氧化物酶活力达到45.86 U/mL,高于本试验中锰过氧化物酶活力的最大值,但漆酶活力为20.60 U/mL,低于本试验菌种。周玥等(2014)利用响应面法对筛选出的裂褶菌F17的固态产酶培养基进行优化,结果表明在培养第6天时,MnP的产量最大为11.18 U/g,与本研究的MnP最大产量13.744 U/mL相比产量较低。
本研究表明,采集到的裂褶菌菌株能够分泌漆酶和锰过氧化物酶,是一种高产漆酶的菌种,可对其进行深层开发利用,发挥其在秸秆降解等多方面的价值。
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,DOI:10.1016/j.carbpol.2011.02.022 URL [本文引用: 1]
Illustrations of pathogenetic wood-decaying fungi in China
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Pathogenic wood-decaying fungi on woody plants in China
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Resources and diversity of wood-decaying fungi in Yunnan
.
Fatigue damage assessment of deep-sea mooring lines under the influence of wave climate change based on neural network model
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Quantitative evaluation of millling effects on lignin structure during the isolation process of milled wood lignin
,DOI:10.1007/s10086-004-0682-7 URL [本文引用: 1]
Isolation of lignin-degrading strain and its enzyme producing charateristics in corn stover degradation
,
Automatic impervious surface area detection using image texture analysis and neural computing models with advanced optimizers
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A comparative study of the optimization of processing technology of conditioning pine board meat by response surface methodology and BP neural network-particle swarm optimization
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Screening of microbial pretreatment process of corn stalk
.
Optimisation of submerged culture condition for the production of mycelial biomass and exopolysaccharide by Pleurotus nebrodenisis
,DOI:10.1007/BF03175078 URL [本文引用: 1]
Biology and biotechnology of white rot fungi
.
Screening of lignin degrading strain BYL-7 and optimization of degradation conditions
,
Comparative studies on laccase production in liquid and solid cultures of Schizophyllum commune
,
Comprehensive treatment and effective utilization of crop straw
,
Screening and identification of a lignin degrading strain and its optimized liquid fermentation conditions
,
Research on the high yield and biodegradation capability to rice straw of Agaricus bisporus cultivation
.
Deep matching prior network: toward tighter multi-oriented text detection//IEEE
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Study on degradation and application of lignocellulose of corn straw by Schizophyllum commune
.
Economic fungi in China
.
A novel lignin degradation bacteria-Bacillus amyloliquefaciens SL-7 used to degrade straw lignin efficiently
,DOI:10.1016/j.biortech.2020.123445 URL [本文引用: 1]
Genome sequence of the model mushroom Schizophyllum commune
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Biodegradation of lignin
,DOI:10.1139/b95-351 URL [本文引用: 1]
Lignin is an aromatic polymer forming up to 30% of woody plant tissues, providing rigidity and resistance to biological attack. Because it is insoluble, chemically complex, and lacking in hydrolysable linkages, lignin is a difficult substrate for enzymatic depolymerization. Certain fungi, mostly basidiomycetes, are the only organisms able to extensively biodegrade it; white-rot fungi can completely mineralize lignin, whereas brown-rot fungi merely modify lignin while removing the carbohydrates in wood. Several oxidative and reductive extracellular enzymes (lignin peroxidase, manganese peroxidase, laccase, and cellobiose:quinone oxidoreductase) have been isolated from ligninolytic fungi; the role of these enzymes in lignin biodegradation is being intensively studied. Enzymatic combustion, a process wherein enzymes generate reactive intermediates, but do not directly control the reactions leading to lignin breakdown, has been proposed as the mechanism of lignin biodegradation. The economic consequences of lignin biodegradation include wood decay and the biogeochemical cycling of woody biomass. Efforts are being made to harness the delignifying abilities of white-rot fungi to aid wood and straw pulping and pulp bleaching. These fungi can also be used to degrade a variety of pollutants in wastewaters and soils, to increase the digestibility of lignocellulosics, and possibly to bioconvert lignins to higher value products. Key words: delignification, white-rot fungi, biobleaching, lignin peroxidase, manganese peroxidase, laccase.
Optimization of decolorization of malachite green dye by Schizophyllum commune G18
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Oxidative mechanisms involved in lignin degradation by white-rot fungi
,DOI:10.1021/cr000115l PMID:11749405 [本文引用: 1]
Resource diversity of Chinese macrofungi: edible, medicinal and poisonous species
,DOI:10.1007/s13225-019-00432-7 [本文引用: 1]
Activities of lignocellulose enzyme and genetic variation of the related genes of three white-rot fungi
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The current research status of biological characteristics and cultivation of Schizophyllum commune
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Culture condition of Schizophyllum for cellulase
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Screening of efficient lignin-degrading fungal strains and their degradation on cornstalk
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Research on laccase from Schizophyllum commune: its enzymatic properties and application to advanced treatment of papermaking wastewater
,
Optimization of manganese peroxides production from Schizophyllum sp. F 17 in solid-state fermentation of agroindustrial residues
,PMID:25007587
Manganese peroxidase (MnP), a crucial enzyme in lignin degradation, has wide potential applications in environmental protection. However, large-scale industrial application of this enzyme is limited due to several factors primarily related to cost and availability. Special attention has been paid to the production of MnP from inexpensive sources, such as lignocellulosic residues, using solid-state fermentation (SSF) systems. In the present study, a suitable SSF medium for the production of MnP by Schizophyllum sp. F17 from agro-industrial residues has been optimized. The mixed solid medium, comprising pine sawdust, rice straw, and soybean powder at a ratio of 0.52:0.15:0.33, conferred a maximum enzyme activity of 11.18 U/g on the sixth day of SSF. The results show that the use of wastes such as pine sawdust and rice straw makes the enzyme production more economical as well as helps solve environmental problems.
木质素降解菌株的分离及其降解玉米秸秆过程中产酶特点
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裂褶菌液体和固体培养产漆酶的比较研究
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裂褶菌菌株G18对孔雀石绿染料的脱色优化
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