毛云芝菌产漆酶液体培养条件的优化

探索了不同的培养条件下毛云芝菌产漆酶的情况,发现该菌漆酶属于组成型漆酶,其酶活力高,产酶周期短,酶的合成与菌体生长没有明显相关性;以漆酶的酶活为指标,采用正交试验设计方法对毛云芝菌液体培养的摇瓶生长条件进行了优化研究.结果表明:优化培养基的组成(质量分数)为麸皮3%,蛋白胨0.2%,牛肉膏0.3%,MgSO4·7H2O 0.05%, KH2PO4 0.3%; TE 6%(体积分数),VB1 20 mg/L;自来水,pH自然.该营养条件下的酶活为1 668 U/mL,约是优化前培养基酶活的16倍,为工业化生产提供了一定的理论依据.     

金属铜、锰离子对粗糙脉孢菌漆酶合成的影响

研究了金属离子铜、锰对粗糙脉孢菌合成漆酶的影响。结果表明：10μmol／L的铜离子产生的漆酶活力最高(4.01 U／ml)，是不加铜离子的2．62倍：150μmol／L的锰离子产生的漆酶活力最高(5．05 U／ml)，是不加锰离子的4．1倍．说明适量的金属离子铜、锰对粗糙脉孢菌合成漆酶是有利的，但是这一合成调控的机制有待进一步深入研究。     

硬毛粗盖孔菌产漆酶条件优化及其对日落黄的降解作用

以硬毛粗盖孔菌(Funalia trogii)为研究对象,考察培养基组成及培养条件等对其发酵产漆酶的影响,并进一步研究漆酶对食用合成色素日落黄的降解脱色效果。结果表明,最适培养基组成及培养条件为:玉米淀粉25 g/L,蛋白胨2 g/L,Cu~(2+)1.5 mmol/L,香兰素1 mmol/L,培养温度30℃、初始p H6.0,摇床转速200 r/min,硬毛粗盖孔菌培养8 d后发酵液漆酶活力为(329±12) U/m L,是初始培养条件下的11.96倍。漆酶粗酶液对日落黄呈现出较高的降解脱色作用并可耐受高浓度底物(800 mg/L),50～200 mg/L日落黄脱色率均可达90%以上,漆酶介体ABTS可提升对日落黄脱色效率及速率,并降低高浓度底物对漆酶的抑制作用,曲酸的加入则抑制了漆酶活性,导致漆酶对日落黄的脱色率仅为7.14%。本研究为漆酶资源在食品行业的深入开发与应用提供理论依据。     

1株耐高温酸性脂肪酶产生菌的筛选鉴定与其酶学特性研究

以菜籽油为唯一碳源,经过富集、驯化、平板分离初筛和复筛,从长期淤积油污的食堂下水道中筛选获得1株脂肪酶产生菌CQNU 3-3。利用16S r DNA序列系统发育分析,结合形态学与生理生化特征确定CQNU 3-3为代尔夫特菌,并将其命名为Delftia tsuruhatensis strain CQNU 3-3。研究发酵条件对菌株产酶的影响发现,该菌株在初始pH为2的酸性条件下,发酵酶活力最高;发酵第1天酶活力即可达到最大,发酵温度为32~37℃较为适宜。对其产生脂肪酶的酶学特性进行分析,表明该酶最适反应pH为7.0,最适反应温度为50℃,Cu~(2+)、尿素、Mg~(2+)和Na+对酶活力有显著的抑制作用,而Zn~(2+)和EDTA可以显著提高酶活力。油脂降解率实验表明该菌株对样品中油脂的降解率可高达73.54%。     

诱导条件下营养因子对粗糙脉孢菌合成漆酶的影响

研究了粗糙脉孢菌合成漆酶的一些重要影响因素，包括诱导剂种类、碳源、氮源。获得了粗糙脉孢菌合成漆酶的较适宜每件：以20g／L葡萄糖为碳源，2g／L硝酸铵为氮源，粗糙脉孢菌静止培养2-3d后，再加入放线菌酮（终浓度2．8μmol／L）诱导培养6—7d后产生的漆酶的最高酶活可达4-5u／mL。     

Trichoderma viride菌生物量测定及其纤维素酶合成特征

利用HPLC法测定Trichodermaviride菌固态发酵曲中的麦角固醇含量。研究了麦角固醇与菌丝体间的关系。该菌固态曲中麦角固醇分离条件以 1∶2 5 (m/v)的丙酮抽提 1 5h为最佳。当固态发酵培养至 69h时 ,曲中的生物量达到最大值 ,为每克干曲中含有 0 5 75 g菌丝体。此时该菌所产生CMC酶和FP酶活力均达到最大值 ,呈现正相关性 ,说明这 2种酶的合成特征均为同步合成型 ,而C1 酶活力高峰滞后 ,出现在 72h。     

紫外和超高压诱导漆酶产生菌变异的对比研究

分别利用紫外线和超高压诱变漆酶产生菌灵芝(Ganoderma.lucidum Karst),发现超高压的诱变幅度较大.经初步筛选和发酵,获得了2株具有较大应用潜力的漆酶超高压突变株G1502和G2001,前者漆酶活力比出发菌株提高了2.83倍,发酵时间缩短了1 d;后者漆酶活力提高了0.89倍,发酵时间缩短了3 d.     

降解β-胡萝卜素双加氧酶的分离纯化及其部分酶学性质

研究了西方许旺酵母菌发酵产生双加氧酶的分离纯化及其酶学特性,并利用该酶降解β-胡萝卜素生成香味物质。通过摇瓶发酵,并对粗酶液进行硫酸铵梯度沉淀、半透膜透析、DEAE-Sepharose离子交换和Sephadax G-100凝胶过滤等处理,得到转化β-胡萝卜素生成香气物质的双加氧酶。实验结果表明,西方许旺酵母菌发酵产生的双加氧酶被纯化了36.43倍,酶活力回收率为21.0%,分子量为55.0 ku。酶的最适温度为40℃,最适p H为8.5;金属离子对降解β-胡萝卜素双加氧酶活力的影响为:Fe~(2+)Mg~(2+)K~+Na~+Mn~(2+)Cu~(2+)Ca~(2+)Zn~(2+)Ag~+。Fe~(2+)和Mg~(2+)能明显增强酶活力,而Zn~(2+)和Ag~+能抑制酶活力;SDS和胃酶抑素对酶活力有显著抑制作用。降解β-胡萝卜素双加氧酶的K_m=8.24×10~(-4)mol/L,V_(max)=2.16×10~(-4)mol/(min·mg)。     

Paecilomyces sp. S152胞外漆酶的合成调控

探索了不同碳氮源营养条件下,Paecilomyces sp.S152胞外漆酶的分泌情况,发现麦芽糖(20 g/L)、大豆蛋白胨(10 g/L)及酵母粉(1 g/L)最适于Paecilomyces sp.S152胞外漆酶的合成,发酵7～8 d后,漆酶酶活达到5 064 U/L.研究发现Paecilomyces sp.S152漆酶在细胞内及细胞外均有分布,添加1.5 g/L的Tween 80可促进胞内漆酶的释放,提高胞外漆酶酶活,缩短发酵周期(6 d),生产强度提高至946 U/(L·d).     

过氧化氢与漆酶的协同催化反应

本文研究了微量过氧化氢(H2O2)存在提高漆酶催化2,2′-连氮-双(3-乙基苯并噻唑-6-磺酸)(ABTS)反应的影响.采用顶空气相色谱技术测定了漆酶/H2O2体系中的O2产生与消耗量.结果表明,H2O2和漆酶之间在较短时间内存在着协同作用;加入一定量的H2O2能提高漆酶催化反应的速率;漆酶能够在较短的时间内催化H2O2的分解,微量H2O2与漆酶蛋白质作用,可使漆酶部分失活,但对漆酶活力的抑制不大.     

利用木质素沉淀物产漆酶的研究

从玉米秸秆的碱处理液中分离木质素沉淀物,进一步用于Coriolus versicolor液态发酵生产漆酶。研究结果表明,木质素沉淀物对漆酶的形成有明显的促进作用,当其添加量为0.6%时,在摇瓶条件下漆酶活力可高达7005.6IU/ml,比对照组的漆酶活力提高了4.04倍。发酵培养基中添加10g/L葡萄糖有利于菌种的前期生长,从而提高漆酶产量。在3.7L发酵罐中进行产酶试验,发酵罐最适通气量为0.75vvm,最适转速为300r/ min,漆酶活力可达到7506.2IU/mL。该试验结果对于降低玉米秸秆碱预处理液的环境污染,促进木质素沉淀物的回收利用以及加速漆酶的规模化生产等均具有重要意义。     

白腐菌木素氧化酶系的检测及其漆酶诱导产生的研究

为筛选生长速度快的高产漆酶菌种,通过平板组织分离法在东北林区的4个自然保护区采集、鉴定、分离纯化得到34个大型真菌菌种,其中25个为白腐菌.用丁香醛连氮法、苯胺蓝平板脱色法对获得的菌种进行木素氧化酶系定性测定,结果发现有14个菌种具有漆酶活性.选择其中生长速度快、漆酶活性高的菌种进行了培养方式、诱导剂诱导对漆酶产生的影响,研究发现白耙齿菌、血红密孔菌诱导、静止培养漆酶产生最高峰值分别达到340、480U/mL,明显高于其他3种培养方式,这说明白耙齿菌、血红密孔菌是生长速度快的高产漆酶菌种,且静止培养可代替振荡培养进行漆酶产生、制备等相关的后续工作.     

杂优-2平菇漆酶的分离纯化及酶学性质

将杂优-2平菇菌丝进行液体培养,发酵液经硫酸铵分级沉淀、DEAE（diethylaminoethyl）-Sepharose fastflow层析和Superdex-200 prep grade层析等方法纯化,获得了电泳纯的杂优-2平菇漆酶,并对纯化的漆酶进行了部分酶学性质研究。结果显示,杂优-2平菇漆酶比活力为115 U/mg,分子质量约为244.0 kD,亚基分子质量约为85.6 kD。最适反应pH值和最适反应温度分别为5.0和55 ℃,在pH 6.0～8.0及40～55 ℃范围内稳定性较好;最适条件下,以2,2’-联氮-二（3-乙基苯并噻唑-6-磺酸）为底物的Km值为2.1 mmol/L,最大反应速率（vmax）为0.117 μmol/（min·L）。Fe2＋、抗坏血酸对该酶活性具有完全抑制作用,乙二胺四乙酸、Ag＋、Mg2＋、Li＋对该酶活性影响较小;草酸、甲醇、正丁醇、K＋、Ca2＋、Ba2＋、Zn2＋、Cd2＋、Pb2＋、Mn2＋、Co2＋对该酶活性有不同程度的抑制作用;Cu2＋激活作用不明显;尿素、乙醇、异丙醇对该酶活性具有激活作用。     

Optimization of Cultivation Conditions for the Production of γ-Cyclodextrin Glucanotransferase by Bacillus macorous

Abstract

The production of γ-cyclodextrin glucanotransferase (γ-CGTase) from Bacillus macorous WSH02-06 was optimized in shake flasks using conventional sequential techniques and statistical experimental design. Effects of nutrients including carbon and nitrogen sources, cation ions, initial pH, and temperature on γ-CGTase production were investigated. Corn starch, peptone, Mn²⁺, and Zn²⁺ were found to be essential for obtaining high γ-CGTase activity and biomass. The promoting effect of manganese and zinc on enzyme production has not been reported previously. According to the results of orthogonal array experiment, the optimal culture medium for high γ-CGTase activity was determined. Maximal γ-CGTase activity obtained in the optimized culture broth was about 250?U/mL which was 10-fold higher than that obtained in the basal medium. Time course of cell growth and γ-CGTase production in a 7 l fermenter showed that enzyme production was growth associated, and that maximum γ-CGTase activity reached 277?U/mL in 17?h.     

产漆酶绿色木霉的筛选、鉴定和产酶条件优化

木质素是稻草秸秆的主要成分之一,要实现稻草秸秆的糖化以达到开发利用的目的,首先要解除木质素的包裹阻碍作用。生物预处理去除木质素因具有温和、低耗和环保等优点而成为研究的热点。采用PDA-愈创木酚法筛选到一株能够高效产漆酶的木霉,鉴定结果为绿色木霉(Trichoderma viride)。对其进行单因素实验优化产纤维素酶发酵条件,最佳产酶发酵条件为:摇床转速120r/min、接种量为6%、pH5.5、培养温度28℃、培养时间3d,在此条件下CMCase、FPA和βG酶活力分别达(2.73±0.08)、(0.95±0.09)、(1.75±0.12)IU/mL。     

Optimization of Cultivation Conditions for the Production of γ-Cyclodextrin Glucanotransferase by Bacillus macorous

The production of γ-cyclodextrin glucanotransferase (γ-CGTase) from Bacillus macorous WSH02-06 was optimized in shake flasks using conventional sequential techniques and statistical experimental design. Effects of nutrients including carbon and nitrogen sources, cation ions, initial pH, and temperature on γ-CGTase production were investigated. Corn starch, peptone, Mn²⁺, and Zn²⁺ were found to be essential for obtaining high γ-CGTase activity and biomass. The promoting effect of manganese and zinc on enzyme production has not been reported previously. According to the results of orthogonal array experiment, the optimal culture medium for high γ-CGTase activity was determined. Maximal γ-CGTase activity obtained in the optimized culture broth was about 250 U/mL which was 10-fold higher than that obtained in the basal medium. Time course of cell growth and γ-CGTase production in a 7 l fermenter showed that enzyme production was growth associated, and that maximum γ-CGTase activity reached 277 U/mL in 17 h.     

培养基及培养条件对Pycnoporus sp. SYBC-L1分泌漆酶的影响

从作者所在实验室保存的3株白腐真菌中筛选到一株液态发酵产漆酶的密孔菌Pycnop-orus sp.SYBC-L1,以漆酶活力为指标,采用正交试验法,优化了Pycnoporus sp.SYBC-L1分泌漆酶的培养基:麸皮水煮液60 g/L,葡萄糖60 g/L,豆粕粉15 g/L,CuSO_4·5H_2O 1.0 mmol/L;培养条件:初始pH 3.0,装液量50 mL/250 mL,30℃、200 r/min培养13 d,漆酶活力达24.95 U/mL,为优化前的36.16倍.     

Improved biomass saccharification by Trichoderma reesei through heterologous expression of lacA gene from Trametes sp. AH28-2

The Trametes sp. AH28-2 laccase gene lacA fused to cellobiohydrolase I signal peptide coding sequence was heterologously expressed in T. reesei. The lacA cDNA was under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter. Native PAGE analysis indicated that two transformants, L8 and L38, were able to secrete recombinant laccase A, and their laccase activities corresponding to ABTS oxidation reached 3.62 IUml(-1) and 1.50 IUml(-1) respectively. Most of the characteristics of the recombinant laccase were similar to those of the native enzyme. Reducing sugar yields of L8 and L38 obtained from saccharification of corn residue by crude enzyme increased by 31.3% and 71.6% respectively compared to the host strain. These results indicated that the engineering strains developed in this work could be potentially used for laccase production and tailoring cellulase properties with laccase proteins through genetic manipulation would be a feasible strategy to improve saccharification efficiency of biomass by cellulase preparation.     

Laccase and Mn-peroxidase production by Coriolus hirsutus strain 075 in a jar fermentor

The white-rot fungus Coriolus hirsutus strain 075 excretes considerable amounts of laccase and Mn-peroxidase into culture broth over a brief production time. The effects of agitation speed, temperature, aeration and inoculum amount on laccase production using a 10-l fermentor were studied. The optimum fermentation conditions were a 15% inoculum, an aeration rate of 0.88 vvm, an agitation speed of 160 rpm, and a temperature of 28 degrees C. By optimizing the fermentation conditions, the laccase activity reached 80+/-3 U/ml in 3 d and the purified enzyme output was 30 mg/l. The laccase and Mn-peroxidase were purified by means of isoelectrofocusing and ion-exchange chromatography. The pIs of the laccase isoenzymes were 4.2 and 4.5. Mn-peroxidase had only one isoenzyme with a pI of 3.2. The optimum pH was 4.5 for laccase with syringaldazine as the substrate and 5.0-5.3 for Mn-peroxidase with Mn(+2) and H2O2 as the substrates. The laccase and Mn-peroxidase retained 50% of their activities at 50 degrees C after 55 h and 12 h of incubation time, respectively.     

Isolation of a novel alkaline-induced laccase from Flammulina velutipes and its application for hair coloring

Laccase is a member of the multi-copper oxidase family and a promising for hair coloring. In this study, we isolated a novel alkaline-induced laccase from the white-rot fungus Flammulina velutipes and studied the possibility to apply the enzyme for hair coloring. Laccase activity detected in the culture supernatant of F. velutipes was found to significantly increase when exchanging the medium to laccase inducing one whose pH was adjusted to 9.0. Three isozymes were detected by activity staining on non-denaturing SDS-PAGE. The major isozyme, Flac1, was purified from the culture supernatant after being induced at pH 9.0 by ion-exchange column chromatography. The N-terminal peptide sequence of Flac1 was determined, revealing clear homology with laccases from other white-rot fungi. Optimum pH of oxidation was found to be around pH 5.0-6.5 regardless of several different substrates used. Oxidation activities of Flac1 to several hair dye agents as substrate showed the higher activity at pH 6.5 than that at pH 9.0. Oxidation activity was also detected at pH 9.0 which was suitable for hair coloring. When the purified Flac1 was applied for hair coloring system without using hydrogen peroxide, effective coloring was observed at the protein amount of 0.25mg/1g of hair used. These results indicated that this alkaline-induced novel laccase isolated from the culture supernatant of F. velutipes might be a useful enzyme for hair color.