以水稻秸秆为基质里氏木霉产酶条件优化

以里氏木霉(Trichoderma reesei)为研究对象,对水稻秸秆进行糖化试验。通过单因素试验及响应面法优化里氏木霉产酶培养基及产酶条件。结果表明,里氏木霉产酶最佳培养基为：水稻秸秆15.0 g/L、(NH₄)₂SO₄ 2.0 g/L、KH₂PO₄ 3.0 g/L、MgSO₄·7H₂O 0.5 g/L、吐温-80 0.5 mL/L、微量元素液10.0 mL/L、FeSO₄·7H₂O 0.005 g/L。此优化条件下,菌株的滤纸酶酶活为0.612 PFU/mL,提高了52.6%。最佳发酵条件为：发酵温度29℃,初始pH 6、接种量5.0%、转速150 r/min、发酵时间8 d。在此优化条件下,滤纸酶酶活为1.12 PFU/mL,提高了83.2%。     

一株木质素降解白腐菌的筛选、鉴定及其产漆酶培养基的优化

为筛选产木质素降解酶的菌株,采用苯胺蓝-PDA平板法和愈创木酚-PDA平板法从铁皮石斛生长的树皮中筛选到菌株SHIHU-X2,经ITS测序分析将SHIHU-X2鉴定为白腐真菌白黄小脆柄菇(Psathyrella candolleana)。应用响应面法优化产漆酶培养基,结果表明SHIHU-X2菌株产漆酶的最优培养基是:去皮马铃薯200 g/L,葡萄糖20 g/L,蛋白胨1.24 g/L,KH2PO4 1 g/L,酒石酸铵0.02 g/L,CuSO4 0.01 g/L,MgSO4 0.56 g/L,MnSO4 0.057 g/L。漆酶活力从初始产酶培养基1.11 U/mL提高到响应面优化后的2.32 U/mL,漆酶活力提高了109 %。     

枝孢菌AY-42产纤维素酶液态发酵优化

为提高枝孢菌AY-42纤维素酶的产量,通过单因素、响应面法优化其培养基成分及产酶条件。采用单因素试验确定了最适碳氮源分别为油菜秸秆粉和酵母粉。采用Plackett-Burman(PB)试验筛选出3个最显著的影响因素:MgSO₄·7H₂O、油菜秸秆粉、KH₂PO₄。进一步采用响应面优化后得到培养基组成:油菜秸秆粉12.5g/L,酵母粉2g/L,MgSO₄·7H₂O 0.75g/L,NaCl 0.5g/L,KH₂PO₄5g/L,FeSO₄·7H₂O 0.01g/L。采用优化后的培养基优化培养条件,结果表明:最佳培养时间为3d,最佳接种量为5%,最适装液量为70mL,最适转速为200r/min,最佳培养温度为28℃。优化后CMC酶活提高到(4.20±0.06)IU,相比优化前酶活3.23IU提高了30.03%,产酶能力得到较大提升。该研究为枝孢菌应用于纤维素酶生产提供了一定理...     

卡拉胶酶的发酵培养基及培养条件优化

以生物量与卡拉胶酶活为指标,研究发酵培养基组成和培养条件对菌株ASY5产卡拉胶酶的影响,优化菌株Pseudoalteromonas carrageenovora ASY5产卡拉胶酶的培养基和培养条件,以提高卡拉胶酶的产量。结果表明,最优培养基和培养条件为:卡拉胶5 g/L,胰蛋白胨5 g/L,Na Cl 20 g/L,Ca Cl₂0.2 g/L,Na H₂PO₄·2H₂O 1.3 g/L,Na₂HPO₄·12H₂O3.8 g/L,温度18℃,初始p H为6.5,接种量2%,装液量70 m L。在优化工艺条件下,菌株ASY5的生物量和卡拉胶酶活比初始条件分别提高了80.4%和52.2%,菌株ASY5发酵产卡拉胶酶的能力大幅度提高。      

解淀粉芽孢杆菌GSB a-1产凝乳酶培养基组成的优化

从甜酒曲中分离筛选得到1株解淀粉芽孢杆菌菌株GSBa-1,为了提高该菌株液态发酵产凝乳酶的能力,采用单因素实验和响应面法优化其产酶培养基组成。通过单因素实验分析了碳源、氮源、金属盐、磷源对菌株GSBa-1产凝乳酶的影响,并采用响应面法对产酶培养基中麦芽糖、蛋白胨和酵母浸粉含量3个主要因素的优化组合进行了定量研究,确定解淀粉芽孢杆菌GSBa-1产凝乳酶的优化培养基组成为:麦芽糖1.93 g/L、蛋白胨10.89 g/L、酵母浸粉2.15 g/L。在此优化培养基培养条件下,该菌株产凝乳酶活力可达(562.57±7.67)Su/m L,接近理论预测值537.10 Su/m L,且平均误差为4.53%。优化后解淀粉芽孢杆菌GSBa-1产凝乳酶活力比基础培养基提高了1.88倍。     

一株乳酸明串珠菌的鉴定及抑菌培养基配方优化

为了开发利用乳酸菌及其代谢产物相关资源,本研究对实验室前期从内蒙古马奶酒中分离保藏的一株乳酸菌进行16S rRNA基因序列分析,确定该菌为乳酸明串珠菌(Leuconostoc lactis),并以此菌株为研究对象,以抑菌圈直径大小为指标,采用单因素实验、响应面分析法对该菌株的培养基配方进行优化。通过单因素实验后,确定蔗糖、蛋白胨分别为最适碳源与最适氮源,利用响应面法优化培养基得到的最佳培养基配方为:蔗糖24.84 g/L,蛋白胨12.72 g/L,无水CH₃COONa 3.95 g/L,K₂HPO₄ 2 g/L,牛肉粉5 g/L,吐温80 1 mL/L,C₆H₁₇N₃O₇ 2 g/L,MnSO₄ 0.05 g/L,MgSO₄ 0.2 g/L,蒸馏水1 L。经过优化后,抑菌圈直径达到了(23.40±1.28)mm,比初始抑菌圈直径提高了2.1倍,与预测值基本一致。     

响应面法优化异硫链霉菌LMZM利用玉米芯粉产木聚糖酶发酵培养基的研究

本研究采用响应面法对异硫链霉菌LMZM产木聚糖酶的发酵培养基进行优化。首先利用Plackett-Burman实验设计筛选出影响产酶的4个显著性因素:玉米芯粉、大豆粉、酵母粉和MgSO——4。在此基础上,研究不显著因素的最低添加量来降低生产成本,然后运用最陡爬坡法逼近最大响应值区域,最后利用中心复合设计确定显著性因素之间的交互作用及最佳组成。结果表明,玉米芯粉27.85 g/L、大豆粉16.25 g/L、酵母粉3.46 g/L、MgSO₄1.18 g/L、K₂HPO₄0.60 g/L、Na₂HPO₄0.40 g/L,木聚糖酶最大理论酶活可达117.80 U/m L,经3次平行实验验证,实际酶活平均值为116.63 U/m L与预测酶活相近,且比优化之前的酶活提高了1.28倍。      

耙齿菌EA-LJS-73产七叶皂苷培养基配方的响应面法优化

单因素实验的基础上应用响应面法对产七叶皂苷菌株耙齿菌EA-LJS-73培养基进行方法设计、优化并验证培养基方案,以提高其产七叶皂苷的能力。得到优化后的培养基(1L)如下:淀粉22.660 g,酵母浸粉4.230 g,MgSO₄ 7.600 g时,此条件下七叶皂苷浓度的理论值可达到1.116 g/L,验证实验的平均浓度为1.105 g/L,比优化前提高了35%。通过发酵动力学分析得出发酵10 d时,菌株生物量和七叶皂苷浓度达到最高,分别为11.376 g(菌体干重)和1.665 g/L。因此优化的培养基配方合理、可行。     

海藻酸钠裂解酶高产菌株的筛选、鉴定及产酶条件的优化

以海藻酸钠为唯一碳源培养基,对广西北海涠洲岛采集的马尾藻进行微生物的筛选和分离,采用 3, 5-二硝基水杨酸法 (DNS)进行海藻酸钠裂解酶相对酶活力测定,获得高产酶菌株M0101;依据形态、生理生化特征及 16S rDNA 序列分析对其进行鉴定,该菌属于弧菌属,命名为Vibrio gangliei M0101;通过单因素实验确定了菌株M0101的产酶优化方案:海藻酸钠50 g/L,(NH₄)₂SO₄ 2 g/L,K₂HPO₄ 1 g/L,NaCl 100 g/L,MgSO₄·7H₂O 1 g/L,FeSO₄·7H₂O 0.01 g/L,培养温度30 ℃,初始pH9.0,200 r/min;优化后菌株 M0101产酶最大活性可达221.90 U/mL,相较于优化前最大酶活36.32 U/mL,优化后酶活力是优化前的6.11倍。表明M0101能高效水解高浓度海藻酸钠,是强耐盐的菌株,具有大规模制备海藻酸钠裂解酶的潜力。     

蜡状芽胞杆菌促进红球菌发酵产甲壳素脱乙酰酶

以胶体甲壳素作为产酶诱导物,利用蜡状芽胞杆菌和红球菌共同发酵提高甲壳素脱乙酰酶(chitin deacetylase, CDA)的产量。通过监测2菌株混合发酵时甲壳素酶和CDA酶活力随时间的变化,验证了双菌株协同发酵产脱乙酰酶的作用。在此基础上,采用单因素实验考察两菌株配比、发酵周期、温度、接种量、装液量和摇瓶转速等因素对脱乙酰酶活力的影响,并进一步设计Plackett-Burman(PB)实验、最陡爬坡实验、中心组合设计实验和响应面优化实验,确定适宜2菌株协同发酵的工艺条件。结果显示,2菌株接种量最佳配比为2∶8,最佳产酶培养基组分(g/L):蛋白胨0.25、K₂HPO₄ 0.6、MgSO₄ 1.5、酵母浸粉3.0、NaCl 8.5、KH₂PO₄ 0.8、胶体甲壳素8.0。优化后菌株产CDA的活力约为优化前的1.96倍,研究结果可为工业化产CDA提供参考。     

产胞外葡萄糖氧化酶菌株的筛选、鉴定及酶学性质初步研究

乳酸脱氢酶（LDH）是乳酸生成途径中的关键酶,敲除乳酸脱氢酶基因,理论上可以减少甚至消除乳酸的产生,提高2,3-丁二醇的产量和得率。该研究采用体外拼接方式构建乳酸脱氢酶基因的同源线性片段ldhL-Cmr-ldhR,将其电转化至Klebisella oxytoca HD79中,通过Red同源重组技术筛选敲除成功的重组菌株,经聚合酶链式反应（PCR）、荧光定量-聚合酶链式反应（FQ-PCR）及2,3-丁二醇产量检测验证。结果表明,重组菌株2,3-丁二醇产量为40.20 g/L、转化率为0.38 g/g和生产强度为0.48 g/（L·h）,相比供试菌株分别提高了26.8%、11.8%和45.5%;而乳酸产量则由4.83 g/L下降至2.45 g/L,相比供试菌株降低了49.3%。该实验为后续进一步提高2,3-丁二醇的产量和扩大菌株选择范围奠定基础。     

Inhibition of food-related pathogenic bacteria by god-transformed Penicillium nalgiovense strains.

Two strains of Penicillium nalgiovense, which carried the god gene of Aspergillus niger and had increased glucose oxidase (GOD) activity compared with the wild-type strain, were tested for their ability to suppress the growth of certain food-related pathogenic bacteria. In contrast to the wild type, which showed no antibacterial effect when grown in mixed culture with different bacteria, the two transformed strains were highly antagonistic. The strain that expressed higher amounts of GOD in general had higher inhibitory activity. Both strains showed antibacterial activity against Listeria monocytogenes, Salmonella Enteritidis, and Staphylococcus aureus. The inhibitory activity was dependent on the glucose concentration in the medium. S. aureus was completely inhibited at 1% glucose in the presence of the higher GOD-producing transformant. In contrast, if arabinose was used as a carbon source, no inhibition occurred. If catalase was added to the medium, the inhibitory activity of the transformants was completely inactivated, indicating that the hydrogen peroxide produced was responsible for the antibacterial activity of the transformants.     

产葡萄糖氧化酶菌株的诱变筛选及遗传稳定性研究

采用平板显色初筛、Underbofler滴定法复筛及紫外-硫酸二乙酯复合诱变的方法,从临沂果园土样中得到一株产葡萄糖氧化酶（GOD）活性较好的菌株,命名为Bla-6,其产GOD酶活为（10.50±0.25） U/mL,与原始菌株LPA-4产GOD酶活（6.75 U/mL）相比提高了55.57%。结合5.8S rDNA-ITS区序列系统进化、菌落形态、生长特性、菌体形态等确定其分类地位。结果表明,所得菌株Bla-6被鉴定为黑曲霉（Aspergillus niger）,经8代传代培养后,GOD酶活维持在10.5 U/mL左右,该菌株遗传稳定性较好。     

OXIDOREDUCTASES FROM TOMATO FRUIT: INHIBITORY EFFECT OF A FUNGAL GLUCOSE OXIDASE

Glucose oxidase (GOD) produced by a strain of Penicillium (Penicillium variabile P16) was employed, in a model system, to control the activities of tomato lipoxygenase (LPO), polyphenoloxidase (PPO), and peroxidase (POD). GOD in-hibited all three enzymes, at the typical pH of tomato fruit (4.3), i.e., the LPO was inhibited up to ca. 88%, the PPO up to ca. 63% and the POD up to ca. 50%. GOD also prevented β-carotene bleaching caused by these enzymes.     

毕赤酵母组成型高效表达启动子的筛选鉴定

为获得高产量、高活性的葡萄糖氧化酶(GOD)酵母表达菌株,优化了GOD的T132S/T56V双突变编码序列,将之克隆到表达载体pGAP_k上得到表达载体pGAP_k-h₂-GOD,以pGAP启动GOD基因的表达。将pGAP_k-h₂-GOD上的pGAP启动子替换为pGCW14和pAOX1,并对pGCW14启动子进行改造,得到3种pGCW14的改造体,最终得到包括pGAP_k-h₂-GOD在内的6种不同启动子的重组表达载体。将这6种载体转化毕赤酵母GS115,建立了一种简便高效的筛选方法初步筛选出GOD重组菌株,再对筛选到的转化子进行发酵培养,测定发酵上清液的GOD酶活力,以此来比较各种启动子启动效率的强弱。结果显示:pGCW14的启动效率是pGAP的3~5倍,改造后的启动子pGCW14+G20A/C-467T(0.767)和pGCW14-UA(0.689)的启动效率较原始的pGCW14(0.574)都有明显的提高,尤其pGCW14+G20A/C-467T启动效率最高,比原始pGCW14提高了32.5%左右。与诱导型启动子pAOX1(1.187)相比,pGCW14+G20A/C-467T(1.109)的启动效率仅比其低6.6%左右。结论:改造后的启动子可望在毕赤酵母组成型高效表达的研究应用中具有一定的前景。     

黑曲霉H1-9b 葡萄糖氧化酶的分离纯化及部分性质研究

采用研磨法破碎黑曲霉H1-9b 菌丝体,经pH5.7 磷酸缓冲液抽提获得粗酶液,粗酶液经DEAE-Sepharose离子交换层析和Superdex-200 凝胶过滤层析,得到葡萄糖氧化酶纯品。该酶的比活力为30569.7U/mg,回收率为30.2%,提纯倍数为41.4 倍,分子质量为94.1kD,为单亚基酶。该酶最适温度为37℃,最适pH 值为5.7,在30～40℃温度范围内稳定性较好,在pH4.0～8.0 范围内活性稳定。以不同浓度葡萄糖为底物在最适条件下测得该酶K_m 值为30.69mmol/L,V_max 为21.88μmol/L。Ag^+、Cu²⁺、Zn²⁺ 对该酶活性有较大抑制作用。结果表明,该酶稳定性较好,有一定的应用价值。     

以黑曲霉生产面包品质改良复合酶的研究

从粮食样品中分离并筛选出 1株可同时产生α 淀粉酶 (FAA)和葡萄糖氧化酶(GOD)的黑曲霉菌株。经试验表明 ,该菌株在适宜的条件下可稳定产生FAA 2 0 0u/mL以上 ,GOD 1 5u/ g以上。产生的 2种复合酶在pH 6～ 7下可表现最高酶活力 ,酶作用的适宜温度为 3 0～ 40℃ ,40℃下保温 3 0min酶活降低 5 0 %左右 ,在 60℃以上 2种酶均基本无活性 ,适宜用作面包制作的品质改良剂     

芽孢杆菌LWM1的分离鉴定及其对蜡样芽孢杆菌的抑制作用

从土壤样品中分离出10株芽孢杆菌（Bacillus spp.）,以蜡样芽孢杆菌（Bacillus cereus）为指示菌株,采用牛津杯扩散法筛选出一株具有较强抑菌活性的菌株LWM1。综合形态学、生理生化试验、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）及16S rDNA基因序列同源性分析,菌株LWM1被初步鉴定为一株解淀粉芽孢杆菌（Bacillus amyloliquefaciens）,并研究了其在马铃薯葡萄糖水（PDW）和营养肉汤培养基（NB）中发酵上清液对蜡样芽孢杆菌的抑制效果。结果表明,PDW培养基更有利于菌株LWM1生长和抑菌物质产生。因此,可利用PDW培养基为主要底物大规模发酵制备菌株LWM1的抑菌物质。     

Activity and stability of glucose oxidase in molecular films assembled alternately with polyions

Anionic glucose oxidase (GOD) was assembled alternately with polycations, namely, poly(ethylenimine) (PEI) and poly(dimethyldiallyl-ammonium chloride) (PDDA), in the preparation of molecular films. Enzymatic activity of the films was investigated by sequential redox reaction with glucose, peroxidase (POD) and DA67 dye. The apparent activity was not influenced by substrate diffusion at up to 5 microg of immobilized GOD (at the area of 5 x 5 mm(2) x 2 faces). This is ascribed to the less dense packing of the alternate molecular film compared with Langmuir-Blodgett (LB) films. Immobilized GOD could be released into solution, and its activity was about 80% of native GOD, indicating that the immobilization did not cause significant denaturation. The enzyme activity of the GOD film was maintained for 14 weeks when stored in buffer and in air at 4 degrees C. Activity measurement after incubation at elevated temperatures showed that significant deactivation was not observed up to 50 degrees C. This shows that GOD in the film has higher thermostability than native GOD. The pH profile of the GOD activity in the film became broad and shifted towards higher pH than that of native GOD. The GOD film was also prepared by the premixing method, in which a GOD-polyion complex was assembled alternately with another oppositely-charged polyion. The enzyme activity of the alternate film obtained by premixing was much higher (maximal enhancement, 67-fold) than that of the conventionally assembled films. Better dispersion of GOD in the premixed film appears to enhance the enzyme activity.     

一株木防己拟盘多毛孢的鉴定及其次生代谢产物分析

目的:研究分离自葡萄状枝瑚菌Ramariabotrytis子实体的16072314菌株分类学地位及其次生代谢产物成分。方法:采用形态学观察结合ITS序列分析法对16072314菌株进行菌种鉴定;利用硅胶柱色谱、凝胶柱色谱、制备薄层色谱和半制备高效液相色谱对16072314菌株固体发酵产物的化学成分进行分离纯化,根据理化性质及波谱数据分析鉴定化合物。结果:经鉴定16072314菌株为木防己拟盘多毛孢Pestalotiopsis cocculi;从16072314菌株的代谢产物中分离纯化了8个化合物,分别鉴定为7-羟基香豆素,阿魏酸,麦角甾醇,对羟基苯甲醛,对羟基苯乙醇,齐墩果酸,β-谷甾醇,过氧麦角甾醇。结论:8个化合物均为首次从木防己拟盘多毛孢中分离得到,阿魏酸为首次从拟盘多毛孢属中分离得到。本研究为拟盘多毛孢属的真菌资源利用和开发奠定了基础。