铜绿假单胞菌产蛋白酶的发酵条件优化

【目的】鉴定一株来源于酱油曲能够分泌蛋白酶的铜绿假单胞菌CAU342A,优化其产蛋白酶的发酵条件。【方法】采用形态学观察、16S r RNA基因序列比对和生理生化方法鉴定菌株CAU342A;通过碳源、氮源、初始pH、温度、表面活性剂及发酵时间的单因素优化和正交试验获得最适发酵条件。【结果】菌株CAU342A被鉴定为铜绿假单胞菌(Pseudomonas aeruginosa),其最适发酵产酶条件为(质量体积比):3%酒糟,1.5%酵母浸提物,0.05%吐温-80,0.5%NaCl,0.7%K_2HPO_4,0.3%KH_2PO_4,0.04%MnSO_4,培养基初始pH 7.5,30°C培养72 h。在最适发酵条件下,该菌株最大产酶水平达到2 653.5 U/m L。蛋白酶酶谱分析表明该菌株能够产生至少4种具有蛋白酶活性的同工酶,其中两个主要酶谱带对应分子量分别为32 k D和50 k D。【结论】铜绿假单胞菌CAU342A高产蛋白酶,具有很大的工业应用潜力。     

Trametes sp. LS-10C固态发酵产漆酶培养基优化及其对双酚A的降解

漆酶是一种绿色高效的多酚氧化酶,在降解双酚A方面具有巨大潜力。为降低发酵产漆酶的成本及考察漆酶在双酚A降解中的能力,本研究以麸皮和柚皮为主要基质,优化了栓菌固态发酵产漆酶条件,对优化后获得的漆酶在双酚A降解中的应用进行了研究。结果表明,在培养基组分为：麸皮和柚皮粉比例为6:4（W/W）、固液比1:2.5（W/V）、铜离子2%（W/W）、蔗糖3%（W/W）、硝酸钾2%（W/W）、稻壳20%（W/W）的条件下,栓菌发酵产漆酶的酶活最高,发酵11d后,酶活可达到38.4U/gds。当双酚A初始浓度为10μg/mL时,在55℃条件下酶解140min后,双酚A基本降解完全。     

棘孢曲霉固态发酵柚皮产柚苷酶的条件优化

【目的】以柚皮为原料,优化棘孢曲霉利用柑橘加工副产物固态发酵柚苷酶的条件。【方法】采用高效液相色谱法检测酶活力,通过单因素试验考察固水比、装样量、接种量、温度对柚苷酶发酵的影响,用正交试验优化发酵条件。【结果】单因素试验结果的显著性分析表明培养基的固水比、装样量和培养温度对柚苷酶产量有显著性影响,而接种量影响不显著;经正交试验确定的优化条件是：固水比1:1 (质量体积比),装样量5 g/250 mL三角瓶,温度为30 °C,接种1 mL孢子悬浮液,发酵8 d。在此优化条件下,柚苷酶酶活力为8.19 IU/g干物质,比初始培养基产柚苷酶活力提高7.38倍。【结论】通过对固水比、装样量和发酵温度进行优化,大幅度提高了棘孢曲霉固态发酵柑橘加工副产物的柚苷酶产量,为柚苷酶的生产提供了一种高产发酵工艺。     

粗毛栓菌产漆酶对考马斯亮蓝的脱色降解

粗毛栓菌粗酶液经聚丙烯酰胺凝胶电泳,在CBBG－250染色后,漆酶带处有一透明圈,经纯化漆酶和CBBG－250溶液直接作用以及菌体的培养结果证实,漆酶对CBBG－250具有一定的脱色降解作用,在漆酶活力达118u/ml时,CBBG－250溶液在595nm波长的光吸收60min内下降了32．4％。该粗毛栓菌产漆酶对工业染料废水也具有一定的降解脱色作用。     

天然介体在产漆酶真菌氧化蒽和芘中的重要作用（英文稿）

【目的】研究了氧化还原介体在产漆酶真菌氧化蒽和芘的作用。【方法】通过非变性电泳和酶活力分析。【结果】发现血红密孔菌Z-1和木蹄层孔菌Z-5只产漆酶,其最大酶产量分别为11.90 U/mL和4.83 U/mL,不产木质素过氧化酶和锰过氧化物酶。木蹄层孔菌Z-5的胞外液尽管具有较低的漆酶活性,但是氧化了74.3%的蒽和12.4%的芘,高于血红密孔菌Z-1对蒽和芘的氧化率,提示天然介体可能存在于真菌胞外液中并且影响了漆酶对多环芳烃的氧化。实验进一步表明,木蹄层孔菌Z-5灭活和不灭活的超滤液以及灭活的胞外液对纯漆酶氧化多环芳烃的促进作用均大于血红密孔菌Z-1,说明木蹄层孔菌Z-5的天然介体比血红密孔菌Z-1能够更为有效地促进多环芳烃氧化。【结论】氧化还原结体在产漆酶真菌降解底物过程中发挥了重要作用,这也解释了木蹄层孔菌Z-5胞外液尽管漆酶活性不高,但是具有较大多环芳烃氧化率的原因。     

天然介体在产漆酶真菌氧化蒽和芘中的重要作用(英文)

【目的】研究氧化还原介体在产漆酶真菌氧化蒽和芘的作用。【方法】通过非变性电泳和酶活力分析。【结果】发现血红密孔菌Z-1和木蹄层孔菌Z-5只产漆酶,其最大酶产量分别为11.90 U/mL和4.83 U/mL,不产木质素过氧化酶和锰过氧化物酶。木蹄层孔菌Z-5的胞外液尽管具有较低的漆酶活性,但是氧化了74.3%的蒽和12.4%的芘,高于血红密孔菌Z-1对蒽和芘的氧化率,提示天然介体可能存在于真菌胞外液中并且影响了漆酶对多环芳烃的氧化。实验进一步表明,木蹄层孔菌Z-5灭活和不灭活的超滤液以及灭活的胞外液对纯漆酶氧化多环芳烃的促进作用均大于血红密孔菌Z-1,说明木蹄层孔菌Z-5的天然介体比血红密孔菌Z-1能够更为有效地促进多环芳烃氧化。【结论】氧化还原结体在产漆酶真菌降解底物过程中发挥了重要作用,这也解释了木蹄层孔菌Z-5胞外液尽管漆酶活性不高,但是具有较大多环芳烃氧化率的原因。     

不同搅拌器类型对齿毛菌发酵产漆酶的影响

【目的】白腐菌Cerrena unicolor Y-G07是一种不产孢子的丝状真菌,其漆酶合成与生长相偶联。应用于单细胞微生物发酵的搅拌器并不适用于白腐真菌。在此,综合考虑溶氧效果和剪切力作用,研究不同搅拌器类型对白腐菌Cerrena unicolor Y-G07发酵生产漆酶产量的影响。【方法】针对Y-G07菌株生长过程需氧量大,且对剪切力敏感的特性,订制5种不同类型的搅拌器(径向流、轴向流等),在通气已控制在设备最大量程及转速优化的前提下,研究Y-G07在发酵生产过程中使用不同类型搅拌器对菌丝生长形态、生长速度、溶氧情况、糖代谢和漆酶合成的影响。【结果】Cerrena unicolor Y-G07菌株对不同类型搅拌器产生的发酵液流态性质和剪切力敏感,表现在菌丝体的生长形态、细胞浓度差异较大,且生长周期改变,从而影响漆酶的合成。采用六折叶DT602搅拌器最有利于该菌株形成致密度合适的网状菌丝体,菌丝体细胞浓度高,断裂少、生长状况好,漆酶的单位产量可达690 U/m L,相对于普通使用的六叶平直叶搅拌器(448 U/m L)提高了54%。【结论】选择合适的搅拌器类型有利于好氧但对剪切力敏感的微生物发酵。     

南极交替单胞菌R11-5产卡拉胶酶的发酵条件优化

【背景】极地寒冷环境中发现了大量具有潜在应用前景的冷适应酶,同时也存在种类繁多的海藻多糖降解菌,因此极端环境微生物是筛选获得新颖、高效多糖降解酶的重要新源泉。由于筛选培养基通常并非野生菌发酵产酶的最优条件,为了使野生菌的产酶效率达到最高,需要对其培养条件进行优化,从而为其深入研究及开发利用提供依据。【目的】对一株产卡拉胶酶的南极菌株进行种属鉴定,并采用响应面法对该菌的发酵产酶条件进行优化。【方法】通过16SrRNA基因对产卡拉胶酶的南极菌株进行种属鉴定,采用响应面法优化南极菌株产酶发酵条件。【结果】该南极菌属于交替单胞菌属(Alteromonas),命名为交替单胞菌R11-5。发酵条件优化结果显示,7个环境因子影响交替单胞菌R11-5的产酶量。利用Design-Expert软件中的Plackett-Burman设计实验,筛选出影响交替单胞菌R11-5产酶量的4个主要因素分别为培养温度、牛肉膏浓度、卡拉胶浓度和Ca~(2+)浓度。通过Box-Behnken设计和响应面分析得到交替单胞菌R11-5最佳产酶发酵条件为:温度15.0°C,牛肉膏浓度11.0 g/L,卡拉胶浓度3.0 g/L,Ca~(2+)浓度5.0 mmol/L。优化后发酵上清液酶产量达到87.193 U/mL,与优化前相比提高了1.8倍。【结论】响应面法提高了南极交替单胞菌R11-5卡拉胶酶的产量,为其开发应用提供了科学依据。     

粗毛栓菌漆酶的诱导及其对中性染料和有机磷农药的降解

研究了不同初始培养pH和培养时间下,不同诱导剂对粗毛栓菌产漆酶诱导特性的影响及漆酶对中性染料、有机磷农药的降解.结果表明： RB-亮蓝、ABTS和邻联甲苯胺对粗毛栓菌产漆酶均有不同程度的诱导作用,其中ABTS的诱导作用最好,其诱导的最适初始pH为4.0,最佳时间为13 d;所产漆酶的适宜反应温度为38 ℃,酶在40 ℃保温20 min仍可保留786%的酶活力,稳定性较好;在ABTS介导下,漆酶降解6种中性染料的最佳温度分别为30 ℃(中性黑、中性枣红、中性桃红、甲基橙)和60 ℃(中性深黄、甲酚红),最适pH分别为6.0(中性黑)、2.0(中性枣红、中性桃红)和4.0(甲基橙、中性深黄、甲酚红),最佳降解时间分别为6 h(甲基橙、中性深黄和甲酚红)、12 h(中性桃红)和24 h(中性枣红).而漆酶降解乐果、毒死蜱、敌百虫、哒嗪硫磷4种有机磷农药的最佳温度均为25 ℃,最佳降解时间为9 h,最佳pH分别为10.0(乐果、毒死蜱、哒嗪硫磷)和8.0(敌百虫).     

响应面设计优化莱氏绿僵菌SZCY固体发酵培养条件及致病力测定

【背景】莱氏绿僵菌(Metarhizium rileyi)对新入侵我国的草地贪夜蛾(Spodoptera frugiperda)具有较强的致病力和田间流行性,因此具备深入开发的价值。【目的】优化莱氏绿僵菌SZCY固态发酵培养条件,测定所产分生孢子对草地贪夜蛾幼虫的毒力,为提高该菌株分生孢子规模化生产奠定基础。【方法】采用单因素试验确定了相对适宜的固态培养基,利用Box-Behnken响应面法优化该菌株的固态培养基和发酵参数,同时评价不同条件下该菌所产分生孢子对草地贪夜蛾幼虫的毒力。【结果】去颖稻谷(rice)为莱氏绿僵菌SZCY菌株固相产孢最佳载体。培养温度、光周期及酵母浸粉含量是影响莱氏绿僵菌SZCY固态发酵产孢量的主要因素。莱氏绿僵菌SZCY固态发酵最佳工艺参数为温度22.83℃、光周期18.68 h L:5.32 h D、酵母浸粉4.98 g/100 g,在此条件下,莱氏绿僵菌在去颖稻谷固态培养基上的产孢量为5.65×10¹⁰孢子/g,用其制备浓度为10⁷孢子/mL的孢子悬浮液,对草地贪夜蛾3龄幼虫的LT₅₀为3....     

木薯粉同步糖化发酵(SSF)产丁二酸

【目的】通过优化产琥珀酸放线杆菌GXAS137同步糖化发酵木薯粉产丁二酸的发酵培养基,提高丁二酸产量,降低生产成本。【方法】在单因素试验的基础上,先利用Plackett-Burman试验设计筛选出影响丁二酸发酵的重要参数,再采用正交试验确定重要参数的最佳水平。【结果】价格低廉玉米浆可用作氮源,影响丁二酸产量的重要参数是木薯粉、玉米浆、碱式碳酸镁和糖化酶浓度。最佳条件为(g/L):木薯粉100,玉米浆14,糖化酶2.0 AGU/g底物,碱式碳酸镁75。优化后丁二酸产量达到69.31 g/L,丁二酸得率为90.01%,生产强度为1.44 g/(L·h)。与初始条件(52.34 g/L)相比,丁二酸浓度提高了32.42%。并利用1.3 L发酵罐对SSF与SHF两种发酵工艺进行了比较,SSF丁二酸产量(72.21 g/L)远高于SHF(56.86 g/L)。【结论】产琥珀酸放线杆菌同步糖化发酵木薯粉丁二酸产量高,生产成本低,具有较好的工业化应用前景。     

羊源丁酸梭菌HDRyYB1发酵工艺的优化

【目的】目前,国内外鲜有关于羊源丁酸梭菌的报道。本课题选用羊源丁酸梭菌HDRy YB1为研究对象,对其发酵工艺进行优化,为该菌株作为饲料添加剂应用于畜牧业生产奠定基础。【方法】采用Plackett-Burman(PB)试验设计法和响应面法分析并优化显著影响HDRy YB1菌株发酵液中芽胞数的培养基成分。【结果】发酵培养基中的面粉浓度、鱼粉浓度和米粉浓度显著影响发酵液中的芽胞数,优化后的发酵培养基组分(质量体积比)为:面粉3.72%、鱼粉0.90%、米粉3.96%、酵母粉0.60%、Na Cl 0.19%、Mg SO4·7H2O 0.19%、KH2PO4 0.01%、Na HCO3 0.01%、Ca CO3 0.48%;培养参数为:37°C,初始p H为7.2-7.4,瓶装量100/250,接种量3%。在此条件下,HDRy YB1菌株发酵完全(18 h)的芽胞数为1.478×108 CFU/m L,是优化前的2.7倍。【结论】HDRy YB1菌株发酵培养基得到了优化,优化后的培养基可用于后期的扩大发酵试验,验证其在实践生产中的应用价值。     

Simultaneous saccharification and fermentation of acid-pretreated rapeseed meal for succinic acid production using Actinobacillus succinogenes

Rapeseed meal was evaluated for succinic acid production by simultaneous saccharification and fermentation using Actinobacillus succinogenes ATCC 55618. Diluted sulfuric acid pretreatment and subsequent hydrolysis with pectinase was used to release sugars from rapeseed meal. The effects of culture pH, pectinase loading and yeast extract concentration on succinic acid production were investigated. When simultaneous saccharification and fermentation of diluted acid pretreated rapeseed meal with a dry matter content of 12.5% (w/v) was performed at pH 6.4 and a pectinase loading of 2% (w/w, on dry matter) without supplementation of yeast extract, a succinic acid concentration of 15.5 g/L was obtained at a yield of 12.4 g/100g dry matter. Fed-batch simultaneous saccharification and fermentation was carried out with supplementation of concentrated pretreated rapeseed meal and pectinase at 18 and 28 h to yield a final dry matter content of 20.5% and pectinase loading of 2%, with the succinic acid concentration enhanced to 23.4 g/L at a yield of 11.5 g/100g dry matter and a productivity of 0.33 g/(Lh). This study suggests that rapeseed meal may be an alternative substrate for the efficient production of succinic acid by A. succinogenes without requiring nitrogen source supplementation.     

响应面法优化多杀菌素发酵培养基的研究

采用响应面分析方法,对刺糖多孢茵（Saccharopolyspora spinosa）H-2产多杀菌素的发酵培养基进行优化研究。运用单因子试验筛选出葡萄糖和棉籽粉为最适碳源和氮源,通过Plack—ett—Burman设计试验,对影响发酵培养基的8个相关因子进行评估并筛选出具有显著效应的4个因子：葡萄糖、棉籽粉、黄豆饼粉及玉米浆。通过最陡爬坡实验逼近以上4个因子的最大响应区域后,采用Box-Behnken响应面分析法,确定发酵产多杀菌素最佳培养基为葡萄糖64．5g,麦芽糖20g,玉米浆2g,大豆油40g,棉籽粉25g,黄豆饼粉2．4g,蛋白胨25g,CaCO35g,定容至1L,pH7．0。培养基优化后多杀菌素产量由278．1mg／L提高到508．7mg／L,比初始多杀茵素产量提高了1．83倍。     

Production of carboxylic acids from hydrolyzed corn meal by immobilized cell fermentation in a fibrous-bed bioreactor

Corn meal hydrolyzed with amylases was used as the carbon source for producing acetic, propionic, and butyric acids via anaerobic fermentations. In this study, corn meal, containing 75% (w/w) starch, 20% (w/w) fibers, and 1.5% (w/w) protein, was first hydrolyzed using amylases at 60 degrees C. The hydrolysis yielded approximately 100% recovery of starch converted to glucose and 17.9% recovery of protein. The resulting corn meal hydrolyzate was then used, after sterilization, for fermentation studies. A co-culture of Lactococcus lactis and Clostridium formicoaceticum was used to produce acetic acid from glucose. Propionibacterium acidipropionici was used for propionic acid fermentation, and Clostridium tyrobutylicum was used for butyric acid production. These cells were immobilized on a spirally wound fibrous matrix packed in a fibrous-bed bioreactor (FBB) developed for multi-phase biological reactions or fermentation. The bioreactor was connected to a stirred-tank fermentor that provided pH and temperature controls via medium circulation. The fermentation system was operated at the recycle batch mode. Temperature and pH were controlled at 37 degrees C and 7.6, respectively, for acetic acid fermentation, 32 degrees C and 6.0, respectively, for propionic acid fermentation, and 37 degrees C and 6.0, respectively, for butyric acid production. The fermentation demonstrated a yield of approximately 100% and a volumetric productivity of approximately 1 g/(1 h) for acetic acid production. The propionic acid fermentation achieved an approximately 60% yield and a productivity of 2.12 g/(1 h), whereas the butyric acid fermentation obtained an approximately 50% yield and a productivity of 6.78 g/(1 h). These results were comparable to, or better than those fermentations using chemically defined media containing glucose as the substrate, suggesting that these carboxylic acids can be efficiently produced from direct fermentation of corn meal hydrolyzate. The corn fiber present as suspended solids in the corn meal hydrolyzate did not cause operating problem to the immobilized cell bioreactor as is usually encountered by conventional immobilized cell bioreactor systems. It is concluded that the FBB technology is suitable for producing value-added biochemicals directly from agricultural residues or commodities such as corn meal.     

Productivity of laccase in solid substrate fermentation of selected agro-residues by Pycnoporus sanguineus

A comparative study on solid substrate fermentation (SSF) of sago 'hampas', oil palm frond parenchyma tissue (OPFPt) and rubberwood sawdust with Pycnoporus sanguineus for laccase production was carried out. Optimal mycelial growth of Pyc. sanguineus was observed on all the substrates studied over a 21 days time-course fermentation. Laccase productivity was highest during degradation of sago 'hampas' and OPFPt and a range from 7.5 to 7.6 U/g substrate on the 11th day of fermentation compared to degradation of rubberwood sawdust with a maximum laccase productivity of 5.7 U/g substrate on day 11 of SSF. Further optimization of laccase production was done by varying the inoculum age, density and nitrogen supplementation. SSF of OPFPt by Pyc. sanguineus gave maximum productivity of laccase of 46.5 U/g substrate on day 6 of fermentation with a 30% (w/w) of 4 weeks old inoculum and 0.92% nitrogen in the form of urea supplemented in the substrate. The extraction of laccase was also optimized in this study. Recovery of laccase was fourfold higher at 30.6 U/g substrate on day 10 of SSF using unadjusted tap water at pH 8.0 as extraction medium at 25+/-2 degrees C compared to laccase recovery of 7.46 U/g substrate using sodium acetate buffer at pH 4.8 at 4 degrees C. Further optimization showed that laccase recovery was increased by 50% with a value of 46.5 U/g substrate on day 10 of SSF when the extraction medium was tap water adjusted to pH 5.0 at 25+/-2 degrees C.     

樟绒枝霉(Malbranchea cinnamomea)固体发酵产木聚糖酶的发酵条件优化

[目的] 研究樟绒枝霉(Malbranchea cinnamomea) CAU521利用农业废弃物固体发酵产木聚糖酶的发酵条件.[方法]采用单因素试验法优化影响菌株产酶的各个条件,包括碳源种类、氮源种类、初始pH、初始水分含量、培养温度及发酵时间共6个因素.[结果]获得的最佳产酶条件为:稻草为发酵碳源、2％(W/W)的酵母提取物为氮源、初始pH 7.0、初始水分含量80％和发酵温度45℃.在此条件下发酵6d后木聚糖酶的酶活力达到13 120 U/g干基碳源.[结论]樟绒枝霉固体发酵产木聚糖酶的产酶水平高,生产成本低,具有潜在的工业化应用前景.     

青霉PT95菌固态发酵产生类胡萝卜素的研究

本文对青霉Ｐｅｎｉｃｉｌｌｉｕｍｓｐ．ＰＴ９５菌株在固态发酵条件下菌核内产生类胡萝卜素进行了初步研究。结果表明,在３种固态发酵培养基中,玉米粉培养基（ＳＭＡ）比麸皮２基和棉籽壳培养基更适合于ＰＴ９５菌株固态发酵产生类胡萝卜素。为了增加菌核干重和提高类胡萝卜素产率,ＳＭＡ中需要添加氮源、碳源和植物油。在所度的各种氮、碳源中,以硝酸钠和麦芽糖效果最佳。通过我试验确定了在培养基盐溶液中添加硝酸钠３ｇ／Ｌ     

Direct Bio-Utilization of Untreated Rapeseed Meal for Effective Iturin A Production by Bacillus subtilis in Submerged Fermentation

The feasibility of using untreated rapeseed meal as a nitrogen source for iturin A production by Bacillus subtilis 3–10 in submerged fermentation was first evaluated by comparison with two different commercial nitrogen sources of peptone and ammonium nitrate. A significant promoting effect of rapeseed meal on iturin A production was observed and the maximum iturin A concentration of 0.60 g/L was reached at 70 h, which was 20% and 8.0 fold higher than that produced from peptone and ammonium nitrate media, respectively. It was shown that rapeseed meal had a positive induction effect on protease secretion, contributing to the release of soluble protein from low water solubility solid rapeseed meal for an effective supply of available nitrogen during fermentation. Moreover, compared to raw rapeseed meal, the remaining residue following fermentation could be used as a more suitable supplementary protein source for animal feed because of the great decrease of major anti-nutritional components including sinapine, glucosinolate and its degradation products of isothiocyanate and oxazolidine thione. The results obtained from this study demonstrate the potential of direct utilization of low cost rapeseed meal as a nitrogen source for commercial production of iturin A and other secondary metabolites by Bacillus subtilis.     

Ethanol production by Saccharomyces cerevisiae in biofilm reactors

Biofilms are natural forms of cell immobilization in which microorganisms attach to solid supports. At ISU, we have developed plastic composite-supports (PCS) (agricultural material (soybean hulls or oat hulls), complex nutrients, and polypropylene) which stimulate biofilm formation and which supply nutrients to the attached microorganisms. Various PCS blends were initially evaluated in repeated-batch culture-tube fermentation with Saccharomyces cerevisiae (ATCC 24859) in low organic nitrogen medium. The selected PCS (40% soybean hull, 5% soybean flour, 5% yeast extract-salt and 50% polypropylene) was then used in continuous and repeated-batch fermentation in various media containing lowered nitrogen content with selected PCS. During continuous fermentation, S. cerevisiae demonstrated two to 10 times higher ethanol production in PCS bioreactors than polypropylene-alone support (PPS) control. S. cerevisiae produced 30 g L⁻¹ ethanol on PCS with ammonium sulfate medium in repeated batch fermentation, whereas PPS-control produced 5 g L⁻¹ ethanol. Overall, increased productivity in low cost medium can be achieved beyond conventional fermentations using this novel bioreactor design. Received 20 May 1997/ Accepted in revised form 29 August 1997