汉逊德巴利酵母发酵葡萄糖生产D-阿拉伯糖醇

从378株耐高渗酵母中,筛选到1株由葡萄糖发酵高产D-阿拉伯糖醇的酵母。通过生理生化和分子生物学的鉴定,证实该菌株为Debaryomyces hansenii,保藏编号CICIM Y 0504。研究该酵母摇瓶发酵的主要影响因素,确定其摇瓶发酵条件为:葡萄糖200 g/L,酵母膏10 g/L,初始pH值3,装液量20 mL/250 mL,温度30℃。在此条件下发酵120 h,D-阿拉伯糖醇浓度达90.37 g/L,转化率45.18%。在15 L发酵罐对该酵母进行扩大培养,结果表明,初始葡萄糖浓度200 g/L的分批发酵产D-阿拉伯糖醇64.07 g/L,转化率33.94%;葡萄糖浓度控制在30～50 g/L的分批补料发酵产D-阿拉伯糖醇125 g/L,转化率37.5%。研究结果对葡萄糖发酵生产D-阿拉伯糖醇工业化的实现具有重要启示。     

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。     

一株产钙调磷酸酶抑制剂链霉菌CZW-15的

【目的】筛选产钙调磷酸酶抑制剂的放线菌菌株。【方法】通过酵母报告基因(CDRE::LacZ)高通量筛选模型与人体钙调磷酸酶试剂盒检测相结合的方法,从土壤放线菌中筛选产钙调磷酸酶抑制剂的菌株;根据形态和培养特征、生理生化实验以及16S rRNA基因序列比对进行菌株鉴定;采用单因素实验和正交实验对发酵条件进行优化。【结果】从土壤中筛选得到一株产高活性钙调磷酸酶抑制剂的放线菌CZW-15,经鉴定属于链霉菌属(Streptomyces)。酵母报告基因检测显示其发酵上清液对依赖酵母钙调磷酸酶的报告基因表达抑制活性IC50为7.4 mL/L,而对照药物FK506的IC50为65 μg/L。该菌的最佳摇瓶发酵配方为(%)：玉米粉2、酵母粉0.1、K2HPO4 0.04、MgSO4 0.04、NaCl 0.05、FeSO4 0.001;发酵条件为：初始pH 7.5,温度28 °C,转速180 r/min,装液量50 mL/250 mL,接种量4%,培养时间4 d。优化后1.5 μL发酵上清液对依赖酵母钙调磷酸酶的报告基因表达抑制活性达到117%,比优化前提高4.5倍。【结论】CZW-15属于链霉菌属,该菌产生高活性的抑制酵母钙调磷酸酶的活性物质。     

一株红景天内生细菌的筛选及初步研究

【目的】从红景天根部筛选并鉴定一株产酪醇的细菌,初步研究其产酪醇特性,为寻找红景天替代资源提供新途径。【方法】用NA培养基从大花红景天根部中分离内生细菌,通过薄层层析(TLC)、高效液相色谱(HPLC)、气相色谱-质谱联用(GC-MS)筛选出产量最大的菌株,经菌落形态分析、革兰氏染色分析及16S rRNA基因序列分析其分类学地位。单因素实验确定初始pH、培养温度、发酵时间及接种量对菌株产酪醇活力的影响。【结果】从大花红景天根部分离出14株内生细菌,其中8株能产酪醇,筛选出酪醇产量最大的菌株B3,经菌落形态分析、革兰氏染色分析及16S rRNA基因序列分析初步鉴定为水生拉恩氏菌(Rahnella aquatilis)。研究其发酵条件,其最适pH为6.0,最适温度为32 °C,最佳发酵时间为42 h,最佳接种量为15%。在最适发酵条件下,用改良NA培养基发酵,B3菌株酪醇的产量为15.68 mg/L。【结论】B3菌株是一株具有产酪醇能力的细菌,在最适发酵条件下酪醇产量达到15.68 mg/L,具有潜在的开发价值。     

高产几丁质酶巴伦葛兹类芽孢杆菌的筛选和发酵条件优化

【目的】鉴定一株来源于中国南海海水样能够分泌多种胞外几丁质酶的类芽孢杆菌CAU904,并优化其产几丁质酶的发酵条件。【方法】采用形态学观察、16S r DNA序列比对及生理生化实验鉴定;通过碳源、氮源、温度、初始p H、表面活性剂种类以及发酵时间的单因素优化实验获得最佳发酵条件。【结果】菌株CAU904被鉴定为巴伦葛兹类芽孢杆菌(Paenibacillus barengoltzii),其最优发酵产酶条件为:0.5%胶体几丁质,0.2%酵母浸提物,0.1%吐温-80,培养基初始p H 7.0,45°C培养72 h。在最优发酵条件下,该菌株最大产酶水平达到8.2 U/m L,比优化前提高了5.4倍。几丁质酶的酶谱分析表明该菌株能够产生多达11种具有几丁质水解活性的同工酶,其中主要酶谱带对应分子量分别为54、47和38 k D。【结论】实验结果为巴伦葛兹类芽孢杆菌几丁质酶的分离纯化和酶的应用提供了基础。     

产黑色素海洋放线菌的分离、鉴定和产色素条件优化

【目的】从连云港高公岛沿岸海底泥样中分离筛选到一株产黑色素的海洋放线菌。【方法】对筛选得到的菌株HT-18所产的色素进行紫外-可见吸收光谱、红外光谱分析。通过形态特征、培养特征、生理生化测定以及16S rRNA序列的系统发育学分析确定菌株HT-18的分类学地位。采用单因素对产色素发酵条件进行优化。【结果】菌株HT-18所产色素为黑色素。鉴定该菌株属链霉菌属(Streptomyces)。最佳产色素条件为:初始pH7.0,装液量为70/250 mL,温度31°C,发酵时间为3 d。【结论】海洋链霉菌HT-18是一株具有研究和应用潜力的产黑色素菌株。     

中条山野生葡萄产香酵母的筛选及其混菌发酵对葡萄酒香气成分的影响

【背景】产香酵母可赋予葡萄酒独特的香气,因此,分离筛选优良产香酵母对酿造具有地域风味的特色葡萄酒具有重要意义。【目的】从中条山野生葡萄中筛选产香酵母,进行种群鉴定和生理生化特性研究,并将其应用于葡萄酒发酵过程,研究其对葡萄酒香气成分的影响。【方法】采用稀释涂布平板法从中条山野葡萄中分离筛选酵母菌,对其进行分子生物学鉴定。优选其中具有显著香气的产香酵母,与酿酒酵母F15进行混合发酵,采用气相色谱质谱联用(gas chromatograph-mass spectrometer,GC-MS)对香气成分进行分析,采用半定量法测定香气成分含量。【结果】共分离获得各种菌株13株,26S rRNA基因D1/D2区序列分析表明它们分布于Issatchenkia、Torulaspora、Pichia、Saccharomyces和Rhodotorula等5个不同属内。优选其中一株香气较为浓郁的酵母菌株Issatchenkia orientalis strain XS-6开展研究,结果发现该菌株最高耐受乙醇浓度为8%,最高耐受NaCl浓度为6%,最适生长温度为38℃。与酿酒酵母F15混菌发酵的葡萄酒中共检测出31种香气成分。香气物质总含量较单菌发酵增加19.8%,其中11种香气成分含量增加明显,尤其是具有玫瑰香气的苯乙醇。醇类与酯类物质含量较单菌发酵增加19.6%,并发现了香草酸乙酯(ethyl vanillate)、邻苯二甲酸二丁酯(dibutyl phthalate)等7种新的酯类物质。【结论】产香酵母XS-6对乙醇、NaCl、温度等具有良好的耐受性,而且与酿酒酵母F15混菌发酵对西拉葡萄酒香气成分具有明显的影响,可能在改善葡萄酒风味方面具有潜在的应用价值。     

产肌醇毕赤酵母细胞工厂的优化

【背景】肌醇是一种B族维生素,广泛应用于食品、医药、饲料等领域。微生物发酵法是最具前景的肌醇生产方法,但使用大肠杆菌生产的肌醇在食品及医药领域中的使用受到限制。毕赤酵母作为生物安全菌株是工业上生产异源蛋白的良好宿主,其本身含有天然的肌醇合成途径,具有被改造成为高效生产肌醇细胞工厂的潜力。【目的】通过代谢工程改造毕赤酵母工程菌株,降低副产物的生成并提高肌醇的产量。【方法】以实验室前期构建的产肌醇毕赤酵母工程菌株为出发菌株,确定副产物阿拉伯糖醇、核糖醇和甘露糖合成相关基因。通过关键基因敲除、发酵液中葡萄糖浓度控制降低副产物的产量。通过过表达甘油转运蛋白、甘油激酶和甘油-3-磷酸脱氢酶基因实现产肌醇毕赤酵母对甘油和葡萄糖的共利用,得到重组菌Z10。经过发酵条件优化,进一步提高Z10的肌醇产量。【结果】在最优条件下,重组菌Z10的肌醇产量达到36.7 g/L,是目前酵母类细胞工厂生产肌醇的最高值,副产物总产量与出发菌株相比降低了63.1%。【结论】在毕赤酵母中建立了降低阿拉伯糖醇、核糖醇和甘露糖合成的有效策略,并通过甘油、葡萄糖共利用及相对应的发酵条件优化提高了肌醇产量,为肌醇及其他高价值生物...     

产胞外多糖酵母菌株的筛选鉴定及发酵产糖

【目的】微生物胞外多糖大多具有良好的功能和特性,但对酵母胞外多糖的研究甚少。本研究从自然界中筛选出产胞外多糖的酵母菌株,并对其发酵产糖条件进行初步研究。【方法】利用平板涂布法从自然界中分离得到酵母菌株,苯酚硫酸法测定菌株胞外多糖的产量,筛选出胞外多糖高产菌株,并对其进行5.8SrDNA分类鉴定,最后优化其产糖培养基组成。【结果】对从葡萄、蜜枣、土壤样品中分离得到的132株酵母进行筛选,最终得到3株高产胞外多糖的酵母菌株Z14、Z20和L25。经5.8S rDNA序列测定及系统发育分析,从左优红葡萄中分离得到的Z14和Z20与东方伊萨酵母(Issatchenkia orientalis)处于同一分支,相似性达到99%以上;从落叶松近表层土壤分离得到的L25与土生隐球酵母(Cryptococcus humicolus)处于同一分支,相似性为98.8%。经优化,利于Z20胞外多糖合成的最优发酵培养基配方为:葡萄糖8%,(NH4)SO40.2%,KH2PO40.1%,酵母浸粉0.1%,CaCl20.01%。在初始pH6.0,发酵温度28℃,摇床转数160 r/min条件下,在此培养基中发酵4 d后胞外多糖产量可达2.046 g/L,比复筛时的产量1.137 g/L提高了79.9%。【结论】文献已报道某些属的酵母可以生产胞外多糖,经本文研究发现Issatchenkia属的酵母也可以合成胞外多糖,并且改变基础产糖培养基的成分可以显着提高Z20胞外多糖的产量。     

酵母产γ-氨基丁酸发酵条件的研究

目的：研究酵母产γ-氨基丁酸的发酵条件,提高其产γ-氨基丁酸的能力。方法：以高产γ-氨基丁酸的酵母突变株为材料,通过单因素实验研究培养温度、摇床转速、接种量、种龄和培养时间等条件对菌株发酵生产γ-氨基丁酸的影响。结果：最适发酵条件为：培养温度30℃,摇床转速220 rpm,接种量4%,种龄为2d的种子菌,培养时间4d。在此发酵条件下,变异菌发酵液中γ-氨基丁酸含量高达2.588 g.L-1,较优化前提高了53%。结论：发酵条件的优化,提高了菌株产γ-氨基丁酸的能力。     

Novel enzymatic method for the production of xylitol from D-arabitol by Gluconobacter oxydans

Microorganisms capable of producing xylitol from D-arabitol were screened for. Of the 420 strains tested, three bacteria, belonging to the genera Acetobacter and Gluconobacter, produced xylitol from D-arabitol when intact cells were used as the enzyme source. Among them, Gluconobacter oxydans ATCC 621 produced 29.2 g/l xylitol from 52.4 g/l D-arabitol after incubation for 27 h. The production of xylitol was increased by the addition of 5% (v/v) ethanol and 5 g/l D-glucose to the reaction mixture. Under these conditions, 51.4 g/l xylitol was obtained from 52.4 g/l D-arabitol, a yield of 98%, after incubation for 27 h. This conversion consisted of two successive reactions, conversion of D-arabitol to D-xylulose by a membrane-bound D-arabitol dehydrogenase, and conversion of D-xylulose to xylitol by a soluble NAD-dependent xylitol dehydrogenase. Use of disruptants of the membrane-bound alcohol dehydrogenase genes suggested that NADH was generated via NAD-dependent soluble alcohol dehydrogenase.     

Very high gravity ethanol fermentation by flocculating yeast under redox potential-controlled conditions

ABSTRACT: BACKGROUND: Very high gravity (VHG) fermentation using medium in excess of 250 g/L sugars for more than 15 % (v) ethanol can save energy consumption, not only for ethanol distillation, but also for distillage treatment; however, stuck fermentation with prolonged fermentation time and more sugars unfermented is the biggest challenge. Controlling redox potential (ORP) during VHG fermentation benefits biomass accumulation and improvement of yeast cell viability that is affected by osmotic pressure and ethanol inhibition, enhancing ethanol productivity and yield, the most important techno-economic aspect of fuel ethanol production. RESULTS: Batch fermentation was performed under different ORP conditions using the flocculating yeast and media containing glucose of 201 [PLUS-MINUS SIGN] 3.1, 252 [PLUS-MINUS SIGN] 2.9 and 298 [PLUS-MINUS SIGN] 3.8 g/L. Compared with ethanol fermentation by non-flocculating yeast, different ORP profiles were observed with the flocculating yeast due to the morphological change associated with the flocculation of yeast cells. When ORP was controlled at [MINUS SIGN]100 mV, ethanol fermentation with the high gravity (HG) media containing glucose of 201 [PLUS-MINUS SIGN] 3.1 and 252 [PLUS-MINUS SIGN] 2.9 g/L was completed at 32 and 56 h, respectively, producing 93.0 [PLUS-MINUS SIGN] 1.3 and 120.0 [PLUS-MINUS SIGN] 1.8 g/L ethanol, correspondingly. In contrast, there were 24.0 [PLUS-MINUS SIGN] 0.4 and 17.0 [PLUS-MINUS SIGN] 0.3 g/L glucose remained unfermented without ORP control. As high as 131.0 [PLUS-MINUS SIGN] 1.8 g/L ethanol was produced at 72 h when ORP was controlled at [MINUS SIGN]150 mV for the VHG fermentation with medium containing 298 [PLUS-MINUS SIGN] 3.8 g/L glucose, since yeast cell viability was improved more significantly. CONCLUSIONS: No lag phase was observed during ethanol fermentation with the flocculating yeast, and the implementation of ORP control improved ethanol productivity and yield. When ORP was controlled at [MINUS SIGN]150 mV, more reducing power was available for yeast cells to survive, which in turn improved their viability and VHG ethanol fermentation performance. On the other hand, controlling ORP at [MINUS SIGN]100 mV stimulated yeast growth and enhanced ethanol production under the HG conditions. Moreover, the ORP profile detected during ethanol fermentation with the flocculating yeast was less fluctuated, indicating that yeast flocculation could attenuate the ORP fluctuation observed during ethanol fermentation with non-flocculating yeast.     

海洋来源链霉菌MY0504产纤溶酶的发酵条件优化

【目的】以发酵液纤溶酶活力为指标,优化海洋来源的链霉菌菌株MY0504的发酵条件。【方法】在菌株生长曲线及单因素试验基础上,采用Plackett-Burman设计筛选影响纤溶酶活性的主要因素,进一步用最陡爬坡试验及Box-Behnken中心组合设计法优化发酵条件。【结果】纤溶酶活性最高的发酵条件为:葡萄糖21.68 g/L,酵母粉25.31 g/L,NaCl5.0 g/L,K_2HPO_4·3H_2O3.0 g/L,MgSO_4·7H_2O 0.5 g/L,FeSO_4·7H_2O 0.02 g/L,装液量50 mL(250 mL摇瓶),接种量10%(体积比),初始pH 7.5,温度24°C,转速200 r/min,培养时间4.5 d。发酵液纤溶酶活性可达2 190.6 U/mL。【结论】确定了MY0504菌株产纤溶酶的最优发酵条件,为该酶的进一步分离纯化及性质研究奠定基础。     

Production of erythritol from glucose by an osmophilic mutant of Candida magnoliae

Candida magnoliae and its mutants were analyzed to produce erythritol from glucose with high yield and productivity. One mutant, M2, showed higher erythritol conversion yield and productivity than the wild strain. The osmophilic mutant produced 25 g erythritol l^–1 after 83 h of a flask culture in a medium containing 10% (w/v) glucose, corresponding to a 25% increase in erythritol and a 30% increase in erythritol productivity compared with the wild type. The fermentation properties were further improved by cultivating the osmophilic mutant in a fermenter containing 20% (w/v) glucose medium with 0.54 g l^–1 h^–1 of erythritol productivity and 43% of erythritol conversion yield based on glucose.     

Production of D-arabitol by Metschnikowia reukaufii AJ14787

A potent producer of D-arabitol was isolated by screening of natural sources and identified as Metschnikowia reukaufii AJ14787. Resting cells of this strain can efficiently produce D-arabitol from D-glucose with a weight yield of more than 60%, and can also produce D-arabitol from several other types of sugars such as polyols, ketoses, and aldoses. To improve productivity, various culture conditions such as temperature and the concentrations of D-glucose and nitrogen sources were examined. Under optimal conditions, 206 g/l of D-arabitol was produced from D-glucose with a weight yield of 52% in 100 hours.     

D-arabitol metabolism in Candida albicans: construction and analysis of mutants lacking D-arabitol dehydrogenase.

Candida albicans produces large amounts of the acyclic pentitol D-arabitol in culture and in infected animals and humans, and most strains also grow on minimal D-arabitol medium. An earlier study showed that the major metabolic precursor of D-arabitol in C. albicans was D-ribulose-5-PO4 from the pentose pathway, that C. albicans contained an NAD-dependent D-arabitol dehydrogenase (ArDH), and that the ArDH structural gene (ARD) encoded a 31-kDa short-chain dehydrogenase that catalyzed the reaction D-arabitol + NAD <=> D-ribulose + NADH. In the present study, we disrupted both ARD chromosomal alleles in C. albicans and analyzed the resulting mutants. The ard null mutation was verified by Southern hybridization, and the null mutant's inability to produce ArDH was verified by Western immunoblotting. The ard null mutant grew well on minimal glucose medium, but it was unable to grow on minimal D-arabitol or D-arabinose medium. Thus, ArDH catalyzes the first step in D-arabitol utilization and a necessary intermediate step in D-arabinose utilization. Unexpectedly, the ard null mutant synthesized D-arabitol from glucose. Moreover, 13C nuclear magnetic resonance studies showed that the ard null mutant and its wild-type parent synthesized D-arabitol via the same pathway. These results imply that C. albicans synthesizes and utilizes D-arabitol via separate metabolic pathways, which was not previously suspected for fungi.     

代谢工程改造大肠杆菌合成丁二酸及发酵罐放大工艺

【背景】Escherichia coli AFP111发酵生产丁二酸时大量副产乙酸,丁二酸得率低。【目的】代谢工程改造EscherichiacoliAFP111,提高丁二酸得率,降低副产物乙酸的生成,建立100 L规模的丁二酸发酵工艺。【方法】一步同源重组敲除乙酸合成途径关键酶基因,改造丁二酸合成途径关键酶启动子实现过表达;单因素优化5L发酵罐培养条件。【结果】敲除乙酸产生途径编码乙酸激酶和磷酸转乙酰酶的基因ackA-pta、苏氨酸脱羧酶和2-酮丁酸甲酸裂解酶的基因tdcDE获得SX02菌株,摇瓶发酵条件下其乙酸产量下降了53.42%,丁二酸得率提高9.85%。在SX02菌株基础上,经启动子改造过表达编码葡萄糖激酶的基因glk后获得菌株SX03,其Glk酶活性提高3.66倍,乙酸产量下降了31.62%,丁二酸得率提高8.28%。SX03菌株发酵生产丁二酸在5 L发酵罐进行放大,其乙酸产量为3.97 g/L,丁二酸得率为1.62 mol/mol葡萄糖,相比出发菌株的乙酸产量下降了75.76%,丁二酸得率提高19.12%。在5L发酵罐上对比研究了中和剂Na2CO3和NaOH混合液替换碱式MgCO3的发酵效果,并优化了发酵pH、搅拌转速和葡萄糖浓度,获得如下最适发酵条件:pH6.8,搅拌转速250r/min,葡萄糖100g/L,发酵结束时乙酸产量为2.24 g/L,丁二酸得率为1.66 mol/mol葡萄糖。中和剂替换优化后乙酸产量下降了20.65%,丁二酸得率提高2.47%。菌株SX03发酵工艺进一步在100 L发酵罐上实现放大,其乙酸产量为1.91 g/L,丁二酸得率为1.30 mol/mol葡萄糖。【结论】通过代谢工程改造的大肠杆菌,其副产物乙酸含量显著下降,丁二酸得率提高,并在5 L和100 L发酵罐上实现了工艺放大,展现出较大的工业化利用潜力。     

Scale-up of erythritol production by an osmophilic mutant of Candida magnoliae

Erythritol production by an osmophilic mutant of Candida magnoliae was performed in fermentations of up 50 l to develop an optimized commercial process. By simultaneous feeding glucose and yeast extract, erythritol productivity of 1.2 g l(-1) h(-1) was reached giving 200 g erythritol l(-1) with a yield of 0.43 g g(-1).     

黑曲霉阿魏酸酯酶在毕赤酵母中的组成型表达

【目的】实现黑曲霉来源的阿魏酸酯酶在毕赤酵母(Pichia pastoris GS115)中的组成型表达。【方法】以黑曲霉(Aspergillus niger)基因组为模板,经重叠延伸PCR扩增得到阿魏酸酯酶基因(AnfaeA),将其与载体pGAP9K相连,构建重组表达载体p GAP9KAnfae A,经SalI线性化后电转入毕赤酵母GS115中,得到重组菌株。高效液相色谱法测定发酵液中阿魏酸酯酶活力,并对重组菌进行了发酵优化。【结果】克隆得到783 bp的阿魏酸酯酶编码基因并实现了其在毕赤酵母中的组成型表达。重组菌发酵84 h后,上清液中酶活达5.72±0.10 U/m L。重组酶(reAnfaeA)经分离纯化后比酶活为59.75 U/mg,大小约为40 k D。发酵优化结果为:葡萄糖40.0 g/L,蛋白胨10.0 g/L,酵母膏30.0 g/L,CaCO_3 0.2 g/L,种龄28 h,接种量3%(体积比),装液量50 m L/250 m L。在此条件下发酵培养,酶活达15.60±0.23 U/m L。【结论】阿魏酸酯酶在毕赤酵母中的组成型表达,对研究毕赤酵母组成型表达系统和阿魏酸酯酶的发酵生产具有一定的借鉴意义。