维生素C二步发酵中巨大芽孢杆菌对氧化葡萄糖酸杆菌生长和产酸的影响

通过测定氧化葡萄糖酸杆菌转化L－山梨糖中成ZKGA的细胞酶活性、摇瓶发酵及中长变化,研究了Vc:步发酵中巨大茅孢杆菌对氧化葡萄糖酸杆菌生长和产酸作用的影响。结果显示：巨大芽孢杆菌胞外液和胞内液均可促进氧化葡萄糖酸杆菌的增殖,主要表现为缩短其中长周期中的延迟期;巨大芽孢杆菌通过所产生的部分生物活性物质增强氧化葡萄糖酸杆菌产酸的细胞酶活性,促进氧化葡萄糖酸杆菌转化L一山梨糖生成2KGA.     

氧化葡萄糖酸杆菌SCB329的培养及保存

氧化葡萄糖酸杆菌SCB329和苏云金芽孢杆菌SCB933是混合发酵产生维生素C前体2－KLG两株主要菌种,本文对氧化葡萄糖酸杆菌SCB329的纯培养,传代及纯小菌的保存及其对产酸的影响作了研究。     

混合培养中巨大芽孢杆菌对氧化葡萄糖酸杆菌的作用

为查明维生素Ｃ二步发酵混合培养中巨大芽孢杆菌与氧化葡萄糖酸杆菌间的关系,通过生长曲线测定、静息细胞实验及摇瓶发酵实验研究了巨大芽孢杆菌对氧化葡萄糖酸杆菌生长和产生２－酮基－Ｌ－古龙酸作用的影响;采用超滤分离、凝胶层析及聚丙烯酰胺凝胶电泳技术对巨大芽孢杆菌胞外液中具有促进氧化葡萄糖酸杆菌产酸作用的活性物质进行了分离和纯化。结果表明,大菌胞内液和胞外液均可促进小菌生长,大菌胞外液中具有该作用的组分分子     

Vc 二步发酵中的微生物生态调控

研究了Vc二步混合菌发酵中氧化葡萄糖酸杆菌与巨大芽孢杆菌的生长和相互作用。结果表明,2株混合菌在发酵中可形成一种协同共生,促进2－酮基－L－古龙酸产生;二菌协同共生的过程及条件不同,促进产酸能力亦不同。环境因子影响二菌协同共生,优化环境因子可显著改善二菌协同共生效率,并提高醇到发酵转化率。     

巨大芽孢杆菌在维生素C二步发酵中的作用

巨大芽孢杆菌培养时期不同其上清液对促进氧化葡萄糖酸杆菌生长和产酸能力不同,在稳定期及衰亡初期显著促进氧化葡萄糖酸杆菌生长与产酸.pH、温度可改变上清液对氧化葡萄糖酸杆菌生长与产酸的影响.上清液中含活性物质具有蛋白质的部分性质,对酸、碱、热敏感.     

Vc二步发酵中的微生物生态调控

研究了Vc二步混合菌发酵中氧化葡萄糖酸杆菌与巨大芽孢杆菌的生长和相互作用.结果表明,2株混合菌在发酵中可形成一种协同共生,促进2酮基L古龙酸产生;二菌协同共生的过程及条件不同,促进产酸能力亦不同.环境因子影响二菌协同共生.优化环境因子可显著改善二菌协同共生效率,并提高醇酸发酵转化率.     

VC三菌种一步发酵方法的构建与研究

就维生素C微生物一步发酵方法进行了探索,构建了酮古龙酸杆菌、氧化葡萄糖酸杆菌和芽孢杆菌三菌混菌一步发酵的方法。研究发现,植物内生芽孢杆菌可以与酮古龙酸杆菌配合,促进酮古龙酸杆菌生长和产酸。在有山梨醇存在的条件下酮古龙酸杆菌及其伴生菌能够快速地生长增殖,植物内生芽孢杆菌在发酵的10h中不断消耗山梨醇。5L的发酵罐中,酮古龙酸杆菌、氧化葡萄糖酸杆菌和植物内生芽孢杆菌三菌混菌一步发酵在恒定的30℃温度,600r/min搅拌速度和1.5vvm通气条件下,补料发酵过程中醇酸质量转化率达到了81.89%,在分批发酵过程中,醇酸质量转化率达到了87.90%,进一步优化了维生素C生产工艺。     

巨大芽孢杆菌（Bacillus megaterium）突变株Bn,B5的生物学特性及在Vc二步发酵中的伴生作用

对选出的巨大芽孢杆菌突变株Bn,B5进行了生物学特性及发酵条件的研究,发现它们具耐低pH和抗高浓2KGA特性.可促进氧化葡萄糖酸杆菌生长,使其延迟期缩短,产酸增加.适宜的通气量下,摇瓶糖酸转化率提高10%～14%;当发酵pH为6.2～6.6时,转化率提高20%～30%.     

维生素c发酵中2－酮－L－古龙酸还原酶的研究

由于2－酮－L－古龙酸还原酶(简称KGR)的存在,理论上造成了Vc合成前体2－酮－L－古龙酸(简称2KLG)部分被还原成L－艾杜糖酸〔1.2〕,影响了Vc二步发酵的产率。而从L－山梨糖到2－酮－L－古龙酸的转化中,除被KGR催化的还原负反应外,均属于氧化反应。所以,从该角度出发,抑制KGR的活性,消除还原负反应,是提高2KLG产酸率,进而提高Vc二步发酵产率的关键。本研究在纯化KGR的基础上⑶,对其物质组成及性质进行了研究,并研制了几种稀土元素化合物,考察了它们对KGR纯酶及在L－山梨糖发酵中对2－KLG产酸率的影响。     

氧化葡萄糖酸杆菌中硫辛酸合成模块对维生素C一步混菌发酵的影响

在由氧化葡萄糖酸杆菌和普通生酮古龙酸杆菌构建的维生素C两菌一步发酵体系中,为了强化氧化葡萄糖酸杆菌对普通生酮古龙酸杆菌生长和产酸的促进作用,文中在氧化葡萄糖酸杆菌中构建硫辛酸合成功能模块。由含硫辛酸功能模块的氧化葡萄糖酸杆菌和普通生酮古龙酸杆菌组成的两菌一步体系,能减轻普通生酮古龙酸杆菌单菌培养时的生长抑制,强化两菌的互作关系,使维生素C前体(2-酮基-L-古龙酸,2-KGA)的产量提高到73.34 g/L(对照组为59.09 g/L),醇酸转化率提高到86.0%。研究结果为进一步优化维生素C两菌一步发酵体系提供了新思路。     

一株芽孢杆菌在维生素C二步发酵中对小菌的促进作用

从土壤中分离到1株能更好促使小菌生长和产酸的芽孢杆菌B601,作为伴生菌与巨大芽孢杆菌相比,在生长过程中,发酵液中B601活菌数小于巨大芽孢杆菌,而其芽孢数则多于巨大芽孢杆菌。对B601组成菌系的发酵条件进行优化,得到如下结果：100g／L L-山梨糖、6g／L尿素、10g/L玉米浆、培养温度30℃和发酵周期44h。与巨大芽孢杆菌组成菌系相比其底物,L-山梨糖质量浓度提高了25％,尿素下降了50％．玉米浆质量浓度下降了33％,温度提高了2℃,发酵周期缩短了4h。结果表明：B601作为伴生菌,与巨大芽孢杆菌相比,该菌株明显提高了发酵效率。     

高渗条件下利用蔗糖提升2-酮基-L-古龙酸生产效率

旨在进一步提升维生素C前体2-酮基-L-古龙酸(2-KLG)的生产效率。在详细考察了2-KLG工业化生产过程中渗透压变化规律的基础上,研究了高渗对混合菌系细胞生长和2-KLG合成的影响,提出蔗糖促进伴生菌巨大芽胞杆菌Bacillus megaterium生长,进而促进普通生酮古龙酸菌Ketogulonigenium vulgare生长和产酸的策略。结果表明,2-KLG的积累和碱性物质的流加使渗透压上升了832mOsmol/kg;高渗抑制了巨大芽胞杆菌的生长(15.4%),从而抑制普通生酮古龙酸菌(31.7%)的生长,导致2-KLG产量和生产强度分别下降67.5%和69.3%(以1250mOsmol/kg为例);蔗糖的添加则显著促进巨大芽胞杆菌的生长,使高渗条件下(摇瓶,1250 mOsmol/kg)2-KLG产量(40.6g/L)提高87%;在3L发酵罐中,补加10mmol/L蔗糖使2-KLG发酵周期缩短10.8%,2-KLG生产强度提高10.4%。研究成果为在环境胁迫下提高混菌生产目标代谢产物的产量提供了潜在的策略。     

山梨糖发酵产生2—酮基—L—古龙酸氮源代谢规律

对山梨糖发酵产生2-酮基-L-古龙酸氮源代谢规律进行了初步研究。通过对这一混合发酵体系蛋白和尿素代谢的研究表明,氮源代谢与单一菌体发酵相比有其特殊性,主要表现在尿素的加入有两个作用,即作为生理碱性物质调节体系pH和为菌体代谢提供部分氮源,而体系的蛋白含量随发酵时间的延续不断增加,其增加的原因是巨大芽孢杆菌由营养体转变成芽孢所致,这是该发酵体系的特点。本文还对该发酵体系各种氨基酸变化规律进行了讨论,将一共17种氨基酸按其变化规律分成了三类,较好地解释了各种氨基酸的变化情况,为进一步深入研究该体系的动力学特性提供了数据基础。     

Integrated proteomic and metabolomic analysis of an artificial microbial community for two-step production of vitamin C

An artificial microbial community consisted of Ketogulonicigenium vulgare and Bacillus megaterium has been used in industry to produce 2-keto-gulonic acid (2-KGA), the precursor of vitamin C. During the mix culture fermentation process, sporulation and cell lysis of B. megaterium can be observed. In order to investigate how these phenomena correlate with 2-KGA production, and to explore how two species interact with each other during the fermentation process, an integrated time-series proteomic and metabolomic analysis was applied to the system. The study quantitatively identified approximate 100 metabolites and 258 proteins. Principal Component Analysis of all the metabolites identified showed that glutamic acid, 5-oxo-proline, L-sorbose, 2-KGA, 2, 6-dipicolinic acid and tyrosine were potential biomarkers to distinguish the different time-series samples. Interestingly, most of these metabolites were closely correlated with the sporulation process of B. megaterium. Together with several sporulation-relevant proteins identified, the results pointed to the possibility that Bacillus sporulation process might be important part of the microbial interaction. After sporulation, cell lysis of B. megaterium was observed in the co-culture system. The proteomic results showed that proteins combating against intracellular reactive oxygen stress (ROS), and proteins involved in pentose phosphate pathway, L-sorbose pathway, tricarboxylic acid cycle and amino acids metabolism were up-regulated when the cell lysis of B. megaterium occurred. The cell lysis might supply purine substrates needed for K. vulgare growth. These discoveries showed B. megaterium provided key elements necessary for K. vulgare to grow better and produce more 2-KGA. The study represents the first attempt to decipher 2-KGA-producing microbial communities using quantitative systems biology analysis.     

微生物混合培养从D山梨醇产生维生素C前体——2-酮基-L-古龙酸

氧化葡萄糖酸杆菌（Gluconobacter oxydans)SCB329以D－山梨醇为底物培养时可产生微量2－酮基－L－古龙酸;而葡萄糖酸杆菌（Gluconobacter sp.)SCB110能将D－山梨醇以较高效率转化为L－山梨糖,但不产2－酮基－L－古龙酸。将两种微生物在以山梨醇为底物的培养基中混合培养,其代谢产物经分离提纯后进行熔点测定、元素分析、红外吸收光谱测定等,确定其主要的代谢产物是2－酮基－L－古龙酸。     

Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain

AIMS: Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. METHODS AND RESULTS: The keratinase gene with and without leader sequence from the chromosomal DNA of Bacillus licheniformis MKU3 was amplified by PCR and cloned into pET30b and transferred into Escherichia coli BL21. The ker gene without leader sequence only expressed in E. coli and the recombinant strain produced an intracellular keratinase activity of 74.3 U ml(-1). The ker gene was further subcloned into E. coli-Bacillus shuttle vector, pWH1520. Bacillus megaterium ATCC 14945 carrying the recombinant plasmid pWHK3 expressed the ker gene placed under xylA promoter and produced an extracellular keratinase activity of 95 U ml(-1). Response surface methodology (RSM) was employed to optimize the fermentation condition and to improve the level of keratinase production by the recombinant strain. A maximum keratinolytic activity of 166.2 U ml(-1) (specific activity, 33.25 U mg(-1)) was obtained in 18 h of the fermentation carried out with an initial inoculum of 0.4 OD600 nm and xylose concentration of 0.75% w/v. CONCLUSIONS: Bacillus licheniformis keratinase was cloned and successfully expressed using T7 promoter in E. coli and xylose inducible expression system in B. megaterium. Response surface methodology was employed to optimize the process parameters, which resulted in a three-fold higher level of keratinase production by the recombinant B. megaterium (pWHK3) than the wild type strain B. licheniformis MKU3. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that B. megaterium is a suitable host for the expression of cloned genes from heterologous origin. Optimization of fermentation conditions improved the keratinase production by B. megaterium (pWHK3) and suggested that this recombinant strain could be used for the production of keratinase.