地衣芽孢杆菌中性β-甘露聚糖酶的分离纯化

采用絮凝、超滤及离子交换对地衣芽孢杆菌中性β-甘露聚糖酶进行了分离纯化,纯化倍数为33倍,酶活收率达42%,所得纯化的β-甘露聚糖酶比活为4341 U/mg蛋白质.本文提出的分离纯化工艺易于工业放大.     

高酶活性β-甘露聚糖酶定向进化及其用于制备甘露寡糖

为了提高β-甘露聚糖酶的活性,本研究采用易错PCR将来源于地衣芽孢杆菌(Bacillus licheniformis)KD-1的β-甘露聚糖酶基因manBl进行分子进化,并在枯草芽孢杆菌(B. subtilis)中进行表达,以定向筛选酶活性提高的β-甘露聚糖酶突变体。筛选得到的突变体ManBl (I91N/L211I),其比酶活性为 15554.7 U/mg,是野生型ManBl的4.2倍,食品级表达的胞外酶产量达17601.3 U/mL。应用AlphaFold2对该酶的三维结构进行预测,结果表明,尽管β-甘露聚糖酶的2个位点(I91N 和 L211I)位于催化中心之外,但在很大程度上影响酶活性。β-甘露聚糖酶ManBl (I91N/L211I)水解魔芋胶产物主要由甘露六糖、甘露三糖和甘露二糖组成。该研究首次报道I91N/L211I 2个位点联合突变能够提高β-甘露聚糖酶活性；ManBl (I91N/L211I)食品级表达,为该酶绿色安全地应用奠定了基础。     

一株β-甘露聚糖酶生产菌细胞培养条件的优化

地衣芽孢杆菌SY-8具有分泌β-甘露聚糖酶的能力.为提高地衣芽孢杆菌SY-8的产酶能力,必须先找到该菌培养的最佳环境条件,因此考查温度、摇床转速、传代浓度、培养pH、渗透压等因素对细胞生长状态的影响,发现当温度为30℃,摇床转速为100 rpm/min,传代浓度为1.25×106 cell/mL,细胞培养pH为6.5,渗透压为360 osmol/g时,能达到SY-8细胞培养的最佳条件,有利于满足大规模培养的需要.     

中性β-甘露聚糖酶分批发酵动力学研究

以魔芋粉为碳源,研究了10 L自控发酵罐中枯草芽孢杆菌TJ-200603分批发酵产中性β-甘露聚糖酶的过程动力学。实验数据表明,菌体生长呈现典型S型曲线,而酶的合成与菌体生长同步进行,属于生长耦联型。基于这些过程曲线的变化规律,构建了β-甘露聚糖酶分批发酵过程的动力学模型。并经非线性拟合和优化,获得了最佳的模型参数值,最终确定了能够较好表征实际发酵过程中菌体细胞生长、产物β-甘露聚糖酶合成以及基质总糖消耗的3个动力学方程。     

丙酸杆菌的两种固定化细胞反应器发酵生产丙酸及其代谢通量分析

根据构建的费氏丙酸杆菌合成丙酸的生化反应网络,利用代谢通量分析法分析了课题组构建的两种固定化细胞反应器对丙酸发酵的影响.结果表明,固定化细胞发酵可以调节葡萄糖-6-磷酸、磷酸烯醇式丙酮酸和丙酮酸节点处的代谢通量分布,从而最终影响生物量和各种有机酸的合成.与游离发酵相比,两种固定化发酵方式戊糖磷酸途径通最都有显著提高,乙...     

压裂液增稠剂降解菌的化学诱变育种研究

为了获得高活性的生物酶破胶剂,降低压裂液的粘度。本研究对压裂液增稠剂降解菌枯草芽孢杆菌进行诱变。枯草芽孢杆菌分泌的β-甘露聚糖酶可以分解增稠剂,常常作为生物酶破胶剂。以Bacillus subtilis D为出发菌株,经2%浓度的甲基磺酸乙酯诱变,经过透明圈初筛和DNS复筛,获得酶活力提高的诱变菌株Bacillus subtilis D12,酶活提高了13.28%。     

外部代谢物模拟在代谢工程中的应用指导

外部代谢物是代谢网络模型中通过添加运输反应来平衡其在菌体内的通量的一类代谢物,比如底物、产物或副产物、信号分子等。首次通过对模型中运输反应的模拟来预测大肠杆菌和枯草芽孢杆菌在不同的生长状态下外部代谢物的优先排出顺序。部分模拟结果已经通过实验和文献数据得到证实。这一分析对应用代谢工程技术提高目标产物的产量有很大的指导意义。     

双水相萃取直接从枯草芽孢杆菌发酵液中提取β-甘露聚糖酶

研究了聚乙二醇(PEG)平均相对分子质量、PEG浓度、(NH4)2SO4浓度、NaCl浓度对β-甘露聚糖酶和总蛋白分配系数、相体积比和萃取率的影响。实验表明PEG100020%(m/m)和(NH4)2SO415%(m/m),NaCl为2%(m/m)组成的双水相体系,室温下直接对含菌体的枯草芽孢杆菌发酵液抽提β-甘露聚糖酶,可纯化2.76倍,萃取率达98.79%。     

新型生物絮凝剂REA-11的代谢模型建立与代谢网络分析

利用代谢通量分析方法,对谷氨酸棒杆菌Corynebacterium glutamicum CCTCC M201005分批发酵不同阶段和不同溶氧浓度下的代谢网络模型进行了计算,考察了代谢节点对絮凝剂REA-11合成的影响,并对C. glutamicum生长代谢过程中能量和还原力的产生及消耗问题进行分析.结果发现,磷酸戊糖途径(PP)通量在整个发酵过程中始终维持在较高的水平;REA-11合成通量随溶氧浓度的增加而降低,菌体合成通量则随溶氧水平的增加而增加;ATP通量的增加可以促进菌体生长,而与REA-11的合成呈负相关.     

克雷伯氏杆菌代谢灵活性分析

微生物对外界环境的变化具有灵活的适应性,通过代谢网络的通量可变性分析可以对代谢灵活性进行量化,得到影响菌体代谢灵活性的因素。使用代谢流分析软件COBRA Toolbox,对克雷伯氏杆菌基因组尺度代谢网络进行通量平衡分析与通量可变性分析,分别得到内部反应的可变性、交换反应的可变性和生长速率的可变性等3个因素对菌体代谢灵活性的影响程度,其中生长速率的可变性是影响代谢灵活性的主要因素,调控菌体至次优模式下生长可使其具有更大的代谢灵活性。通过进一步研究生长速率对通量可变性的影响,找到提高代谢灵活性的两个必要条件,从而增强菌体应对外界环境改变的能力。     

代谢通量分析在酶合成过程中的应用研究进展

代谢通量分析可以有效预测微生物的代谢状况,指导生物合成过程,已在多个领域得到成功应用。本文概括分析了三大代谢通量分析技术（基于物料平衡、基于同位素示踪技术以及基于基因组尺度规模网络）在工业生物技术领域的应用,阐述了相关技术的优势及局限性。在此基础上重点论述了基于物料平衡及同位素示踪这两类通量分析技术在酶合成领域的研究进展,分析了代谢通量分析应用于复杂代谢产物可能存在的问题,指出如何将信号转导、转录调控等信息纳入代谢网络将成为未来酶合成过程代谢分析的研究重点。     

克雷伯氏杆菌发酵生产1,3-丙二醇的代谢通量优化分析

以克雷伯氏杆菌为研究对象,系统研究了1,3-丙二醇厌氧和微氧代谢途径.采用代谢通量分析法建立了代谢通量平衡模型,通过线性规划对1,3-丙二醇厌氧和微氧发酵进行了优化分析,得到其最优代谢通量分布和最大理论得率,并分析了比生长速率、底物浓度以及乙醇生成速率、氧气消耗速率、呼吸商对1,3-丙二醇得率的影响.     

Analysis of Data on Xanthan Fermentation in Stationary Phase Using Black Box and Metabolic Network Models

The xanthan fermentation data in the stationary phase was analyzed using the black box and the metabolic network models. The data consistency ls checked through the elemental balance in the black box model. In the metabolic network model, the metabolic flux distribution in the cell is calculated using the metabolic flux analysis method, then the maintenance coefficients is calculated.     

Hydrolysis of freshly prepared wheat starch fractions and commercial wheat starch using α-amylase

The enzymic hydrolysis of commercial wheat starch and freshly prepared wheat starch fractions was studied in batch and flow systems. Fresh starch was prepared by wet separation of wheat flour into starch milk and gluten, followed by processing the starch milk using a serially connected hydrocyclone system to produce underflow and overflow streams. The underflow stream consisted mainly of the larger (> 30 w m) granules, while the overflow stream contained only the smaller (< 10 w m) granules. In batch system, the hydrolysis behavior of wheat starch fractions was investigated under the action of Sigma f -amylase ( Bacillus licheniformis ), either as soon as they were prepared or after spray drying. The two fractions of fresh wheat starch showed different hydrolysis behavior, the difference getting larger as the separation improves. Underflow streams were found to be much more susceptible to hydrolysis than overflow streams, regardless of whether the hydrolysis was carried out right away or on dried samples, and also regardless of whether rinsing was applied. In flow system, dried underflow stream and commercialwheat starch were hydrolyzed using Orbamil-T ( Bacillus licheniformis ), Orbamil-BHT ( Bacillus stearothermophilus ), and Sigma f -amylase ( Bacillus licheniformis ). Hydrolysis of fresh starch was found to be significantly faster than commercial wheat starch. Commercial f -amylase Orbamil-T was found to be almost as effective as the purified Sigma f -amylase.     

Dynamic Modeling and Metabolic Flux Analysis for Optimized Production of Rhamnolipids

A comprehensive metabolic network based on the fundamental pathways representing the central metabolism of rhamnolipid by Pseudomonas aeruginosa is proposed and a dynamic model compatible with the underlying metabolic network is developed involving the macro-reactions derived from the elementary flux modes of the reaction network. The experimentally validated mathematical model is then coupled with a global optimization technique called differential evolution (DE) to optimize the medium composition as well as the extracellular and intracellular fluxes of the metabolic network. The analysis of the results shows the usefulness of the integrated approach involving the development of a dynamic model based on the metabolic network structure and model-based optimization of the medium composition and metabolic fluxes by an efficient evolutionary optimization technique to enhance the productivity of rhamnolipid.     

产朊假丝酵母分批发酵生产谷胱甘肽的代谢通量分析

根据产朊假丝酵母利用葡萄糖作为惟一碳源生物合成谷胱甘肽的代谢网络,利用代谢通量分析方法分析了谷胱甘肽分批发酵不同阶段各代谢途径碳通量的分布和变化规律.在此基础上,通过分阶段温度控制和L-半胱氨酸添加等策略,调节谷胱甘肽分批发酵过程中的代谢通量分布,谷胱甘肽生物合成的产量明显提高,最大增加幅度分别达到23%和91%.同时对这些策略下谷胱甘肽的过量合成机理进行了解释.     

通过通量平衡分析计算经验和真实维持系数

The stoichiometric matrix of a simplified metabolic network in Bacillus Subtillis was contructed from the flux balance equations, which were used for reconciliation of the measured rates and determination of the inner metabolic rates. Thus more reliable results of the true and empirical maintenance coefficients were obtained. The true maintenance coefficient is linearly related to the specific growth rate and changes with the P/O ratiol. The neasured biomass yield of adenosine triphosphate (ATP) is also linearly related to the P/O ratio.     

Studies on the Applicability of Artificial Neural Network (ANN) in Continuous Stirred Ultrafiltration

An artificial neural network model of a continuous stirred ultrafiltration process, is proposed in the present study, which is able to predict permeate volumetric flux and permeate concentration at different bulk concentration, stirrer speed, pressure and time. Because of the complexity in generalization of the phenomenon of ultrafiltration by any mathematical model, the neural network proves to be a very promising method for the purpose of process simulation. The network uses the Back‐propagation Algorithm for evaluating the connection strengths, representing the correlations between inputs (bulk concentration, stirrer speed, pressure and time) and output (permeate concentration and flux). The network employed in the present study uses four input nodes corresponding to the operating variables, and two output nodes corresponding to the measurement of the performance of the network (flux and permeate concentration). Experiments were performed to constitute the learning databases for the continuous stirred ultrafiltration process using PEG‐6000 solute, and cellulose acetate membrane of 5000 MWCO. The network employed in the present study uses two hidden layers, with the optimum number of nodes being thirty and twenty. A leaning rate of 0.3, and momentum factor of 0.4 was used. The results predicted by the model were in good agreement with the experimental data, and the average deviations for all the cases are found to be well within ±10 %.     

利用基元模式分析酿酒酵母的葡萄糖厌氧发酵过程以提高乙醇产量

Elementary flux mode （EFM） analysis was used in the metabolic analysis of central carbon metabolism in Saccharomyces cerevisiae based on constructed cellular network. Calculated from the metabolic model, the ethanol-producing pathway No. 37 furthest converts the substrate into ethanol among the 78 elementary flux modes. The in silico metabolic phenotypes predicted based on this analysis fit well with the fermentation performance of the engineered strains, KAM3 and KAMll, which confirmed that EFM analysis is valid to direct the construction of Saccharomyces cerevisiae engineered strains, to increase the ethanol yield.