高密度培养重组E-coli产胆固醇氧化酶的乳糖诱导策略

研究了乳糖诱导重组大肠杆菌表达胆固醇氧化酶。结果表明,乳糖不仅可以作为诱导剂诱导外源蛋白的合成,而且能作为碳源促进菌体的生长。通过对诱导条件的优化,实现了重组大肠杆菌的高密度培养,最高密度达68.9(OD600);在此基础上确定了最佳的诱导时机为发酵中期,菌体产酶水平达6005.66U/L,生产强度为200.19U/L.h,实现了胆固醇氧化酶的高效生产。     

重组Microdochium nivale还原糖氧化酶用于制备乳糖酸方法的研究

Microdochium nivale(M.nivale)还原糖氧化酶可以催化乳糖转化为乳糖酸,作者采用实验室保存的重组M.nivale还原糖氧化酶基因工程菌X33-p PICZαA-Mn CO,通过诱导在毕赤酵母中表达并制备了重组M.nivale还原糖氧化酶,通过单因素和正交实验考查了乳糖浓度,重组M.nivale还原糖氧化酶用量,反应温度,反应时间对乳糖酸转化率的影响。结果表明:重组M.nivale还原糖氧化酶催化乳糖制备乳糖酸的最适转化条件为:乳糖浓度5 mmol/L,酶的添加量为1.5 U/ml,反应温度为55℃,反应时间为48 h,最终反应乳糖酸的转化率接近100%。     

重组大肠杆菌γ-谷氨酰转肽酶的pET载体的选择及乳糖诱导作用的初步研究

采用PCR技术以E.coliK-12基因组DNA为模板扩增出γ-谷氨酰转肽酶基因,克隆至原核表达载体pET28(a)和pET32(a)中,经测序鉴定后转化大肠杆菌BL21(DE3),借助SDS-PAGE分析方法,对于用乳糖诱导,由T7 lac启动子控制的重组目的产物蛋白的表达规律进行了优化研究.在对诱导条件进行优化控制的前提下,分析比较了乳糖诱导pET28(a)/]-ggt和pET32(a)/γ-ggt两种重组质粒表达大肠杆菌γ-谷氨酰转肽酶蛋白的可溶性及酶活.实验结果表明,对于T7lac启动子控制的重组目的产物,乳糖作为诱导剂也可以起到很好的诱导表达效果,低温培养下,乳糖诱导pET32(a)/γ-ggt重组质粒表达γ-谷氨酰转肽酶具有更高的蛋白可溶性和酶活. 杆茵BL21(DE3),借助SDS-PAGE分析方法,对于用乳糖诱导,由T7 lac启动子控制的重组目的产物蛋白的表达规律进行了优化研究.在对诱导条件进行优化控制的前提下,分析比较了乳糖诱导pET28(a)/]-ggt和pET32(a)/γ-ggt两种重组质粒表达大肠杆菌γ-谷氨酰转肽酶蛋白的可溶性及酶活.实验结果表明,对于T7lac启动子控制的重组目 产物,乳糖作为诱导剂也可以起到很好的诱导表达效果,低温培养下,乳糖诱导pET3     

乳糖诱导极耐高温木聚糖酶基因B在大肠杆菌中的表达

以融合极耐高温木聚糖酶基因B(XynB)的大肠杆菌(E coli)BL2 1(DE3)为研究对象,研究了以IPTG和乳糖作为诱导剂时重组目的产物的诱导表达规律。在摇瓶发酵条件下研究了诱导剂浓度、诱导时机、诱导培养时间、诱导培养温度及初始pH对目标蛋白表达的影响。实验结果表明,IPTG诱导时酶活力达到16 18U/ 10 0mL。乳糖诱导的最优发酵条件为:初始培养基pH 7 0 ,当OD60 0 为2 5时加入0 5 %的乳糖,在37℃条件下诱导培养10h ,收获时OD60 0 达3 5 5 ,酶活力为2 5 91U/ 10 0mL。     

乳糖诱导葡萄球菌肠毒素A基因在大肠杆菌中的表达

以乳糖作为诱导剂代替传统方法中的IPTG,分别从菌龄、诱导剂浓度、诱导时间及诱导剂的添加方式等方面,对乳糖诱导金黄色葡萄球菌肠毒素A(staphylococcal enterotoxins A,SEA)基因在大肠杆菌BL21(DE3)中的表达进行了研究。结果表明,重组大肠杆菌表达SEA的优化条件为:在对数生长期(OD600约为0.6),一次性添加终浓度为0.5 mmol/L的乳糖诱导6 h。目标蛋白的表达量占菌体总蛋白质的36.9%,略低于以IPTG为诱导剂时38.1%的表达量。乳糖价格仅为IPTG的1%左右,因此,在SEA的大规模制备中,使用乳糖作为诱导剂可以大大节约成本。     

乳糖诱导D-塔格糖3-差向异构酶基因在大肠杆菌中的表达

以D-塔格糖3-差向异构酶高效表达菌株BL21(DE3)/pET22b-cbdte为研究对象,考察乳糖作为诱导剂诱导D-塔格糖3-差向异构酶在大肠杆菌BL21(DE3)中表达的可行性.在摇瓶发酵条件下,从诱导时机、诱导剂浓度、诱导时间及诱导温度等四个方面,研究诱导条件对目的蛋白表达的影响.结果表明,工程菌培养至对数生长中期OD值为1.0左右后,加入终浓度为5g/L的乳糖,于20℃的条件下诱导7h,可获得最大酶活.与异丙基-β-D-硫代吡喃半乳糖苷(IPTG)最适诱导条件相比,优化后的乳糖诱导酶活提高82.7％,可溶性目的蛋白的表达量提高99.5％,菌体生物量提高30.5％.研究结果为乳糖作为诱导剂应用于D-塔格糖3-差向异构酶的工业化生产提供有益的参考和借鉴.     

乳糖诱导对重组大肠杆菌表达CGTase的影响

以重组环糊精葡萄糖基转移酶(CGTase)表达菌株BL21(DE3)作为研究对象,比较IPTG和乳糖诱导对重组大肠杆菌表达CGTase量的影响,确定优化诱导方式。以Geobacillus CHB1的基因组DNA为模板,采用PCR技术克隆CGTase基因;利用分子操作技术构建携带有大肠杆菌Omp A信号肽的分泌型表达质粒p EASY-E2-Omp A-CGT,重组质粒热激转化至Escherichia coli BL21中进行表达;在摇瓶发酵条件下,分别以IPTG和乳糖作为诱导剂,通过SDSPAGE分析和测定胞外酶活,确定优化诱导方式。CGTase基因在Escherichia coli BL21中实现表达,且具有一定量的重组CGTase分泌至胞外;乳糖诱导的蛋白表达量优于IPTG诱导,不易形成包涵体;乳糖最佳的诱导起始菌浓度OD600为1.4,诱导浓度、温度分别为0.5%和25℃,分批流加乳糖5次和一次性加入,重组CGTase表达量无明显差异;经初步优化,胞外酶活达19.87 U/m L。Geobacillus CHB1 CGTase基因在大肠杆菌中成功实现胞外表达,乳糖可替代IPTG诱导重组CGTase。     

乳糖诱导大肠杆菌产褐藻胶裂解酶的条件优化

本文研究了使用乳糖替代IPTG(异丙基硫代半乳糖苷)诱导褐藻胶裂解酶在重组大肠杆菌中表达的可行性,通过摇瓶实验,分别对诱导终浓度、诱导温度、诱导时机、诱导时间等方面进行了单因素实验并利用响应面对条件进行了优化设计,并使用Ni-NTA亲和柱对粗酶液进行了纯化得到纯酶。结果表明,诱导乳糖终浓度22 mmol/L,诱导温度29℃,诱导22 h,酶活可达到8.552 U/m L。说明乳糖可以作为基因工程菌的高效诱导剂。     

两种甜叶菊废渣提取物对D-半乳糖致衰老小鼠的抗氧化作用

目的:研究两种甜叶菊废渣提取物对D-半乳糖致衰老小鼠的抗氧化作用。方法:80只小鼠随机被分为正常组、D-半乳糖组、两种甜叶菊废渣提取物低、中、高剂量组(100,200,500 mg/kg bw)。采用颈背部皮下注射D-半乳糖(500 mg/kg bw)构建衰老模型,正常组颈部皮下注射等体积的生理盐水,样品组同时给予不同剂量的甜叶菊废渣提取物。持续11周后,测定小鼠血清、肝脏和脑组织中的超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)的活性和丙二醛(MDA)的含量,以及脑组织中抗氧化酶基因的相对表达量。结果:与D-半乳糖组相比,两种甜叶菊废渣提取物均在不同程度上提高小鼠血清、肝和脑抗氧化酶(SOD和GPx)的活力,降低MDA含量,且在高剂量时(500 mg/kg bw)效果最为显著。两种甜叶菊废渣提取物高剂量组小鼠脑组织中抗氧化酶基因(Nrf-2,SOD1,GPx1,HO-1)的相对表达量显著增加。结论:两种甜叶菊废渣提取物通过促进抗氧化酶基因表达,增加D-半乳糖致衰老小鼠体内相关抗氧化酶的活力并减少氧化损伤产物MDA的含量,具有潜在的延缓衰老的作用。     

蔗糖异构酶基因在Escherichia coli BL21(DE3)中的表达及重组菌的细胞固定化

分别利用IPTG和乳糖两种诱导物诱导蔗糖异构酶(SIase)基因在E.coliBL21(DE3)中实现表达,对诱导温度、诱导时机、诱导物浓度、诱导持续时间进行比较分析并优化,确定了二者的最佳诱导条件,在E.coli培养3h后(OD600约为0.9)添加终浓度为0.8mmol/L的IPTG(0.5mmol/L乳糖)在20℃(24℃)条件下诱导14h(12h)能获得最高的蛋白表达量及SIase酶活。在最优条件下以IPTG为诱导物时目的蛋白占总蛋白的41.6%,单位体积培养液中SIase酶活为12.37U/mL,以乳糖为诱导物时分别为27.2%,14.72U/mL,从收获酶活角度考虑可见乳糖作为诱导物的优势;而后利用海藻酸钠包埋法固定化重组菌,转化初始浓度为500g/L的蔗糖溶液,转化10～11h后异麦芽酮糖平均得率在83%以上,蔗糖平均转化率大于99%,固定化细胞能够连续稳定转化25批次,转化效率相对于原始菌提高了近55%。     

Integrated production of whey protein concentrate and lactose derivatives: What is the best combination?

In order to model and analyze the techno-economic feasibility of a whey processing unit for the production of whey protein concentrate (WPC) integrated with processing of lactose, the present study utilized the software SuperPro Designer® for modeling of the processes, including risk analysis and study of reduced pollution impacts. Six models were constructed for the production of WPC and processing of lactose, which were (1) WPC 34, (2) WPC 34 and lactose powder, (3) WPC 34 and hydrous ethanol fuel, (4) WPC 80, (5) WPC 80 and lactose powder, and (6) WPC 80 and hydrous ethanol fuel. The economic evaluation was performed by analysis of the Payback Period (PP), Net Present Value (NPV), Breakeven Point (BP) and Internal Rate of Return (IRR). Probability distributions obtained by fitting of historical data for whey prices and the final products were used to perform the risk analysis, submitted to a Monte Carlo simulation using the @Risk software. The project showed to be feasible due to the elevated IRR and NPV values, coupled with low BP and PP. When evaluating the individual production of ethanol, it was verified that the production cost of this product was superior to the sale price, making independent production of ethanol from lactose present in the whey uneconomical. Plants with production of lactose powder were more economically attractive and also presented greater reduction of Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD). The financial indices suggested greater feasibility of WPC 80 compared to WPC 34.     

Bulking in aerobic biological systems treating dairy processing wastewaters

The aerobic biological treatment of dairy processing wastewaters is associated with poor sludge settling bulking, and this has been largely linked to the high soluble organic component in these wastewaters, and high chemical oxygen demand COD: NP ratios. Strategies to minimize sludge bulking in activated sludge systems or sequencing batch reactors have been identified and include the operation of these systems under extended aeration conditions, and the incorporation of an anaerobic or anoxic zone to facilitate the degradation of the readily metabolized lactose in the wastewater. Filamentous bacteria linked with sludge bulking in the industry have been associated with various operating conditions.     

Production of galactooligosaccharides using a hyperthermophilic β-galactosidase in permeabilized whole cells of Lactococcus lactis

Galactooligosaccharides (GOS) are novel prebiotic food ingredients that can be produced from lactose using β-galactosidase, but the process is more efficient at higher temperatures. To efficiently express the lacS gene from the hyperthermophile Sulfolobus solfataricus, in Lactococcus lactis a synthetic gene (lacSt) with optimized codon usage for Lc. lactis was designed and synthesized. This hyperthermostable β-galactosidase enzyme was successfully overexpressed in Lc. lactis LM0230 using a nisin-controlled gene expression system. Enzyme-containing cells were then killed and permeabilized using 50% ethanol and were used to determine both hydrolysis and transgalactosylation activity. The optimum conditions for GOS synthesis was found to be at pH 6.0 and 85°C. A maximum production of 197 g/L of GOS tri- and tetrasaccharides was obtained from 40% initial lactose, after 55 h of incubation. The total GOS yield increased with the initial lactose concentration, whereas the highest lactose conversion rate (72%) was achieved from a low lactose solution (5%). Given that a significant proportion of the remaining lactose would be expected to be converted into disaccharide GOS, this should enable the future development of a cost-effective approach for the conversion of whey-based substrates into GOS-enriched food ingredients using this cell-based technology.     

乳糖替代凝乳酶制作大豆干酪技术初探

对加酶发酵制作干酪的传统方法进行了改进,用添加乳糖的方法来代替添加酶进行发酵.试验综合考察了影响混合豆乳(豆乳和牛乳)干酪凝乳的几个因素,即豆乳添加量、发酵剂添加量、乳糖添加量和乳酸钙添加量,并确定混合豆乳干酪的最佳凝乳工艺参数.试验结果表明,最佳工艺参数为:80%豆乳 3%发酵剂 3%乳糖 0.1%乳酸钙.     

IPTG与乳糖联合诱导重组大肠杆菌右旋糖酐蔗糖酶表达

研究异丙基硫代-β-D-呋喃半乳糖苷（isopropyl-β-D-1-thiogalactopyranoside,IPTG）与乳糖联合诱导重组大肠杆菌右旋糖酐蔗糖酶表达的效果。在利用IPTG和乳糖分别作为诱导剂对右旋糖酐蔗糖酶工程菌Escherichia coliBL21（DE3）/pET28-dexYG进行诱导表达的基础上,尝试将此两种诱导剂联合使用,在降低成本的同时获得较好的表达效果。在获得最佳培养基的基础上,考察菌体IPTG与乳糖的联合加入量、菌体浓度、诱导时间对右旋糖酐蔗糖酶表达的影响。在菌浓（OD600 nm）达到3.0时,加入0.1 mmol/L IPTG 95 μL＋2.5 g/L乳糖,25 ℃混合诱导培养4 h,酶活力最高,达到40.44 U/mL。IPTG与乳糖联合诱导重组大肠杆菌右旋糖酐蔗糖酶表达可行。     

Lactose recovery from Panela cheese whey: Effect of whey acidification on spontaneous and stirring-induced crystallisation

This work aimed to investigate the kinetics, yield, size and composition changes in lactose crystals when Panela cheese whey was acidified (pH 3.80) or slightly alkalinised (pH 6.89) before being subjected to spontaneous or stirring-induced crystallisation. At pH 3.8, the crystallisation was accelerated, and the incorporation of salts and proteins in lactose crystals decreased. Nevertheless, these conditions induced the formation of amorphous lactose. In contrast, at pH 6.89 and under stirring conditions, the amorphous lactose was not formed and gave a high crystallisation yield (∼25 g of lactose per Kg of whey) with ∼80% of lactose recovery.     

热稳定性过氧化氢酶工程菌株发酵条件的研究

重组大肠杆菌UM2 1携带来自嗜热脂肪芽孢杆菌过氧化氢酶的基因 ,在以IPTG作为诱导物时 ,在IPTG浓度为 0 75mmol/L、起始 pH6 5、装液量 5 0mL/2 5 0mL三角瓶的条件下 3 7℃诱导 3h ,酶的表达水平最佳 ;在以乳糖作为诱导物时 ,在乳糖质量浓度为 10 g/L、起始 pH7 5、装液量 5 0mL/2 5 0mL三角瓶的条件下 ,3 7℃诱导 5h ,酶的活力最高。乙酸的存在能够抑制酶的表达。工程菌在最适条件下酶活力可达 3 0 0 0 0～ 3 5 0 0 0U/L以上 ,约是原始菌株嗜热脂肪芽孢杆菌的 10倍。工程菌在无选择压力的条件下连续传代 60代 ,基本保持稳定 ,连续传代 10 0代 ,仍有 80 %左右的菌株携带重组质粒。