固定化桔青霉产生核酸酶P_1的研究

利用吸附固定在多孔聚酯载体上的桔青霉(Penicillium citrinum)菌丝细胞,在摇瓶中以分批发酵方式合成核酸酶P_1.试验结果表明:在固定化细胞产酶的条件下,培养液中葡萄糖和蛋白胨的最适浓度分别为10g/L和1g/L,摇瓶转速以180~200r/min为宜.固定化细胞经过48h的产酶周期,培养液中的核酸酶P_1活力可高达513.3U/ml,其产酶效率是游离菌丝的3.6倍,而葡萄糖和蛋白胨的用量仅为游离菌丝产酶的五分之一.在重复分批发酵试验中,固定化菌丝细胞形状稳定,连续28批(共56d)的产酶结果基本一致,每批的平均酶活力为507.4U/ml,在经济上和工艺上均显示了明显的优越性.     

麦芽糖诱导软化芽孢杆菌α-环糊精葡萄糖基转移酶在枯草杆菌中的表达

通过PCR扩增软化芽孢杆菌α-环糊精葡萄糖基转移酶基因,将基因片段克隆到大肠杆菌-枯草杆菌穿梭载体pGJ103中,转化枯草杆菌WB600得基因工程菌进行外源表达。在1.5%的麦芽糖初始发酵培养基上摇瓶培养,48 h后重组枯草杆菌产酶活性为6.1U/ml。通过单因素分析和响应面分析对重组枯草杆菌产CGT酶摇瓶发酵条件进行优化。分析得到培养基关键组分麦芽糖,玉米淀粉和酵母粉三者最佳浓度分别为:15.5g/L,13g/L和20g/L。在此条件下,摇瓶培养36h后α-CGT酶活性为17.6U/ml,5L罐分批发酵30h后酶活达到20U/ml (水解活性为1.4×10⁴ IU/ml)。     

海藻酸钙固定赤霉菌菌丝产素的研究

将赤霉菌丝固定在海藻酸钙微球中进行连续发酵,考察产赤霉素情况。对海藻酸钠和钙盐浓度固定赤霉菌菌丝的微球稳定性进行初步研究,讨论了固定不同菌龄的赤霉菌微球在不同葡萄糖浓度下的产素能力及菌丝生长能力。实验表明:菌丝微球较稳定的固定条件是菌丝8 g/L、海藻酸钠浓度3 g/L和钙离子浓度3 mol/L;摇瓶发酵72 h,90 h的菌丝微球中菌丝营养生长基本停止,当培养液葡萄糖浓度为2 g/L时,赤霉素终浓度为1 145.5 μg/ml,比生产速率为4.61×10^-3/h;在该条件下固定菌丝球的床层式连续发酵,赤霉素比生产速率为4.82×10^-3/h,是相应分批发酵过程中最大赤霉素生产速率的1.87倍。     

卡拉胶固定粘质赛氏菌产碱性蛋白酶的研究

将粘质赛氏菌(Seratia marcescens)包埋于卡拉胶中,发现2．5％的卡拉胶适于固定该菌产碱性蛋白酶。固定化细胞在其较适宜产酶培养基中发酵,酶活力一般可达400u/ml,在卡拉胶中添加3％玉米粉和1%豆饼粉或2％砂子制备固定化细胞,其产酶能力分别提高了25%和23．9％;固定化细胞颗粒越小,其产酶能力越高。采用摇瓶半连续发酵。其产酶半衰期为14次(24小时为一个周期);而用环流器进行半连续发酵,其产酶半衰期为52次(12小时为一个周期),产酶效率分别比游离细胞摇瓶发酵的产酶效率高11．8％和45．07％,而环流器半连续发酵的产酶效率比摇瓶半连续发酵高29．7％。     

多拷贝毕赤酵母重组菌表达GCL摇瓶优化和高密度发酵

目的:构建高效表达白地霉脂肪酶的毕赤酵母重组菌株,并对筛选得到的菌株进行摇瓶发酵条件优化和分批补料高密度发酵工艺研究。方法:将诱导型表达载体pPIC9K-gcl电转化至毕赤酵母GS115。通过橄榄油-罗丹明B平板和摇瓶发酵筛选高脂肪酶活力的重组菌株,运用基于TaqMan探针的实时荧光定量PCR 法确定其拷贝数,并对菌株进行摇瓶发酵条件优化。在此基础上,研究重组菌在3L 发酵罐中的高密度发酵工艺。结果:筛选得到一株具有3 个白地霉脂肪酶基因拷贝的菌株GS115/pPIC9K-gcl 78#,初始酶活力为220 U/ml。当摇瓶发酵条件为甲醇诱导96 h,每24 h甲醇添加量1 %,接种量2 %,培养基初始pH 7.0,500 ml摇瓶装液量50 ml,甲醇诱导温度25℃ 时酶活力达735 U/ml。3L 发酵罐高密度发酵176.5 h,酶活力达到3360 U/ml,总蛋白含量达到4.30 g/L,且发酵过程中细胞活性一直保持在96 % 以上。结论:基因拷贝数与重组菌株的产酶水平呈正相关,摇瓶优化可显著提高重组菌株的产酶能力,为白地霉脂肪酶的工业化生产奠定了技术基础。     

产糖化酶黑曲霉的固定化研究

采用多孔聚酯材料作为固定化载体,考察并比较了载体吸附固定化黑曲霉菌丝细胞的条件,当菌丝体细胞与载体预培养的条件为pH值5.0、孢子浓度为105个/ml、固液比为1/75时,有利于菌丝体的生长、吸附固定及发酵产酶.在产糖化酶的发酵过程中,与游离菌丝体细胞相比,发酵过程持续产酶时间有一定程度的延长,产糖化酶活力始终高于游离菌丝体.     

应用固定化里氏木霉糖化玉米秆纤维素的研究

采用多孔聚酯材料固定里氏木霉（TrichodermareeseiRutC30）菌丝细胞,将固定化细胞在生长限制条件下重复分批培养,使纤维酶的合成与玉米秆纤维原料的酶解糖化耦合在一个反应器中同时进行。在30℃、初始pH4.8、摇瓶转速150r／min的条件下,连续重复进行12次分批培养试验。每批玉米秆用量为60g／L,培养周期4．5d,共54d。培养液中含滤纸酶活力平均为0.70IU／ml,还原糖26．41g／L,糖化率达到理论值的89.11％。固定化菌丝形态正常,菌量保持在10g／L左右。在间歇添料条件下,玉米秆原料的总量可提高到120g／L,7d后还原糖浓度达52.81g／L,糖化率为89．20％。利用固定化里氏木霉同时产酶和糖化植物纤维原料,工艺简便、成本低廉、易于连续自动化操作,是一条有效利用可再生纤维素资源的新途径。     

Relations between gibberellin hyphae and secondary metabolic product GA3 by resting culture method

针对赤霉菌生长与产次级代谢产物赤霉素的关系进行摇瓶静息培养研究.在不同菌龄及不同底物浓度的条件下,菌丝产素能力有较大不同.实验数据表明:在菌龄96 h,葡萄糖浓度为5g/L时,菌丝静息培养72 h后,培养液中赤霉素含量最高,达1100 μg/mL;同时单位时间单位菌丝产率也最大,为7.1μg/mg·h;在该菌龄条件下,当葡萄糖浓度为1g/L时,赤霉素转化效率最高,为0.8484.该研究有助于进一步优化发酵工艺,降低赤霉素生产成本.     

酿酒酵母by1.1b产D-(-)-扁桃酸脱氢酶的发酵条件优化

对一株产D-(-)-扁桃酸对映选择性脱氢酶的酿酒酵母菌(Saccharomyces cerevisiae sp.strain by1.1b)发酵产酶条件进行了优化.研究各种碳源、氮源及无机盐对产酶的影响,应用正交试验优化发酵培养基组成,结果为:蛋白胨60 g/L,麦芽糖30 g/L,MgSO4 0.5 g/L,ZnSO4 0.01 g/L,KCl 1.0 g/L.优化后酶产量提高了7.9倍(由2.56 U/mL增至20.21 U/mL).摇瓶培养最佳条件为:装液量40%,发酵pH 6.5,接种量10%,发酵温度30℃.考察了细胞生长及产酶的时间进程,最佳培养时间为25 h.     

漆酶高产菌株的诱变选育及其产酶条件

以粗毛栓菌Trametesgallica为出发菌,通过紫外诱变处理其担孢子、PDA-RBBR平板变色法初筛、ABTS法测定培养液漆酶酶活力复筛,获得1株漆酶高产诱变菌株SAH-12。用高氮低碳无机盐培养液(LM3)培养时,其峰值酶活力比出发菌株高出4倍,达到5002.6U/L,且产酶稳定。对SAH-12液体培养产酶条件的研究表明:以纤维二糖和蔗糖为碳源明显优于麦麸、淀粉和葡萄糖,其最高酶活分别达18526U/L和13436U/L;有机氮源较无机氮源更有利于SAH-12漆酶的分泌,以蛋白胨、大豆粕和胰化蛋白胨为氮源时其峰值酶活分别达到20544U/L、19671U/L和16180U/L;适宜初始培养pH为4.0;ABTS、单宁酸、没食子酸对产酶均有明显的诱导作用,其中ABTS和单宁酸的诱导效果相对更好,愈创木酚和吐温80对产酶有一定的抑制作用。     

Production of pullulanase by free and immobilized cells of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NRC22 in batch and continuous cultures

The production of extracellular pullulanase by Bacillus licheniformis NRC22 was investigated using different fermentation modes. In batch culture maximal enzyme activity of 18 U/ml was obtained after 24 h of growth. In continuous fermentation by the free cells, maximal reactor productivity (4.15 KU/l/h) with enzyme concentration of 14.8 U/ml and specific productivity of 334.9 U/g wet cells/h was attained at a dilution rate of 0.28/h, over a period of 25 days. B. licheniformis NRC22 cells were immobilized on Ca-alginate. The immobilization conditions with respect to matrix concentration and cell load was optimized for maximal enzyme production. In repeated batch operation, the activity of the immobilized cells was stable during the 10 cycles and the activity remained between 9.8 and 7.7 U/ml. Continuous production of pullulanase by the immobilized cells was investigated in a packed–bed reactor. Maximal reactor productivity (7.0 KU/h) with enzyme concentration of 16.8 U/ml and specific productivity of 131.64 U/g wet cells/h was attained at dilution rate of 0.42/h. The enzyme activity in the effluent started to decline gradually to the level of 8.7 U/ml after 25 days of the operation.     

Selection of mutants of Aspergillus niger showing enhanced productivity of citric acid from starch in shaking culture

Mutants with enhanced citric acid production from soluble starch were induced from Aspergillus niger WU-2223L. After UV-irradiation of a conidial suspension of strain WU-2223L, mutants were selected on modified starch-methyl red agar plates on the basis of higher amylolytic activity and acid productivity. The 8 mutants selected showed enhanced citric acid production from soluble starch in shaking culture. Among them, a representative mutant strain, 2M-43, produced 48.0gg/l of citric acid from 120 g/l of soluble starch in 9 d of cultivation in shaking culture, whereas strain WU-2223L produced 35.1 g/l. Glucoamylase activities in the culture filtrates of strains 2M-43 and WU-2223L reached maximum levels of 3.62 U/ml and 2.11 U/ml, respectively, both at 3 d of cultivation, and thereafter decreased.     

利用重组大肠杆菌生产α-环糊精葡萄糖基转移酶

将来源于软化类芽孢杆菌（Paenibacillus macerans）的α-环糊精葡萄糖基转移酶（α-CGT）基因插入含pelB信号肽的质粒pET-20b（＋）中,构建了表达载体pET-20b（＋）／cgt,并将其转化表达宿主E.coli BL21（DE3）。对重组菌E．coli BL21／pET-cgt进行摇瓶发酵条件的优化,确定了其胞外表达α-CGT酶的最适条件：葡萄糖8g/L,乳糖0．5g/L,蛋白胨12g/L,酵母膏24g/L,K2HPO472mmol／L,KH2PO417mmol／L,CaCl2 2．5mmol／L;初始pH为7．0,诱导温度为25℃。在该条件下培养90h后最终α-CGT酶的胞外比活达到22．1u／mL,与来源菌Pmacerans所产天然酶比活相比提高了42倍,实现了α-CGT酶的高效生产。将基因工程菌在上述条件下于3L发酵罐中发酵,90h后胞外酶比活达到22．6U／mL,证实了工业化放大的可能性。     

Acrylamide synthesis using agar entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34 in a partitioned fed batch reactor

The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10°C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10°C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4°C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.     

Manganese peroxidase production by immobilized Phanerochaete chrysosporium BKM-F-1767 in a cell bioreactor

Manganese-dependent peroxidase (MnP) production was performed in an immobilized cell bioreactor in which Phanerochaete chrysosporium BKM-F-1767 was immobilized on polystyrene foam. The immobilized cell culture yielded significantly greater MnP activity than the conventional stationary liquid culture. Cultivation was carried out in batch mode; the effect of glucose concentration was investigated and growth kinetics parameters were found as, micromax=0.59 day(-1), Ks=0.33 g/L and Kss=14.5. Batch operation led to maximum MnP (770.82 U/L) in the culture medium containing 0.05% Tween 80, 10 g/L glucose, and 174 microM Mn2+ at 37 degrees C and pH 4.5. Enzyme productivity was obtained as 110.12 U/day/L.     

根瘤菌BK-20固定化细胞生产L(+)-酒石酸

顺式环氧琥珀酸水解酶(CESH)是根瘤菌BK-20生产L(+)-酒石酸的关键酶。为提高其生产效率和生产稳定性,首先优化根瘤菌BK-20的产酶条件,然后利用固定化细胞连续生产L(+)-酒石酸。结果显示,优化后游离细胞酶活达(3 498.0±142.6)U/g,较优化前提高643%。固定化细胞酶活达(2 817.2±226.7)U/g,其最适包埋剂、菌体浓度和凝胶浓度分别为海藻酸钠,10%(W/V)和1.5%(W/V)。固定化细胞连续反应10批后,其形状和酶活均无明显改变,单批次转化率达98%以上,具有良好的生产稳定性。     

Immobilization of oxalate decarboxylase to Eupergit and properties of the immobilized enzyme

Oxalate decarboxylase, an oxalate degradation enzyme used for medical diagnosis and decreasing the oxalate level in the food or paper industry, was covalently immobilized to Eupergit C. Different immobilization parameters, including ratio of enzyme to support, ammonia sulfate concentration, pH, and incubation time, were optimized. Under the condition of enzyme/support ratio at 1:20, pH 9, with 1.5?mol/L (NH(4))(2)SO(4), room temperature, and shaking at 30?rpm for 24?hr, activity recovery of immobilized Oxdc reached 90% with an apparent specific activity of 0.44?U/mg support. The enzymatic properties of immobilized Oxdc were investigated and compared with those of the soluble enzyme. Both shared a similar profile of optimum conditions; the optimum pH and temperature for soluble and immobilized Oxdc were 3.5 and 50°C, respectively. The immobilized enzyme was more stable at lower pH and higher temperatures. The kinetic parameters for soluble and immobilized enzyme were also determined.