用于腈纶表面改性的Corynebacterium nitrilophilus 腈水合酶的摇瓶发酵优化

通过毛细管效应、扫描电镜、染色实验等方法,考察了Corynebacterium nitrilophilus腈水合酶(nitrilre hydratase,NHase)在腈纶表面改性中的应用。结果表明,C. nitrilophilus腈水合酶处理后的腈纶纤维的润湿性及染料可染性分别比对照提高了43.5%和85.7%,说明该腈水合酶具有较好的腈纶纤维表面改性性能。为了提高用于腈纶表面改性的C. nitrilophilus腈水合酶的产量,采用单因素实验及正交实验在摇瓶上对碳源、氮源、诱导剂、金属离子等进行考察,获得较优的摇瓶发酵生产腈水合酶的条件:碳源采用葡萄糖,15 g/L;氮源采用酵母粉与氯化铵复配,浓度分别为3 g/L、1 g/L;诱导剂尿素的最适剂量为10 g/L;由于该腈水合酶是钴型酶,所以需在发酵过程中添加氯化钴,浓度为0.07 g/L。经过摇瓶优化,酶活由最初的16.2 U/ml提高到45.7 U/ml,提高了2.8倍。     

海洋真菌岩藻多糖酶的固态发酵条件研究*

本文对海洋真菌Dendryphiella arenaria(TM94)岩藻多糖酶的固态发酵条件进行了研究,主要内容包括碳源、氮源、添加物、起始pH、接种量及温度等.固态发酵最佳培养基组成麸皮7.5g,葡萄糖0.5g,海带粉0.6g,NaNO3为4g/L最佳培养条件为培养温度28℃,起始pH6,接种量3ml(孢子浓度为106个/ml).在28℃培养24h,酶活力可达35.5IU/g干培养基,比活力为1.39IU/mg蛋白质.对岩藻多糖酶酶学性质也进行了研究.     

对羟基苯乙腈水合酶高产菌株的选育

从全省各处采集的50多份土样筛选到一株产腈水合酶能力较高的菌株E10a,该菌株产生的腈水合酶为非诱导酶,产酶条件优化实验结果表明,产酶培养基组成:麦芽糖20 g/L,酵母膏5 g/L,尿素7.5 g/L,味精0.75 g/L,K2HPO40.5 g/L,KH2PO40.5 g/L,MgSO40.5 g/L,FeSO4.7H2O 10 mg/L,CoCl210 mg/L,微量元素母液0.8 ml/L;最佳培养条件为:培养温度28℃,摇床转速150 r/min,培养基起始pH 7.0,培养时间5 d,在优化培养条件下,1 h可将1 g/L质量浓度的底物对羟基苯乙腈全部转化为对羟基苯乙酰胺。     

低温脂肪酶的产酶条件优化及其酶学性质

利用单因素筛选和正交试验对Burkholderia sp. SYBC LIP-Y发酵产酶的液体培养基和发酵条件进行了优化,其优化配方为：可溶性淀粉10 g/L、牛肉膏15 g/L、NaNO3 0.252 g/L、橄榄油40ml/L、Triton x-100 10ml/L、初始pH 7.5、接种量10%(V/V),脂肪酶酶活达到85.23U/ml,是优化前的3.63倍。通过对双水相纯化得到的脂肪酶进行酶学性质研究,确定该酶反应的最适pH为10.0,最适温度为30℃,40℃下保温60min酶活性还有80%以上,该脂肪酶为低温脂肪酶,热稳定性好,具有一定的耐醇性,应用前景广阔。     

表皮短杆菌ZJB-07021产R-酰胺酶培养条件的优化

采用响应面分析法(RSM)对R-酰胺酶产生菌Brevibacterium epidermidis ZJB-07021的发酵培养基进行了优化.首先运用了单因子试验筛选出了发酵培养的最佳pH与温度,在此基础上采用Plackett-Burman(PB)设计法,对 8 种影响产酶的因素进行评价,实验结果表明,葡萄糖、酵母粉与乙酰胺含量对菌株产酰胺酶的活力具有显著的影响.通过旋转中心组合实验考察了葡萄糖、酵母粉和乙酰胺这三个主要因素对菌株所产酰胺酶活力的影响.发酵培养基优化结果为葡萄糖 17.00 g/L,酵母粉 15.74 g/L,乙酰胺 7.05 g/L,采用优化后的发酵培养条件进行摇瓶发酵培养,酰胺酶的酶活达到 72.14 U/L,比优化前的初始发酵培养条件下的酶活提高了73.3%.     

响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基

对枯草芽孢杆菌(Bacillus subtilis)CICC20034利用合成培养基液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适诱导剂为三丁酸甘油酯,氮源为尿素,碳源为葡萄糖,无机盐为MgSO4。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的三丁酸甘油酯、尿素、KH2PO4和培养基起始pH值4个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,获得最适合成培养基组分为:葡萄糖8g/L,尿素8.57g/L,三丁酸甘油酯2.62%,KH2PO42.59g/L,MgSO4.7H2O0.5g/L,TritonX-1000.5g/L,pH9.47。优化后的B.subtilis CICC 20034胞外脂肪酶活力达0.483U/ml,比初始酶活力0.072U/ml提高了6.7倍。     

均匀设计法优化烟管菌产漆酶培养基

应用均匀设计、二次多项式逐步回归分析对烟管菌(Bjerkandera adusta)WZFF.W-Y11产漆酶液态发酵培养基进行优化。结果表明,培养基组成为麸皮水解液1%、淀粉24.0g/L、葡萄糖24.0g/L、豆饼粉4.8g/L、NH4Cl3.2g/L、KH2PO43.2g/L、MgSO4.7H2O0.2g/L、CuSO4.5H2O0.006g/L,起始pH6.5,在28℃、150r/min、250mL的摇瓶培养条件下可以稳定地获得9672U/L的漆酶活力。     

α-氯丙酸脱卤酶发酵条件研究

以从厌氧污泥中分离筛选获得的对α-氯丙酸有高效脱卤能力的微生物菌株W20为出发菌株,对其发酵生产脱卤酶的工艺进行了研究。其产脱卤酶培养基组成为:葡萄糖20.0 g/L,尿素1.0 g/L,酵母膏0.5 g/L,Na2HPO4.12H2O 3.2 g/L,KH2PO41.5 g/L,无水MgSO40.098 g/L,微量元素液10 mL/L,维生素溶液5.0 mL/L。产酶条件为:接种量10%,培养基初始pH7.0,培养温度30℃,装液量80 mL/250 mL摇瓶,摇床转速180 r/min。在以上获得的培养基和培养条件下培养48 h后测酶活,脱卤酶活力达到8.76 U/g干菌体,比在原始条件下提高约10倍。     

嗜热网球菌纤维二糖差向异构酶在枯草芽孢杆菌中的表达及发酵优化

通过化学方法合成嗜热网球菌(Dictyoglomus thermophilum)来源的纤维二糖差向异构酶基因ce,将其引入到载体pBSuL3-ce,构建重组质粒pBSuL3-ce并转化进枯草芽孢杆菌,发酵48h后测定胞内酶活为7. 5U/ml。酶学性质结果表明:该酶的最适pH为8. 5;最适温度为85℃,85℃的半衰期为120min。为降低发酵成本,对发酵培养基进行优化:以35g/L豆粕粉为氮源、5g/L甘油为碳源时,酶活力最高可达12. 3U/ml。依据摇瓶优化的条件在3L发酵罐中扩大培养,胞内酶活达到56U/ml,比摇瓶培养酶活提高了8倍。利用发酵所得酶制备乳果糖,在乳糖浓度为400g/L、反应温度为85℃、初始pH 8. 5、加酶量为20U/ml的条件下,乳果糖转化率可达51%。     

褐藻胶裂解酶产生菌的分离鉴定及产酶发酵优化

对一株从腐烂海带中筛选得到的产褐藻胶裂解酶的菌株进行鉴定,并对其产酶条件进行发酵优化。经形态学、生理生化特征和分子生物学鉴定,将其鉴定为盐单胞菌属,并命名为Halomonas sp. WF6。通过在摇瓶培养水平上进行单因素和多因素正交试验,确定褐藻胶裂解酶产生菌WF6的最适产酶培养基为：褐藻酸钠6.0 g/L,蛋白胨5.0 g/L,酵母粉2.5 g/L,NaCl 30 g/L,K⁺ 5 mmol/L。进而采用最适培养基进行产酶条件的优化,优化后的发酵产酶条件为：初始pH 8.0,培养温度25℃,接种量为2%,摇瓶装液量30 ml/250 ml,培养时间39 h。优化后的褐藻胶裂解酶酶活达117.66 U/ml,是优化前的2.1倍。该酶对褐藻酸钠的酶解产物主要由聚合度为二和三的褐藻寡糖组成。     

Efficient inducible expression of nitrile hydratase in Corynebacterium glutamicum

Nitrile hydratases are important industrial catalysts to produce valuable amides. In this study, we describe a comprehensive and systematic approach to the development of an inducible expression system for enhanced nitrile hydratase expression in Corynebacterium glutamicum. Through promoter engineering, codon optimization and design of ribosome binding site sequences, the nitrile hydratase activity toward 3-cyanopyridine was improved from 0.33 U/mg DCW to 12.03 U/mg DCW in shake-flask culture. By introduction of the novel inducible mmp expression system, the nitrile hydratase activity was further elevated to 14.97 U/mg DCW. Finally, a high nitrile hydratase yield of 1432 U/mL was achieved in a fed-batch fermentation process and used for nicotinamide production. These results provide new insights for the development of heterologous protein expression systems in C. glutamicum.     

Optimization of Culture Conditions of Brevibacterium SP. A4 for the Production of Nitrile Hydratase

Optimum culture conditions of Brevibacterium sp. A4 for production of nitrile hydratase were determined by two mathematical methods: the Hadamard method and graphic analysis of response areas. A minimal medium was optimized and the basic roles of Fe²⁺ and Mg²⁺ were clearly shown. The influence of physico-chemical factors (pH, temperature and light conditions) on the culture and on nitrile hydratase were also studied. Various results permit the production of Brevibacterium sp. A4 cells with low protease and high nitrile hydratase contents.     

内切中性纤维素酶EGⅡ的发酵条件优化

对毕赤酵母基因工程菌EIM-50-eg2产内切中性纤维素酶的主要影响因子进行研究,考察氮源、pH、温度、微量元素PTM1和甲醇浓度等对工程菌产酶的影响。单因素实验和正交实验结果表明,优化后的培养基组成及培养条件:磷酸氢二铵40 g/L,甲醇15 mL/L,硫酸镁10 g/L,磷酸二氢钾9 g/L,初始pH 6.0,培养温度28℃,PTM1添加量0.02%,甲醇诱导浓度1.5%。优化后内切葡聚糖酶活力可达4 158 U/(mL.min)是优化前1 449 U/(mL.min)的2.86倍。     

Cobalt-dependent transcription of the nitrile hydratase gene in Rhodococcus rhodochrous M8

Abstract The effects of cobalt ions on the activities of Rhodococcus rhodochrous M8 enzymes for nitrile utilization, nitrile hydratase and amidase, were investigated. In contrast to amidase, synthesis of nitrile hydratase and its activity required cobalt ions in the growth medium. Northern blot analysis showed that in the presence of cobalt ions, the level of mRNA for nitrile hydratase genes was several times higher than that under cobalt-limited conditions. It was assumed that the low nitrile hydratase activity in cells grown in the absence of cobalt ions is connected either with the weak expression of nitrile hydratase genes or with the rapid degradation of nitrile hydratase mRNA.     

Optimization of Culture Conditions of Brevibacterium SP. A4 for the Production of Nitrile Hydratase

Optimum culture conditions of Brevibacterium sp. A4 for production of nitrile hydratase were determined by two mathematical methods: the Hadamard method and graphic analysis of response areas. A minimal medium was optimized and the basic roles of Fe²⁺ and Mg²⁺ were clearly shown. The influence of physico-chemical factors (pH, temperature and light conditions) on the culture and on nitrile hydratase were also studied. Various results permit the production of Brevibacterium sp. A4 cells with low protease and high nitrile hydratase contents.     

DA-F127水凝胶包埋固定化含腈水合酶细胞

腈水合酶是一类可催化腈类化合物转化生成相应酰胺类物质的酶。含腈水合酶的游离细胞催化水合反应存在酶容易失活、细胞无法重复利用、分离纯化困难等缺陷,细胞固定化技术可有效解决这些问题。为探索合适的固定化方法,以含腈水合酶的重组E.coli细胞为研究对象,以固定化酶活回收率和批次反应情况为评价指标,筛选比较了几种常用的包埋固定化方法。结果表明,DA-F127水凝胶包埋固定化细胞不仅具有较高的酶活回收率,而且稳定性也很好。对该方法进行了固定化条件和操作稳定性优化,当DA-F127浓度为15%、UV光源距离为20cm、光照时间为6min、菌体含量为20mg/g 固定化细胞时,酶活回收率为89.74%,并且可以催化9批次150g/L的3-氰基吡啶完成转化,第九批次转化率可达98.26%。与游离细胞催化过程相比,单位质量游离细胞的烟酰胺产量提高了12倍,具有良好的工业应用前景。     

响应面-满意度函数优化重组大肠杆菌产NMN转移酶发酵条件

重组大肠杆菌Escherichaia coli能高效表达NMN转移酶,以此为出发菌株,以菌体生长量OD600和NMN转移酶的活力为响应值,对重组大肠杆菌产NMN转移酶的发酵条件进行优化.首先以Plackett-Burman实验设计优化筛选出3个主要影响因子:胰蛋白胨、甘油、MgSO4;随后以Box-Behnken中心组合设计建立上述3个因子对OD600和NMN转移酶活力水平的数学模型;最后通过满意度函数获得最佳发酵条件为:酵母粉30 g/L,胰蛋白胨10.5 g/L,甘油3.49 mL/L,MgSO40.45 g/L,K2 HPO440.5 g/L,KH2 PO46.0 g/L,NH4 Cl 1.5 g/L,NaCl 0.6 g/L,接种量1.5%,诱导时间12 h.在该优化条件下,菌体生长和产酶水平均获得了显著的提升.重组NMN转移酶的活力水平从8.85 U/mg提高到15.48 U/mg,菌体生长量OD600从4.85提高到6.01,提高幅度分别为74.92%和23.92%.     

休哈塔假丝酵母HDYXHT-01利用木糖生产乙醇的发酵工艺优化

采用Plackett-Burman (PB) 方法和中心组合设计 (Ccentral composit design,CCD) 对休哈塔假丝酵母Candida shehataeHDYXHT-01利用木糖发酵生产乙醇的工艺进行优化。PB试验设计与分析结果表明：硫酸铵、磷酸二氢钾、酵母粉和接种量是影响木糖乙醇发酵的4个关键因素,以乙醇产量为响应目标,采用CCD和响应面分析法 (Response surface methodology,RSM),确定了木糖乙醇发酵的最佳工艺为：硫酸铵1.73 g/L、磷酸二氢钾3.56 g/L、酵母粉2.62 g/L和接种量5.66％,其他发酵条件为：木糖80 g/L,MgSO4·7H2O 0.1 g/L,pH 5.0,培养温度30 ℃,装液量100 mL/250 mL,摇床转速140 r/min,发酵时间48 h,在该条件下发酵液中乙醇产量可以达到26.18 g/L,比未优化前提高了1.15倍。     

Application of statistical experimental designs for the optimization of medium constituents for the production of citric acid from pineapple waste

Statistical experimental designs were applied for the optimization of medium constituents for citric acid production by Yarrowia lipolytica NCIM 3589 in solid state fermentation (SSF) using pineapple waste as the sole substrate. Using Plackett-Burman design, yeast extract, moisture content of the substrate, KH(2)PO(4) and Na(2)HPO(4) were identified as significant variables which highly influenced citric acid production and these variables were subsequently optimized using a central composite design (CCD). The optimum conditions were found to be yeast extract 0.34 (%w/w), moisture content of the substrate 70.71 (%), KH(2)PO(4) 0.64 (%w/w) and Na(2)HPO(4) 0.69 (%w/w). Citric acid production at these optimum conditions was 202.35 g/kg ds (g citric acid produced/kg of dried pineapple waste as substrate).