酿酒酵母和米曲霉混合发酵生产a-半乳糖苷酶和转化酶

本研究首次利用酿酒酵母Z-06和米曲霉L-09作为混合菌株, 并通过对液态发酵培养基成分的优化, 使α-半乳糖苷酶和转化酶活力分别达到85.22 U/mL和 563.44 U/mL, 比单菌株发酵产酶活分别提高了5.9倍和8.3倍。     

酿酒酵母和米曲霉混合发酵生产α-半乳糖苷酶和转化酶

本研究首次利用酿酒酵母Z-06和米曲霉L-09作为混合菌株,并通过对液态发酵培养基成分的优化,使α-半乳糖苷酶和转化酶活力分别达到85.22 U/mL和563.44 U/mL.比单菌株发酵产酶活分别提高了5.9倍和8.3倍.     

一株产几丁质酶菌株的筛选及其产酶条件的研究

采用平板透明圈法从土壤中分离筛选到一株产几丁质酶放线菌株L12,用250mL摇瓶发酵初筛和复筛,酶活力为0.63U/mL。通过产酶条件实验,初步确定了该菌株较适产酶培养基和摇瓶发酵条件。条件优化后,30℃、250mL摇瓶发酵48h,几丁质酶活力达到1.06U/mL。     

南极海冰细菌中产低温 α-淀粉酶菌株的发酵和酶学特性的初步研究

从147株南极海冰细菌中筛选出一株高产低温a-淀粉酶的菌株Pseudoalteromonas sp.AN175。生长发酵单因素试验表明,菌株AN175在1%麦芽糖、0.5%酵母粉、0.01%磷酸铁、0.5%氯化钙、1%可溶性淀粉、5%NaCl、pH 6.0的培养基,10℃,30%装液量,130 r/min条件下振荡培养9 d可达到最大酶活132.5 U/mL,为优化前的2倍多。生长和产酶试验表明,该菌生长较常温菌缓慢,酶分泌时间滞后。粗酶特性研究显示,该酶催化的最适条件为温度30℃,pH值8.0。     

α-葡萄糖苷酶菌株的选育及发酵条件的研究

从2株芽胞杆菌中通过筛选获得了一株产α-葡萄糖苷酶活力较高的嗜热脂肪芽孢杆菌U2,以嗜热芽孢杆菌U2为菌种,优化发酵培养基后,在温度45℃、初始pH6.8、转速200r/min和10%接种量条件下,发酵20h,菌株U2产酶水平可达到2.62U/mL,比出发菌提高了4倍。     

1株褐藻胶降解菌的筛选、鉴定及产酶条件优化

为获得可产生褐藻胶裂解酶并高效降解褐藻胶的菌株,以海藻酸钠为唯一碳源配制培养基,以透明圈法进行初筛,DNS法复筛,从海洋生物中筛选得到1株高酶活力褐藻胶降解菌株B12,经16S rDNA序列分析、生理生化试验、电镜观察,确定该菌为弧菌属(Vibrio sp.)。通过单因素试验及响应面优化试验对影响菌株生长和产酶条件的5个因素(发酵初始pH值、发酵温度、NaCl质量浓度、接种量和装液量)进行优化。得到该菌株最佳产酶条件:pH 6.52,发酵温度28.2℃,NaCl质量浓度20.1 g/L,接种量2.1%,装液量59.5 mL。在最佳发酵条件下,B12菌株酶活力可达91.68 U/mL,相比于优化前提高了38.5%。菌株开始产酶时间提前6 h, 4℃冷藏酶活力稳定性较好。     

重组Bacillus subtilis产B.stearothermophilus环糊精葡萄糖基转移酶的发酵优化

利用本研究室已构建的重组菌Bacillus subtilis/pBSMuL3-α/β-CGTase对产B.stearothermophilus环糊精葡萄糖基转移酶的发酵产酶进行了优化,考察了培养基中重要成分:碳源、有机氮源、无机氮源、有机与无机氮源质量比、碳源与氮质量比、金属离子种类等单因素对该重组菌产α/β-CGTase的影响,并采用正交实验对发酵培养基进行优化,对优化结果分析可知,重组菌B.subtilis/pBSMuL3-α/β-CGTase发酵产α/β-CGTase的最优培养基成本为:葡萄糖5 g/L,氮源(鱼骨蛋白胨∶NH4Cl=3∶1)25 g/L,1 mmol/L Mg^2+。在最优条件下发酵培养,α/β-CGTase的酶活由原来TB发酵培养基的9.20 U/mL提高至20.32 U/mL,是优化前酶活的2.2倍,为α/β-环糊精葡萄糖基转移酶的工业应用提供了理论支持。     

黑曲霉产菊粉酶菌株的选育及培养基优化

为获得高产菊粉酶的黑曲霉菌株,以Aspergillus niger YH-1为出发菌株,经过亚硝基胍(NTG)诱变,以高温高菊芋粉相结合的方式进行梯度驯化,选育出一株产菊粉酶菌株YH-3,并运用响应面实验方法对该菌株的培养基进行优化。确定了最佳培养基组成:菊芋粉25.2 g/L、豆饼粉40 g/L、蔗糖酯4.9 g/L、NaCl 5.5 g/L。发现内切菊粉酶活力(I)由60.9 U/mL提高到165.0 U/mL,比出发菌株提高了1.7倍。研究证明蔗糖酯对于黑曲霉YH-3发酵产菊粉酶是一种有效的促进剂。     

纤维素降解菌青霉T24-2的分离及产酶特性

从稻田腐烂秸秆中分离到一批纤维素分解菌株。通过滤纸崩解测试、刚果红纤维素平板识别,以及产酶鉴定,筛选得到一株分解纤维素能力较强的真菌。经形态观察和18S r DNA基因片断分析,鉴定该菌株为青霉。对菌株的液态发酵条件进行研究,该菌株培养基含3%稻草粉、0.25%尿素和无机盐营养液,最佳产酶条件为:自然pH,30℃,130r/min发酵4d。该菌株的CMC酶活和滤纸酶活最高分别达到45.01I U/mL和6.89I U/mL。随后对该菌酶解稻草粉进行研究,糖化率达到40.17%。研究表明,青霉T24-2菌株在秸秆综合利用上具有良好的应用前景。     

产低温脂肪酶菌株CZW001发酵培养基优化研究

【目的】研究产低温脂肪酶菌株CZW001发酵培养基。【方法】在单因素试验的基础上, 采用Plackett-Burman (P-B)设计, Box-Behnken (B-B)设计和响应面试验设计(RSM), 在20 °C、pH 8.0、160?r/min发酵2 d条件下, 对发酵培养基进行优化。【结果】该菌株最适产酶培养基为(g/L): 葡萄糖7.68, 橄榄油21.93, 硫酸铵2.0, 磷酸二氢钾1.0, 硫酸镁0.27, 氯化钙0.3, 氯化钠20.0, 吐温-80 1.0。其最高酶活为62.8 U/mL, 比优化前提高了3.14倍。【结论】通过对产低温脂肪酶菌株CZW001发酵培养基优化研究, 明显提高低温脂肪酶活力。     

Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation

The kinetics of β-fructofuranosidase (Ffase) production by Aspergillus niger in submerged (SmF) and solid-state fermentation (SSF) systems was investigated. The maximum productivity of Ffase (81.8 U/l per h) was obtained in SSF for 72 h while it was 18.3 U/l per h in SmF for 120 h. The productivity of extra cellular Ffase produced in SSF was 5-fold higher than in SmF. Optimization of fermentation medium for Ffase production was carried out using De Meo's fractional factorial design with seven components such as (NH₄)₂SO₄, KH₂PO₄, FeSO₄, MgSO₄ · 7H₂O, sucrose, urea and yeast extract. The media designed for SmF after two steps of optimization supported the growth of A. niger and higher productivity of Ffase (58.3 U/l per h) than with the medium before optimization. The optimized medium of SmF when used in SSF, did not improve the Ffase productivity and therefore medium for SSF was optimized independent of SmF. After two optimization steps, the media was defined for SSF which supported the growth and high level of Ffase productivity (149.1 U/l per h) in SSF compared to the medium before optimization (81.8 U/l per h) and optimized medium for SmF (58.3 U/l per h). Our results suggested that the optimized media for SmF and SSF for the production of Ffase have to be different.     

粗壮假丝酵母(Candida valida 20231)产脂肪酶发酵条件的研究

对粗壮假丝酵母(Candida valida 20231)产脂肪酶所需的培养基成分和发酵条件进行了优化。结果表明,粗壮假丝酵母在接种后3 h即进入对数期,18 h后转入稳定期,66 h后进入衰退期;发酵36 h达到最大酶活,至72 h仍然无明显下降趋势。最适合的培养基成分为:4.0%大豆粉,2.0%菜籽油,0.3%葡萄糖,0.3%NH_4NO_3,1.2%K_2HPO_4,0.42%NaH_2PO_4,0.4%MgSO_4·7H_2O。使用优化的培养基,最佳产酶的条件为:装液量80 mL(250 mL摇瓶),初始pH 6,接种量为2.0%,种子菌龄为12 h。在最佳产酶条件下,脂肪酶活力达到32.6 U/mL。     

Optimization of fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05

The optimal fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05 were investigated. The medium components and fermentation conditions were optimized according to the one at a time method, while the concentration of medium components was determined by the orthogonal matrix method. The results showed that the optimal fermentation medium was as follows: sucrose 3.5% (w/v), peptone 0.5%, yeast extract 0.2%, K(2)HPO(4) 0.1%, and MgSO(4) 0.05%. The suitable fermentation conditions were as follows: initial pH 7.0, temperature 25 degrees C, medium volume 75 mL/250 mL, inoculum volume 5% (v/v), time 5d. In such optimal nutrition and environmental conditions, the maximal mycelial yield was 2.124 g/100 mL after 4 day's fermentation, while maximal water-soluble exo-polysaccharides production reached 2.144 g/L after 5 day's fermentation.     

产脂肪酶重组枯草芽胞杆菌的发酵优化

笔者所在实验室前期筛选到1株产脂肪酶粘质沙雷氏菌,克隆其脂肪酶基因,构建重组枯草芽胞杆菌Bacillus subtilis 168/pMA5-lipA,成功实现了来源于粘质沙雷氏菌的脂肪酶基因在枯草芽胞杆菌中的表达。基于以上工作基础上,对B.subtilis 168/pMA5-lipA进行了摇瓶水平上的产酶发酵优化。首先通过单因素和正交试验确定了有利于产脂肪酶的最佳培养基成分,并对发酵条件进行了优化。结果表明:优化后的培养基组分为蔗糖35 g/L,玉米浆27.5 g/L,(NH4)2SO41.25 g/L,CaCl24 g/L,pH 7.0。在最优发酵培养基的条件下,37℃、160 r/min摇床培养33 h,每毫升发酵液中重组菌脂肪酶酶活可达98.6 U,是优化前的3倍。     

尖孢镰刀菌生产蒽醌色素的液体发酵条件研究

优化了尖孢镰刀菌液体发酵生产蒽醌类红色素的发酵条件。通过单因素实验和正交优化实验,确定最佳产色素发酵培养基为：可溶性淀粉30％,(NH4)2SO4 3％,MgSO4 0.3％,KH2PO4 4％,pH 6.0。产色素最适培养条件为：初始pH6.0,装液量20％,接种量10％,吐温-80添加量1％,温度28℃,摇床转速200r/min,发酵周期120h。此条件下,色素效价即可达到8.184U/ml,比优化前提高了1.8倍。国内首次对尖孢镰刀菌所产蒽醌色素进行研究,为其进一步应用奠定基础。     

假丝酵母E-2产生物表面活性剂摇瓶发酵条件的优化

从扬子石化的废水淤泥中筛选到1株能发酵液体石蜡产脂肽类生物表面活性剂的假丝酵母Candida E-2.通过单因子实验和正交试验,得到了最佳发酵培养基组成(g/L):牛肉膏3.0,蔗糖2.0,酵母膏0.25,KH2PO4 12.5,MgSO4 0.3,NaCl 1.5,CaCl,0.05,尿素0.5 5;液体石蜡10％(体积分数).最佳培养条件:初始pH7.0,接种量0.12g/L,装液量为200mL三角瓶30mL,培养时间为5 d.最终产量提高了2.7倍,达1.582g/L.     

Optimization of bio-hydrogen production from biodiesel wastes by Klebsiella pneumoniae

Biodiesel wastes containing glycerol were utilized by Klebsiella pneumoniae DSM 2026 to produce hydrogen. The optimization of medium components was performed using both Plackett-Burman and uniform design methods. Using the Plackett-Burman design, glycerol, yeast extract, NH(4)Cl, KCl and CaCl2 were found to be the most important components, which were further investigated by uniform design and second-order polynomial stepwise regression analysis. The optimized medium containing 20.4 g.L(-1) glycerol, 5.7 g.L(-1) KCl, 13.8 g.L(-1) NH(4)Cl, 1.5 g.L(-1) CaCl(2) and 3.0 g.L(-1) yeast extract resulted in 5.0-fold increased level of hydrogen (57.6 mL/50 mL medium) production compared to initial level (11.6 mL/50 mL medium) after 24 h of fermentation The optimization of fermentation condition (pH, temperature and inoculum) was also conducted. When the strain grew in the optimized medium under optimal fermentation condition in a 5-L stirred tank bioreactor for batch production, hydrogen yield and production reached 0.53 mol/mol and 117.8 mmol/L, respectively. The maximum hydrogen evolution rate was 17.8 mmol/(L.h). Furthermore, 1,3-propanediol (6.7 g.L(-1)) was also obtained from the liquid medium as a by-product.     

短小芽孢杆菌(Bacillus pumilus) WHK4以羽毛粉为底物产蛋白酶条件的优化

探索Bacillus pumilusWHK4以羽毛粉为底物产酶的最佳条件和最佳培养基组成。以羽毛粉发酵培养基为基础,首先采用单因子试验考察底物浓度、初始pH、接种量、外加碳源、外加氮源对WHK4产酶活力的影响。在单因子试验的基础上采用正交试验设计对底物浓度、温度、初始pH、接种量、外加(NH4)2SO4、外加麦芽糖进行优化。结果显示:Bacillus pumilusWHK4最佳的产酶条件为初始pH7.38,菌龄16 h,接种量5%,37℃。最佳的培养基组成为:1 L基础发酵培养基,40.0 g羽毛粉,10.0 g(NH4)2SO4和10.0 g麦芽糖。在优化的条件下Bacillus pumilusWHK4 24 h产蛋白酶活力为每毫升90 U。对培养条件和培养基的优化为Bacillus pumilusWHK4产蛋白酶的分离纯化奠定了基础。     

植物乳杆菌CLP0279发酵生产低温SOD培养基的优化

【目的】提高植物乳杆菌CLP0279发酵生产低温超氧化物歧化酶(superoxide dismutase,SOD)的能力。【方法】在单因素实验基础上,采用Plackett-Burman (PB)设计、Box-Behnken (BB)设计和响应面分析法(RSM),对发酵培养基进行优化。【结果】植物乳杆菌CLP0279产低温SOD最佳发酵培养基(g/L)：玉米粉25.000,磷酸二氢钾2.600,磷酸氢二钾1.830,硫酸铜0.011,硫酸锌0.014。在最佳培养基条件下产酶活力达到194.82 U/ml,是优化前的1.36倍。【结论】通过响应面分析,对植物乳杆菌CLP0279发酵生产低温SOD的培养基进行优化,明显提高了产酶能力。确定了磷酸氢二钾、硫酸铜和硫酸铵为发酵培养基中影响酶活的3个关键因子。研究结果为SOD的发酵放大提供了依据。