天山一号冰川沉积层低温脂肪酶产生菌的筛选及初步鉴定

从新疆天山一号冰川西支尾部的底部沉积层中分离筛选出高产低温脂肪酶菌株,进行初步鉴定及产酶探讨。使用罗丹明-B、吐温80、维多利亚蓝选择培养基以及三丁酸甘油酯酶活检测板,结合棕榈酸对硝基苯酯(p-NPP)比色法,筛选出产低温脂肪酶酶活较高的菌株,研究其生长特性及常规生理生化实验,并根据16S rRNA基因序列初步确定其所属菌属。共分离筛选到5株产低温脂肪酶酶活较高的菌株,经初步鉴定其中4株为假单胞菌属(Pseudomonas sp.),1株为芽孢杆菌属(Bacillus sp.)。该芽孢杆菌属产脂肪酶的能力略高于其余菌株。分离得到的5株菌均属于耐冷菌,有较好的耐盐性,能在低温条件下发酵产酶,其中4株在36h左右达到产酶高峰,为开发冰川环境下丰富的低温酶资源奠定了基础。     

窖泥中产丁酸菌的筛选、鉴定及生长性能研究

通过对川南某酒厂的老窖泥富集培养、分离纯化,经丁酸发酵实验,筛选出1株高产丁酸的梭菌。经16Sr DNA鉴定,该菌为丁酸梭状芽孢杆菌(Clostridium butyricum),命名为丁酸菌YD-4。经对该菌株生长性能条件研究,其最适生长温度为37℃,最适p H值为7.0,最适接种量为2%,菌体浓度达1.64×108个/m L,产丁酸量可达到3.44 g/L。     

海洋青霉碱性脂肪酶液态发酵和部分酶学性质研究

从海洋菌中分离筛选到1株碱性脂肪酶产生菌H-19,对该菌产脂肪酶的液态发酵条件进行了优化,优化后的培养基组成(%):葡萄糖0.5,大豆油1.0,蛋白胨1.0,(NH4)2SO40.5,NaCl 2.75,KCl 0.1,MgCl20.5,MgSO4.7H2O 0.2,CaCl20.05;发酵培养基起始pH8.0,发酵温度28℃,摇床转速180 r/min,发酵周期48 h,脂肪酶的活力可达17.43 IU/mL。酶的最适反应pH为9.4,最适温度36℃,半失活温度为60℃。     

基于高含油废水降解的产脂肪酶菌株的 筛选及发酵工艺优化

为了筛选高产脂肪酶菌株并探究其对高含油废水的降解能力,采用传统的微生物筛选法从餐厨废水中分离筛选菌株,考察菌株对高含油废水（油脂含量20 g/L）的油脂降解率和产脂肪酶酶活,并对产脂肪酶酶活较高的菌株进行鉴定。采用单因素实验和响应面实验对鉴定菌株发酵产脂肪酶的工艺条件进行优化。结果表明：从餐厨废水中筛选出3株细菌、2株酵母菌,其中2株细菌可在高含油废水中快速生长代谢,油脂降解率和产脂肪酶酶活均较高,经鉴定1株为解淀粉芽孢杆菌（Bi）,1株为鲁氏不动杆菌（Bu）;Bi产脂肪酶最优发酵工艺条件为pH 6.69、氯化钠含量301 g/L、蛋白胨含量6.01 g/L;Bu产脂肪酶最优发酵工艺条件为pH 5.07、氯化钠含量6.10 g/L、蛋白胨含量9.38 g/L;在最优发酵工艺条件下,Bi和Bu产脂肪酶酶活分别为（222.83±0.91）U/mL和（231.50±2.28）U/mL,优化后的Bu产脂肪酶酶活甚至高于初始脂肪酶酶活更高的Bi。综上,2株细菌具有高含油废水降解能力,具备广阔的应用前景。     

一株毛霉菌产脂肪酶特性及发酵条件研究

文中观察了一株产脂肪酶毛霉(Mucor sp.)菌株在橄榄油罗丹明B鉴定平板上的菌落形态,对其产脂肪酶进行了酶学特性研究,并探讨了该菌株产脂肪酶的发酵条件,优化了产酶发酵培养基和产酶发酵条件,得出了该菌产酶的最佳发酵条件为以1%全脂大豆粉为氮源,1%玉米浆为碳源,添加0.5%橄榄油和0.1% Tween-80,控制起始pH值为6.0.     

不同微生物菌株产脂肪酶发酵条件优化

从石油污染土壤中分离、筛选出4株产脂肪酶高的微生物菌株,采用正交试验对4株菌产脂肪酶发酵条件进行了初步优化。结果表明,碳源种类和油含量对各种菌株产酶的影响效果较大;其中D4菌株产酶活最高,达到51.667U/mL;而A6菌株产酶活最低,为23.334U/mL。为进一步工业化生产脂肪酶奠定理论基础。     

一株产耐有机溶剂脂肪酶伯克霍尔德氏菌的分子鉴定及酶学性质分析北大核心CSCD

为筛选具有潜在应用价值的产脂肪酶菌株,采用三丁酸甘油酯固体培养基从黄山松树林土壤中筛选产脂肪酶菌株,利用16S rDNA测序对筛选的菌株进行鉴定,并对其产脂肪酶酶活力参数以及酶学性质进行分析。结果表明:筛选的产脂肪酶菌株HSU-7为伯克霍尔德氏菌(Burkholderia sp. HSU-7);菌株HSU-7所产脂肪酶的最适反应温度为45℃,最适pH为5,为一种耐酸性的中温脂肪酶;菌株HSU-7发酵5 d,所产脂肪酶酶活力最大,为72.22 U/mL。同时,K^(+)、Ca^(2+)和Mg^(2+)对菌株HSU-7所产脂肪酶酶活力具有显著的促进作用,而Ni^(2+)、Cu^(2+)、Fe^(3+)和Zn^(2+)具有一定的抑制作用,该脂肪酶可耐受有机溶剂甲醇、乙醇、异丙醇和二甲基亚砜,但Triton X-100可显著抑制其酶活力;此外,该脂肪酶能耐受65℃的高温。综上,菌株HSU-7可高产脂肪酶,且该脂肪酶有较强的热稳定性,可耐受多种有机溶剂,具有很好的工业应用价值。     

脂肪酶产生菌Bacillus subtilis FS321的分离鉴定及其产酶条件的初步优化

从北方堆肥试样中分离筛选得到1株中度耐热脂肪酶产生菌,编号为FS321。对该菌株进行产酶条件的初步优化,得到了摇瓶发酵条件:产酶培养基M4,发酵周期为48～60 h,摇瓶发酵温度为37℃,通气量为25 mL/250 mL锥形瓶。所产脂肪酶在50℃,pH 9.0时表现最高活性。为了鉴定该菌株,分析该菌的细胞脂肪酸谱,克隆测序了其16S rDNA基因序列,系统进化树及细胞脂肪酸组分分析均表明,FS321与Bacillus subtilis具有最紧密的亲缘关系。     

窖泥中高产丁酸梭菌的筛选、鉴定及其生长耐受性研究

从浓香型白酒窖泥中通过传统微生物分离手段筛选高产丁酸的梭菌,并对分离菌株进行形态学观察、分子生物学鉴定及生长耐受性分析研究。结果表明,通过发酵产物检测获得两株高产丁酸菌株,编号为AB2RH027和AB2RH033,经过菌落观察、革兰氏染色及分子生物学鉴定两株菌均为丁酸梭菌（Clostridium butyricum）。通过耐受性实验,确定菌株AB2RH027最适生长pH值为8,最适生长温度为40 ℃,酒精耐受性为15%vol,发酵24 h丁酸产量为5.29 g/L;菌株AB2RH033最适生长pH值为8,最适生长温度为35 ℃,酒精耐受性为18%vol,发酵24 h丁酸产量为5.23 g/L。该研究筛选得到的两株高产丁酸的菌株在白酒品质提升方面具有一定应用价值。     

丁酸梭菌的生物学特性分析及发酵培养基优化

该研究以四株丁酸梭菌（Clostridium butyricum）为出发菌,通过测定4株菌产酸性能、产酶活力、抑菌性能和抗逆性等,筛选生物学性能最优的丁酸梭菌,并以活菌数为响应值,通过Plackett-Burman试验、最陡爬坡试验和响应面试验对其发酵培养基组成进行优化。结果表明,筛选得到生物学性能最优的菌株为丁酸梭菌BLCC1-0022,其最优发酵培养基组成为：可溶性淀粉2.42%、蛋白胨3.27%、牛肉浸膏3.31%、CaCO3 2.47%、MgSO4·7H2O 0.025%、MnSO4·H2O 0.025%,pH 7.0±0.2。在此最优培养基组成下,按照2%（V/V）接种量接种丁酸梭菌发酵液,37 ℃静置培养24 h,丁酸梭菌BLCC1-0022活菌数达6.721×108 CFU/mL,芽孢率为98.21%,与原培养基相比,分别提高了3.22倍、17%。     

一株酸性脂肪酶高产菌株的筛选与鉴定

目的分离筛选出一株酸性脂肪酶高产菌株并对其进行鉴定。方法通过溴甲酚紫酸碱指示剂进行平板初筛,甘油三丁酸酯透明圈法以及摇瓶培养复筛,筛选获得了一株酸性脂肪酶产生菌株;利用平板透明圈法、橄榄油乳化液滴定法和对硝基苯酚比色法3种方法对所筛菌株进行酶活力测定。结果分离获得一株具有高产酸性脂肪酶活力的菌株,将其命名为菌株WY19。经过检测,其粗酶活力达到11000 U/L。通过对菌株WY19进行形态学鉴定、生理生化实验以及分子生物学鉴定,结合系统发育树分析,确定本研究获得的酸性脂肪酶产生菌为克雷伯氏菌。结论菌株WY19所产脂肪酶为酸性脂肪酶,在食品、医药、油脂加工等领域方面具有较好的应用前景。     

碱性脂肪酶高产菌株Fusariumsp.N4-2的筛选与产酶条件研究

从碱湖鱼内脏中分离筛选出1株碱性脂肪酶的高产菌株N4-2,初步鉴定为镰孢霉属(Fusarium)。摇瓶产酶实验表明,该菌株最适产酶培养基的组成为(g/L):橄榄油30,酵母膏5,(NH4)2SO43,MgSO40·75,CaCl20·2,K2HPO44。最适产酶温度30℃,最佳产酶pH为10·0,生长高峰出现在第43h,产酶高峰出现在第52h,碱性脂肪酶的活力高达605·15U/mL,此菌株生长周期短,产酶活力较高,在洗涤制剂、印染、制革等行业有良好的应用前景。     

Isolation and analysis of lipase-overproducing mutants of Serratia marcescens

We have isolated a lipase-overproducing mutant, GE14, from Serratia marcescens 8000 after three rounds of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutant GE14 produced 95 kU/ml of extracellular lipase in the lipase medium, which was about threefold higher than that of produced by the original strain 8000. Enzymatic characteristics including specific activity of purified lipases from culture supernatants of GE14 and 8000 were almost same. The lipase gene (lipA) of GE14 contained two base substitutions; one in the promoter region and another in the N-terminal region of the lipA gene without an amino acid substitution. Promoter analysis using lipA-lacZ fusion plasmids revealed that these substitutions were responsible for the increase in the lipA expression level, independently. In contrast, no base substitution was found in the genes encoding the lipase secretion device, the Lip system. In addition, the genes coding for metalloprotease and the cell surface layer protein which are both secreted through the Lip system and associated with extracellular lipase production, also contained no base substitution. The strain GE14 carrying a high-copy-number lipA plasmid produced a larger amount of the extracellular lipase than the recombinant strains of 8000 and other mutants also did, indicating that GE14 was not only a lipase-overproducing strain, but also an advantageous host strain for overproducing the lipase by a recombinant DNA technique. These results suggest that the lipase-overproducing mutant GE14 and its recombinant strains are promising candidates for the industrial production of the S. marcescens lipase.     

Lipase production by solid fermentation of soybean meal with different supplements

The aim of this work was to study the production of extracellular lipases by solid state fermentation in soybean meal with different supplements. Lipase production by two microorganisms, screened by their potential for lipase production, was followed in terms of hydrolytic activity at different pH values(4, 7 and 9). The supplementation of the medium with urea and soybean oil significantly increased the enzyme production for all studied pH and microorganisms. Microorganism Penicillium P58 and P74 showed the possibility of production of different lipases with alkaline and acidic characteristics. In soybean meal supplemented with urea and soybean oil, this microorganism yielded 139.2 and 140.7U lipase/g of dry substrate in 48 h of fermentation, in alkaline and acidic conditions, respectively. Different behavior was observed when the enzyme extract was evaluated in neutral conditions, which yielded 180.0U lipase/g in 72 h. A new promising lipase producer strain was testedon soybean meal with different supplements. The strain produced a lipase with high activities by solid state fermentation of soybean meal supplemented with urea and soybean oil.     

高产脂肪酶菌株的筛选鉴定及产酶条件优化

采用罗丹明B平板法和脂肪酶活力测定从张弓老酒大曲中分离筛选出一株高产脂肪酶菌株E-11,经形态学、生理生化试验和16S rDNA序列分析,鉴定该菌株为枯草芽孢杆菌（Bacillus subtilis）。通过单因素和响应面试验设计对该菌株的产酶条件进行优化,结果表明,最佳产酶条件为葡萄糖3%、蛋白胨4%、初始pH值为8.0。优化后的脂肪酶活力达15.09 U/mL,是优化前产酶能力的1.2倍。     

Burkholderia sp.JXJ-16低温耐有机溶剂脂肪酶产酶条件优化及粗酶酶学性质

以Burkholderia sp.JXJ-16为出发菌株,对其低温耐有机溶剂脂肪酶的产酶条件进行单因素实验并研究其粗酶酶学性质。该菌株的最佳产酶条件为:蔗糖3.75 g/L、尿素11.25 g/L、K₂HPO₄2 g/L、（NH4）2SO₄1 g/L、Mn SO₄0.25 g/L、猪油乳化液体积分数2.5%,初始p H9.0、培养温度30℃、装样量20 m L/250 m L、接种量3%、发酵时间20 h。JXJ-16脂肪酶粗酶对中链对硝基苯酚酯有最大水解活力,最适底物为对硝基苯酚辛酸酯;该粗酶在35℃、p H8.5⁹.0时酶活力最高,且具有较好的温度（30⁶0℃）和p H（5.0¹0.5）稳定性;Na⁺、Mg²⁺、Ca²⁺、Mn²⁺、EDTA对粗酶活力具有激活作用,Zn²⁺、Cu²⁺、Fe³⁺对粗酶活力具有抑制作用,K⁺对粗酶活力没有显著性影响;除乙醇和乙腈外,该粗酶在一定体积分数的异丙醇、甲醇、丙酮、乙酸乙酯、三氯甲烷、二甲苯和正己烷中具有良好耐受性,且处于激活状态。综上,该菌株能利用廉价易得的培养基原料达到最佳产酶效果,其所产脂肪酶为低温碱性脂肪酶,具有较好的温度和p H稳定性,对多数供试有机溶剂具有良好耐受性。      

Isolation and some properties of extracellular heat-stable lipases from Pseudomonas fluorescens strain AFT 36

Aeration increased the growth and lipase production in milk by Pseudomonas fluorescens strain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented approximately 71% of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8.0 and 35 degrees C; it had a Km on tributyrin of 3.65 mM and was inhibited by concentrations of substrate greater than approximately 17 mM. The enzyme was very stable over the pH range 6-9; it was relatively heat-labile in phosphate buffer in the temperature range 60-80 degrees C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100-150 degrees C: the D values at 150 degrees C were approximately 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the corresponding Z values in the temperature range 100-150 degrees C were approximately 40 and approximately 42 degrees C and the Ea for inactivation were 7.65 X 10(4) J mol-1 and 6.97 X 10(4) J mol-1 respectively.     

镇江香醋醋醅中优势高产酸醋酸菌菌株的筛选

镇江香醋是中国传统发酵香醋之一,具有较高的营养价值。本实验对镇江香醋醋醅中高产酸的优势醋酸菌进行了筛选和分离鉴定。最终分离得到15株产酸菌,通过16S r DNA序列分析,鉴定出8株醋酸菌。8株醋酸菌产酸分析结果表明,醋酸菌D-3-4的产酸量达到60 g/L,为高产酸菌株。对8株醋酸菌进行耐酒精、耐温度、耐乙酸性能测试后发现:在酒精浓度不超过9%时,醋酸菌D-3-4的产酸量最高;当酒精浓度大于9%时,醋酸菌C-3-2-1的产酸量最高,酒精转化率最高;当温度为42℃时,醋酸菌D-3-4和R-4-2仍有30 g/L的产酸量;当乙酸浓度大于30 g/L时,醋酸菌D-3-4与C-3-2-2的产酸量在各菌株中最高;综合各性能比较得出醋酸菌D-3-4性能最优,为优势高产酸醋酸菌。      

D-核糖高产菌种的代谢控制育种

本文综述了核糖高产菌的生物合成途径、高产菌种的生化特征及育种思路,认为芽孢杆菌属的菌株具有核糖的生产能力,转酮酶缺陷是选育核糖高产菌的关键,提高葡萄糖酸激酶或葡萄糖脱氢酶的活性和丧失孢子形成能力,则有利于核糖的大量积累,此外,发酵培养基和培养条件对核糖的积累也非常重要。     

碱性脂肪酶产生菌的筛选及产酶条件的优化

通过固体平板法从含油脂土样中筛选出一株产碱性脂肪酶活力较高的菌株 ,鉴定为解淀粉芽孢杆菌。此菌最佳产酶条件为 :1%淀粉为碳源 ,2 %黄豆粉和 2 %玉米粉为氮源 ,培养基起始 pH 7.0 ,3 0℃培养 72h。对发酵液性质进行初步研究发现 ,此酶最适反应温度为3 7℃ ,最佳反应 pH为 8.5。 0 .0 1mol/LCa2 + 和K+ 对酶有激活作用 ,而Cu2 + 和Fe3 + 则对该酶有抑制作用。