脂肪酶产生菌NJY-1-7的选育及发酵条件的研究

从云南省福贡县的玉米地土壤样品中筛选到一株耐高温产碱性脂肪酶的菌株NJY-1-7,通过16SrDNA鉴定该菌株为伯克氏菌(Burkholderia cenocepacia)。对其发酵条件进行研究结果表明:该菌株产酶的最适培养时间为48h,酶促反应的最适作用pH值为9.0,最适作用温度为50℃,摇瓶发酵最适产酶条件为橄榄油1%,MgSO4.7H2O 0.05%,可溶性淀粉0.3%,酵母膏0.5%,单蒸水70mL。在此条件下发酵脂肪酶酶活力为42.00U/mL。     

一株耐热脂肪酶产生菌XD-23的发酵条件优化

优化脂肪酶产生菌XD-23的发酵条件,采用单因素发酵实验和正交试验,研究其菌株最佳发酵条件,从而提高该菌株所产脂肪酶的活力。通过单因素发酵试验得知,该菌株最佳发酵时间为48 h;最优碳源为橄榄油,其添加量为2.5%;最优氮源为蛋白胨,其添加量为3.0%;最适装液量为80 mL。通过正交试验优选出最佳组合,得出该菌株最适产酶条件为:MgSO4.7H2O 0.05 g,K2HPO4 0.1 g,CaCO2 0.25 g,橄榄油2.5 g,蛋白胨3.5 g,装液量50 mL,发酵培养时间为48 h,在此条件下酶活可提高到258.55 nKat。     

耐高温酸性脂肪酶菌株NJY-1-3的选育及发酵条件的研究

从云南省福贡县的玉米地土壤样品中筛选到一株耐高温产酸性脂肪酶的菌株NJY-1-3,通16SrDNA鉴定该菌株为伯克氏菌(Burkholderia pseudomallei)。对其发酵条件进行研究结果表明:该菌株产酶的最适培养时间为72h,酶促反应的最适作用pH4.0,最适作用温度为65℃,摇瓶发酵最适产酶条件为:橄榄油1%,MgSO4·7H2O0.05%,K2HPO40.1%,酵母膏1.0%,蔗糖0.8%,60mL单蒸水。在此条件下发酵脂肪酶酶活力可达到50.50IU/mL。该酶学特性非常适用于食物的处理、医药工业及生物柴油的制备等。     

脂肪酶产生菌的筛选及其产酶条件优化

通过罗丹明B固体平板显色法初筛和橄榄油乳化法测酶活力复筛,从富油土壤中筛选到1株脂肪酶产生菌HF45,并对该菌株的部分产酶条件进行了初步的研究。在单因素的基础上,采用正交实验对HF45菌株的发酵产酶条件进行优化,并对优化前后的水解产物进行检测。结果表明,该菌株产脂肪酶的最佳培养基组成是:麦芽糖3.0%,豆饼粉2%,KH2PO40.05%,最佳反应pH值为6。在此条件下,脂肪酶最高酶活力达到17.54 U/mL,且其水解甘油三酯的主要产物为1,3-甘油二酯。     

产脂肪酶菌株的筛选鉴定及产酶条件优化

以橄榄油为唯一碳源,采用油脂同化平板从食堂废弃物中筛选出一株产脂肪酶菌株HFE722。通过测定与分析该菌株16S rRNA基因序列,鉴定该菌株为芽孢杆菌（Bacillus sp.）。菌株HFE722在初始条件（发酵温度30 ℃,接种量为1%,自然pH,装液量为100 mL/250 mL,摇床转速为200 r/min）下培养36 h,测得发酵液上清液脂肪酶酶活为2.17 U/mL。优化后所得菌株HFE722产酶的最适发酵条件为：发酵温度30 ℃,发酵周期为36 h,接种量为1%（V/V）,初始pH 7.0,装液量为50 mL/250 mL,摇床转速为160 r/min。在最佳发酵条件下,发酵液上清液酶活可达到5.8 U/mL,酶活较优化前提高了167.28%。     

天祝牧区牦牛乳酥油中产脂肪酶菌株筛选及产酶条件的研究

从甘肃省天祝牧区牦牛乳酥油中分离筛选产脂肪酶微生物.经初筛、复筛后得菌株S122产脂肪酶活力较高;形态学观察和26S rDNA序列比对分析表明该菌为Geotrichum sp.S122.对菌株Geotrichum sp.S122发酵产酶条件进行研究表明,其最适的发酵培养基组成成分(%):蔗糖1.0、蛋白胨2.0、硫酸镁0.1、磷酸氢二钾1.0、橄榄油乳化液1.0;最适发酵产酶条件:培养基初始pH8.0,培养温度28℃,接种量4%,装液量60mL/250mL,摇床转速200r/min,发酵周期96h,优化后脂肪酶活力最高达59U/mL.     

一株产耐高温碱性脂肪酶菌株的筛选及产酶条件优化

用Tween平板法从采集自河北省的122份土样中筛选到一株脂肪酶活性较高的菌株Lipa1318。综合其形态学和生理生化特征以及16S r DNA的序列比对结果,确定该菌株为粘质沙雷氏菌(Serratia marcescens)。其所产胞外脂肪酶的最适反应温度为60℃,最适p H为8.0,是一种耐热的碱性脂肪酶。摇瓶发酵实验证明该菌株的最适产酶条件为:葡萄糖1.0%,牛肉膏1.0%,Triton X-100 1.5%,橄榄油0.50%,Ca Cl20.50mmol/L,发酵液初始p H为8.0,30℃培养96h,通过发酵条件的优化使酶活提高了3.6倍。     

高效产脂肪酶菌株Burkholderia cepacia C1产酶条件优化

从油菜地土壤中分离到一株高效产脂肪酶菌株C1,经鉴定为Burkholderia cepacia。为了进一步提高C1 脂肪酶的产量,对其产酶的发酵条件进行优化。首先采用单因子试验筛选出最佳碳源为麸皮,最佳氮源为蛋白胨。通过Plackett-Burman 设计对发酵产酶的11 个相关因子进行试验,筛选出3 个主效因子,即蛋白胨质量浓度、橄榄油质量浓度及装液量。利用Box-Behnken 试验设计和响应曲面法分析确定主效因子的最优水平,得出产脂肪酶菌株C1 的最优产酶条件：1.50g/100mL 麸皮、1.05g/100mL 蛋白胨、1.63g/100mL 橄榄油、0.2g/100mL K2HPO4、0.05g/100mL MgSO4、初始pH 值为7.0、装液量为30mL。在优化条件下30℃,180r/min 培养72h,脂肪酶活力达到89.65U/mL,比未优化前的28.50U/mL 提高了2.15 倍。     

产碱性脂肪酶热带假丝酵母LYSC-3的筛选、鉴定及发酵条件的优化

以橄榄油为唯一碳源,以溴钾酚紫为显色剂,采用琼脂平板法从富含油脂的土壤中筛选到一株产碱性脂肪酶野生型菌株LYSC-3。经形态学、生理生化和分子生物学的鉴定,确定菌株LYSC-3为热带假丝酵母(Candida tropicalis)。经发酵条件优化,菌株LYSC-3的最佳产碱性脂肪酶的发酵条件为:橄榄油10g·L-1、蛋白胨5g·L-1、蔗糖20g·L-1、(NH4)2SO40.5g·L-1、MgSO4·7H2O0.2g·L-1,K2HPO40.2g·L-1,pH8.0,35℃,150r·min-1摇床振荡培养3d,获得碱性脂肪酶最高酶活力达38.6U·mL-1。     

内生黑曲霉产脂肪酶条件研究及其粗酶酶学特性

针对新疆酿酒葡萄中获得的1株产脂肪酶的内生黑曲霉菌株C2J6,研究了该菌株的产酶条件及酶学特性。结果表明,该菌适宜的产酶条件为1%的乳糖为碳源,1%的蛋白胨为氮源,培养基初始pH值为8.0,培养温度35℃,培养时间约72h,此时所产脂肪酶的活力可达18.75U/mL。该菌所产脂肪酶粗酶液的最适反应温度为40℃,最适反应pH值为7.0,为中温中性酶;在50℃保温1h酶活力保留54.55%,具有良好的热稳定性;在pH值3.0~7.0范围内较稳定,有一定的耐酸性。金属离子Mn2+对酶活力有促进作用,Zn2+、Fe2+、Cu2+对酶活力有抑制作用,K+、Ca2+、Na+、Mg2+对酶活力影响不大。该酶在以葵花油和谷物调和油为底物时,酶活分别为228%和180%,明显高于橄榄油和油烟机废油。     

Production of active lipase by Rhizopus oryzae from sugarcane bagasse: solid state fermentation in a tray bioreactor

This study deals with production of lipase in solid state fermentation by Rhizopus oryzae from sugarcane bagasse. A tray bioreactor was designed for the extracellular enzyme production. Daily, lipase production was evaluated at several incubation temperatures. Furthermore, the influence of temperature and humidity of the cabinet, depth of solid bed, particle size, initial moisture content and supplementary substrate (olive oil) as carbon source was investigated. The obtained results showed that bioreactor temperature of 45 °C, humidity of 80%, solid bed depth of 0.5 cm, particle size in the range of 0.335–1 mm, substrate initial moisture content of 80% for the top tray and 70% for the middle tray and supplementary substrate of 8% (v/w) olive oil led to maximum lipase production. Under optimum fermentation conditions after 72‐h incubation, maximum lipase activities for the top, middle and bottom trays were 215.16, 199.36 and 52.64 U gds^?1, respectively.     

粗状假丝酵母产脂肪酶发酵条件的优化

利用粗状假丝酵母发酵生产脂肪酶,通过对粗状假丝酵母胞外脂肪酶发酵过程的培养基和发酵培养条件的优化,得出产脂肪酶的最佳条件:碳源为聚乙烯醇乳化的橄榄油和葡萄糖,氮源为蛋白胨和尿素,酵母浸出液4.5g/L,MgSO4.7H2O0.4g/L,K2HPO43.5g/L;反应温度30℃,培养基初始pH7.0,接种量为10%,摇床转速180r/min,培养24h。在此发酵条件下,发酵液的最高酶活达到12U/mL。     

Lipase production by yeasts from extra virgin olive oil

Newly produced olive oil has an opalescent appearance due to the presence of solid particles and micro-drops of vegetation water from the fruits. Some of our recent microbiological research has shown that a rich micro-flora is present in the suspended fraction of the freshly produced olive oil capable of improving the quality of the oil through the hydrolysis of the oleuropein. Present research however has, for the first time, demonstrated the presence of lipase-positive yeasts in some samples of extra virgin olive oil which can lower the quality of the oil through the hydrolysis of the triglycerides. The tests performed with yeasts of our collection, previously isolated from olive oil, demonstrated that two lipase-producing yeast strains named Saccharomyces cerevisiae 1525 and Williopsis californica 1639 were able to hydrolyse different specific synthetic substrates represented by p-nitrophenyl stearate, 4-nitrophenyl palmitate, tripalmitin and triolein as well as olive oil triglycerides. The lipase activity in S. cerevisiae 1525 was confined to the whole cells, whereas in W. californica 1639 it was also detected in the extracellular fraction. The enzyme activity in both yeasts was influenced by the ratio of the aqueous to the organic phase reaching its maximum value in S. cerevisiae 1525 when the water added to the olive oil was present in a ratio of 0.25% (v/v), whereas in W. californica 1639 the optimal ratio was 1% (v/v). Furthermore, the free fatty acids of olive oil proved to be good inducers of lipase activity in both yeasts. The microbiological analysis carried out on commercial extra virgin olive oil, produced in four different geographic areas, demonstrated that the presence of lipase-producing yeast varied from zero to 56% of the total yeasts detected, according to the source of oil samples. The discovery of lipase-positive yeasts in some extra virgin olive oils leads us to believe that yeasts are able to contribute in a positive or negative way towards the organological quality of the olive oil.     

耐有机溶剂脂肪酶产生菌的筛选及其粗酶酶学性质

以体积分数2%苯为筛选压力,利用罗丹明B平板显色法和摇瓶发酵法,从采集的花生地土壤样品中分离筛选得到1株中度耐热、耐碱脂肪酶产生菌,编号为H2。通过形态观察、生理生化特性实验及其16S rDNA基因序列对菌种进行鉴定。结果表明,H2菌株与短小芽孢杆菌(Bacillus pumilus)的亲缘关系最紧密。通过研究得到该菌株的摇瓶发酵条件:产酶培养基为:蛋白胨3%、酵母膏1%、NaCl 0.5%、橄榄油1%,pH7.0,摇瓶发酵温度为28℃,摇床转速为180r/min,发酵周期为48～60h。所产脂肪酶在40℃、pH9.0时酶活性最高,对pH值和温度的适应范围较宽,pH6.0～10.0比较稳定,35～50℃具有较高酶活性。     

高产脂肪酶菌株的分离筛选与培养基优化

实验进行了高产脂肪酶菌株的分离筛选与发酵条件优化的研究。通过对35份富含油脂等样品的富集培养、分离筛选,获得320株产脂肪酶的细菌、酵母菌和霉菌,其中细菌X-13产酶活力最高,约为3.5U/mL;X-13发酵产脂肪酶的最佳碳源和氮源分别为可溶性淀粉、牛肉膏,Mg2+、Ca2+、Co2+对X-13菌株产酶有促进作用,Fe2+、Mn2+、Cu2+抑制菌株产酶;正交试验设计优化的培养基成分为：可溶性淀粉6g/L、牛肉膏4g/L、酵母粉0.5g/L、MgSO4 0.2g/L、聚乙二醇（PEG400）0.6mL/L、K2HPO4 1g/L、橄榄油乳化液20mL/L、pH值为7.0。在此优化培养条件下,细菌X-13培养72h的产脂肪酶活力达到9.28U/mL,较优化前提高2.65倍。     

Isolation and characterization of a lipid-degrading bacterium and its application to lipid-containing wastewater treatment

To construct an efficient lipid-containing wastewater treatment system, microorganisms that degrade lipids efficiently were isolated from various environmental sources. Strain DW2-1 showed the highest rate of degradation of 1% (w/v) salad oil among the isolated strains. Strain DW2-1 was identified as Burkholderia sp. and designated Burkholderia sp. DW2-1. The rate of degradation of salad oil, olive oil, sesame oil, and beef tallow by strain DW2-1 were 96.7%, 92.3%, 90.1% and 77.4%, respectively, during a 48-h cultivation. Strain DW2-1 grew well in a synthetic wastewater medium (>1 x 10(10) colony forming unit [CFU]/ml) between 20 degrees C and 38 degrees C, and its rate of degradation of salad oil was above 90% after a 48-h cultivation. The lipase and biosurfactant (BSF) activities of strain DW2-1 after a 48-h cultivation were 1720 U/l and 480 U/ml, respectively. In continuous cultures for lipid-containing wastewater treatment, DW2-1 was stably maintained and degraded more than 90% of salad oil during a 7-d cultivation.     

发酵生产脂肪酶的菌株筛选及其应用

采用从大庆日月星油厂附近的土壤中筛选得到产脂肪酶的菌株xjA,在固态发酵的条件下生产脂肪酶,并将所产脂肪酶应用于合成共轭亚油酸甘油酯。本实验利用单因素和正交试验方法确定产脂肪酶的最佳固态发酵条件：在种龄36h的情况下,碳源(麸皮)与氮源(豆粕)质量比(m麸皮:m豆粕)为1:4、V培养基:V加水量 1:1、接种量0.3%、发酵温度29℃、发酵时间3d。在此条件下进行固态发酵,固态培养基中脂肪酶的活力最高,可达1450U/g;所得脂肪酶用于合成共轭亚油酸甘油酯,产物中共轭亚油酸(CLA)接入率可达到13.6%。     

利用伯克霍尔德氏菌脂肪酶富集裂壶藻油脂中的二十二碳六烯酸

目的：研究伯克霍尔德氏菌胞外脂肪酶对裂壶藻油脂中二十二碳六烯酸（docosahexaenoic acid,DHA）的富集作用。方法：用三丁酸甘油酯平板筛选法,筛选产脂肪酶的细菌;以对硝基苯月桂酸酯为底物,对筛选得到的伯克霍尔德氏菌脂肪酶的酶学性质进行研究;用该脂肪酶水解裂壶藻油脂,通过气相色谱法测定酶解后水相和有机相的脂肪酸组成来研究其对裂壶藻油脂DHA的富集作用。结果：从广州的泥土样品中分离获得一株高产胞外脂肪酶的伯克霍尔德氏菌,在产酶培养基中30 ℃、200 r/min的条件下培养40 h后,脂肪酶酶活力达到最大值70 U/mL。脂肪酶粗酶液酶活性的最适温度为45 ℃,最适pH值为8.5。该菌的脂肪酶对裂壶藻油脂中的DHA有一定的富集作用,能够使油脂中DHA占总脂肪酸的质量分数从最初的30%升高到40%。结论：筛选得到一株能够产生选择性富集DHA的脂肪酶的细菌,拓宽了可用于多不饱和脂肪酸分离纯化的脂肪酶的范围。