产低温脂肪酶非极端细菌的筛选、产酶发酵及粗酶性质研究

目的：筛选产低温脂肪酶非极端细菌菌株,扩大脂肪酶的应用范围。方法：利用维多利亚蓝B平板显色法和摇瓶发酵法,从土壤中筛选产脂肪酶菌株,通过菌落形态和菌体特征观察初步对菌种进行鉴定,并对该菌株的产酶发酵培养基进行了优化。结果：得到一株产低温脂肪酶非极端细菌菌株sybc—li一1,该菌株适宜产酶培养基（％）为淀粉1、牛肉浸膏1、NaNO3 0．08、CaCl2 0．04、MgSO4 0．04、橄榄油2和OP1;初始DH8、30℃、200r／min培养72h,脂肪酶活力可高达到30．2U／mL;所产脂肪酶粗酶最适作用温度20℃,最适pH9．5,0℃时仍能保持70％的酶活性,属于低温酶;该酶与目前报道的低温脂肪酶相比,有较好的热稳定性,粗酶在pH8．5、70℃条件下保温60mla,酶活力损失30％。结论：该菌株为自然环境中筛选的非极端细菌,所产脂肪酶为低温脂肪酶,在开发应用上有良好的前景。     

热稳定碱性脂肪酶产生菌的筛选及发酵条件的优化

从渤海湾盐碱地被油污染的土样中分离筛选出6株产热稳定碱性脂肪酶菌株。其中菌株1-7产脂肪酶能力较强,其最高酶活为8.67U/mL。根据其16S rDNA序列分析和Biolog生理生化分析,初步鉴定为醋酸钙不动杆菌(Acinetobacter calcoaceticus)。初步酶学性质研究表明该菌所产脂肪酶具有较好的热稳定性,最适作用pH为9.0。摇瓶实验表明,该菌株最适产酶培养基为(g/L):玉米粉10,黄豆饼粉20,K_2HPO_4 1,NaNO_3 5,橄榄油10。最适产酶条件为:初始pH 8.0,培养温度37℃,培养时间29 h。接种量为2%,250 mL摇瓶装液量为30 mL。     

枯草芽胞杆菌甲壳素脱乙酰酶的筛选及酶学性质*

从海洋泥土中分离出产甲壳素脱乙酰酶菌株,确定该菌株为产碱属芽孢杆菌,其产酶适宜培养条件为：pH4．0,添加金属离子Ca^2 ,培养时间为80h,温度为350℃。所得甲壳素脱乙酰酶作用的最适温度为40℃～50℃,最适pH为4．5-5．0之间。     

脂肪酶产生菌Geofrichum sp. AS 2.1135产酶条件及酶一般性质的研究

在92株能同化油的地霉属菌株中,Geotrichum sp. AS2．1135菌株产脂肪酶活力为50—60u／ml,对其产酶条件的研究表明,不饱和长链脂肪酸和油类有利于酶的形成。在4％豆饼粉作为有机氮源的培养基中,加入0．2％尿素,酶活力显著增加,酶活达150u／ml。用聚乙二醇橄榄油乳化系统测定酶的作用最适pH为8．0,最适温度为4O一42℃。在pH4一9时5℃下存放24小时,或在pH 5和8时45℃保持15分钟,酶活力不变。     

醋酸钙不动杆菌产生的耐热碱性脂肪酶的研究

从福建省福州市郊区土壤中分离筛选到一株活力强的作用温度高的碱性脂肪酶野生型菌株：醋酸钙不动杆菌F-1903。该菌产酶培养基组成(％)：大豆粉2.O、玉米浆1.0、糊精1.0、磷酸氢二钾0．5、硝酸钠0．5。产酶最适条件：发酵培养基起始pH9.0,温度26℃,周期2。小时。酶作用最适温度54℃,最适pH9．2。Ca2+件对酶有激活作用,EDTA有抑制作用。     

Aspergillus sp.脂肪酶发酵条件优化及酶学性质的研究

作者为了得到一种热稳定性较好的脂肪酶新酶种,通过研究分离白极端环境的Aspergillus sp．的最佳产酶条件及其所产脂肪酶的酶学性质,得出了该菌产酶的最佳发酵条件为：以1％黄豆饼粉为氮源、0．2％玉米淀粉为碳源,1．5％橄榄油为诱导物,起始pH6．0左右。装量10mL（250mL三角瓶。摇瓶转速180r／min）、发酵时间为96h。在最佳发酵条件下可得最大发酵酶活36U/mL。Aspergillus sp．所产的脂肪酶的酶学性质是：最适pH为9．0,在pH5．0—10．0于20℃下放置24h后,残余酶活仍保持在起始酶活的90％以上;该酶的最适温度为50℃,50℃保温60min后仍保留70％以上的酶活。Aspergillus sp．所产脂肪酶的热稳定性较好。     

高产耐高温脂肪酶生产菌的筛选与鉴定

从小笼包蒸屉垫中筛选得到了两株脂肪酶高产菌株J2和J3,经形态观察以及26S rRNA基因(26S rDNA)序列比对鉴定,两株菌分别属于Aureobasidium属的两个变体。200 r/min、30℃下摇瓶发酵3-5 d后,以对硝基苯酚棕榈酸酯(p-NPP)作为底物,用分光光度法测得J2和J3发酵上清液中的脂肪酶酶活分别为10.61 U/m L和14.43 U/m L。对两株菌所产脂肪酶的耐热特性研究显示,菌株J2产脂肪酶的最适反应温度为50℃,并且酶液在50℃保温5 h无酶活损失;另一株菌J3所产脂肪酶的最适反应温度为60℃,酶液在50℃保温5 h后酶活剩余42.19%,在40℃保温5 h没有酶活损失。这表明J2和J3菌株所产脂肪酶具有较好的热稳定性和较高的最适反应温度。     

四株冰川雪细菌产酶能力的初步研究

目的:从青藏高原冰川雪中筛选出一菌多酶的菌株.方法:对恢复出的4个细菌,通过平板透明法研究其产淀粉酶、脂肪酶和蛋白酶的特性.结果:LHG-C-9为惟一可以产淀粉酶的菌株,所产脂肪酶活性最高.4个菌株均不产蛋白酶.结论:LHG-C-9最适生长温度为15℃,属于耐冷菌.对该菌所产淀粉酶和脂肪酶的性质进行了初步研究,其随产淀粉酶的最适作用温度为50℃;最佳产酶pH值为7.0,该pH值所产酶活为83.9U/mL;在60℃的高温下温浴10min后酶活为0%.该菌株所产脂肪酶的最适作用温度为20℃;最佳产酶pH值为7.0,该pH值所产酶活为9.2U/mL;50℃温浴1h后酶活力不足34%.     

极端微生物产碱性蛋白酶菌株的筛选及发酵条件研究*

从深海的 3 8份水样和泥样中筛选到一株产蛋白酶的菌株DY A ,其最适生长温度为1 0℃ ,能适应较大范围的pH值和盐度。此菌株只在酵母膏存在的情况下产酶 ,不利用单一氮源 ,最适产酶条件为 :pH1 0 0 ,1 0℃ ,接种量 0 5 % ,2 0 0r/min摇床培养 48～ 72h。粗酶液的最适作用温度为 40℃ ,最适pH值为 1 0 ,在pH9～ 1 2内稳定 ,是一碱性蛋白酶。     

中国冰川1号产适冷蛋白酶耐冷菌的分离鉴定及产酶条件

从中国冰川 1号样品分离获得一株产适冷蛋白酶耐冷菌株SYP- A2 - 3,鉴定为蜡状芽孢杆菌 (Bacilluscereus)。该菌生长温度范围为 0～ 38℃ ,最适生长温度 2 5℃ ,而最适产酶温度为 15℃。所产蛋白酶为中性金属蛋白酶 ,最适催化温度为 4 2℃ ,低温催化活力较高 ,适宜作用pH为 7. 0～ 8 .5 ,SDS PAGE测定的分子量为 34 2kD。SYP A2 3产酶条件的研究结果显示酪蛋白是较好的氮源 ,葡萄糖、淀粉是较好的碳源 ,产酶最佳pH为 6. 5～ 7. 0 ,在优化的条件下 ,15℃摇瓶产酶达到 380 0U mL ,5L发酵罐通气培养产酶达 4 80 0U mL。     

Optimization of extracellular psychrophilic alkaline lipase produced by marine Pseudomonas sp. (MSI057)

An endosymbiotic Pseudomonas sp. (MSI057), which could produce high yields of lipase, was isolated from marine sponge Dendrilla nigra, collected from the peninsular coast of India. Maximum production of enzyme was obtained in minimal medium supplemented with 1% tributyrin. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 30 degrees C and 9.0, respectively. The enzyme exhibited maximum activity in pH range of 8-9 with an optimum pH 9.0. The activity of purified enzyme was optimum at 37 degrees C and showed 80% activity at 20 degrees C and the enzyme activity decreased dramatically above 50 degrees C. Based on the present findings, the enzyme was characterized as psychrophilic alkaline lipase, which can be developed for industrial applications.     

Studies on the enhanced production of extracellular lipase by Staphylococcus epidermidis

Staphylococcus epidermidis isolated from spoiled frozen marine fish samples exhibited optimum lipase activity of 8.1 U within 72 h in batch fermentation. Inducible effect of different sugars, nitrogen sources, salts and metal ions were studied on enzyme production. Trybutyrin induced the enzyme production by twofold. Addition of lactose in the production medium further improved lipase production. Sodium chloride increased lipase production whereas the presence of metals in the media had an inhibitory effect. Cells of immobilized S. epidermidis in agar beads (3%) increased lipase production compared with free cells. The optimum temperature and pH for enzyme activity was 20 degrees C and 7.0 respectively. Lipase retained its 85% stability at pH 6.0 and at 40 degrees C. Immobilized cells with high lipolytic activity and stability may provide commercial advantages over conventional methods of lipase production.     

Lipolytic activity from Halobacteria: screening and hydrolase production

Strains of Halobacteria from an Algerian culture collection were screened for their lipolytic activity against p-nitrophenyl butyrate (PNPB) and p-nitrophenyl palmitate (PNPP). Most strains were active on both esters and 12% hydrolyzed olive oil. A strain identified as Natronococcus sp. was further studied. It grew optimally at 3.5 M NaCl, pH 8 and 40 degrees C. An increase in temperature shifted the optimum salt concentration range for growth from a wider range of 2-4 M, obtained at 25-30 degrees C, to a narrower range of 3.5-4 M, obtained at 35-40 degrees C. At 45 degrees C the optimum salt concentration was 2 M. These results show a clear correlation between salt and temperature requirement. The optimum conditions for the production of hydrolytic activity during growth were: 3.5 M NaCl and pH 8 for PNPB hydrolytic activity and 4 M NaCl and pH 7.5 for PNPP hydrolytic activity; both at 40 degrees C. The clear supernatant of cells grown at 4 M NaCl showed olive oil hydrolysis activity (in presence of 4 M NaCl) demonstrating the occurrence of a lipase activity in this strain. To our knowledge, this is the first report of a lipase activity at such high salt concentration.     

黑曲霉F044脂肪酶的分离纯化及酶学性质研究

黑曲霉F044脂肪酶发酵上清液经硫酸铵沉淀、透析、DEAESepharoseFastFlow阴离子交换层析和SephadexG-75凝胶过滤层析得到电泳纯的脂肪酶,纯化倍数为73·71倍,活性回收率为34%。对纯化脂肪酶性质研究表明:该脂肪酶分子量约为35~40kD,水解橄榄油的最适温度和最适pH分别为45℃和7·0,在60℃以下和pH2·0~9·0之间有很好的稳定性。该脂肪酶的水解活性对Ca2 表现明显的依赖性,而Mn2 、Fe2 和Zn2 对脂肪酶则有显著的抑制作用。在最适条件下水解pNPP的Km和Vmax分别为7·37mmol/L和25·91μmol/(min·mg)。其N-端的15个氨基酸序列为Ser(Glu/His)-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln。     

Production, properties and application to nonaqueous enzymatic catalysis of lipase from a newly isolated Pseudomonas strain

A potent bacterium for lipase production was isolated from soil and identified as Pseudomonas species. It produced lipase constitutively. A mutant of this strain with a lipase productivity 3.25-fold higher was obtained by treatment with ultraviolet (UV) and nitrosoguanidine (NTG). Its fermentation condition was optimized to a lipase yield of 87.5 U/ml. The lipase had maximum activity at pH 9.0 and 45 degrees C. It was stable at pHs from 7.0 to 11.0 and below 60 degrees C. The effects of metal ions, surfactants and bile salts were also studied. The lipase was 1,3-specific. In organic solvents, the thermal stability of the lipase was significantly enhanced. Its optimum temperature was also slightly increased. The optimum water activity was found between 0.5 and 0.6. The lipase was successfully applied in organic phase to catalyze the glycerolysis of palm oil for monoglyceride (MG) production, and the enantioselective esterification of (R,S)-2-octanol. The enantioselectivity of the lipase could be enhanced substantially by treatment with an amphipathic.     

Characterization of extracellular esterase and lipase activities from five halophilic archaeal strains

A total of 118 halophilic archaeal collection of strains were screened for lipolytic activity and 18 of them were found positive on Rhodamine agar plates. The selected five isolates were further characterized to determine their optimum esterase and lipase activities at various ranges of salt, temperature and pH. The esterase and lipase activities were determined by the hydrolysis of pNPB and pNPP, respectively. The maximum hydrolytic activities were found in the supernatants of the isolates grown at complex medium with 25% NaCl and 1% gum Arabic. The highest esterase activity was obtained at pH 8-8.5, temperature 60-65 degrees C and NaCl 3-4.5 M. The same parameters for the highest lipase activities were found to be pH 8, temperature 45-65 degrees C and NaCl 3.5-4 M. These results indicate the presence of salt-dependent and temperature-tolerant lipolytic enzymes from halophilic archaeal strains. Kinetic parameters were determined according to Lineweaver-Burk plot. The KM and V (max) values were lower for pNPP hydrolysis than those for pNPB hydrolysis. The results point that the isolates have higher esterase activity comparing to lipase activity.     

An inducible,intracellular, alkalophilic lipase inAspergillus flavipes grown on triacylglycerols

An intracellular lipase was induced inAspergillus flavipes grown on various triacylglycerols at pH 6.0 and at 30°C, with maximum activity with sunflower oil. The lipase had an optimum pH for activity of 8.8 and retained 30% of its activity at pH 10.0. It had an optimum temperature for activity, measured over 30 min, of 45°C. It was completely inactivated at 60°C within 10 min.     

Optimization of a thermostable lipase from Bacillus stearothermophilus P1: overexpression, purification, and characterization

An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open reading frame of 1254 nucleotides coding a 29-amino-acid signal sequence and a mature sequence of 388 amino acids. The expressed lipase was isolated and purified to homogeneity in a single chromatographic step. The molecular mass of the lipase was determined to be approximately 43 kDa by SDS-PAGE and mass spectrometry. The purified lipase had an optimum pH of 8.5 and showed maximal activity at 55 degrees C. It was highly stable in the temperature range of 30-65 degrees C. The highest activity was found with p-nitrophenyl ester-caprate as the synthetic substrate and tricaprylin as the triacylglycerol. Its activity was strongly inhibited by 10 mM phenylmethanesulfonyl fluoride and 1-hexadecanesulfonyl chloride, indicating that it contains a serine residue which plays a key role in the catalytic mechanism. In addition, it was stable for 1 h at 37 degrees C in 0.1% Chaps and Triton X-100.     

Optimization of extracellular thermotolerant alkaline protease produced by marine <Emphasis Type="Italic">Roseobacter</Emphasis> sp. (MMD040)

Marine endosymbiontic Roseobacter sp. (MMD040), which produced high yields of protease, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in Luria-Bertani broth. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 37 degrees C and 7.0, respectively. The enzyme exhibited maximum activity in pH range of 6-9 with an optimum pH of 8.0 and retained nearly 92.5% activity at pH 9.0. The enzyme was stable at 40 degrees C and showed 89% activity at 50 degrees C. Based on the present findings, the enzyme was characterized as thermotolerant alkaline protease, which can be developed for industrial applications.     

Ester synthesis at extraordinarily low temperature of -3 degrees C by modified lipase in benzene

The lipoprotein lipase from Pseudomonas fluorescens was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine. The modified lipase in which 55% of the amino groups in the enzyme molecule were coupled with polyethylene glycol was found to be soluble in benzene and catalyzed the reactions of ester synthesis, ester exchange, aminolysis and ester hydrolysis in benzene. The modified lipase had an extraordinary temperature-dependency: enzymic activity for methyl laurate synthesis from methyl alcohol and lauric acid increased with decreasing temperature and attained the maximum at the extremely low temperature of -3 degrees C. The optimum temperature for hydrolysis of methyl laurate was as low as -4 degrees C.