微球菌的脂肪酶合成调节

本文介绍了微球菌１５０４胞外脂肪酶的代谢调节模式。发现在发酵培养基中,通过补加长链脂肪酸及其油脂,能提高脂肪酶的生产水平,而中、短链脂肪酸及其酯抑制产酶能力;高浓度的葡萄糖抑制产酶能力;补加不同浓度的橄榄油,适宜补加时间也应不同。水洗细胞脂肪酶的合成受橄榄油和油酸诱导,受葡萄糖和甘油阻遏,并发现脂肪酶的分解代谢阻遏主要发生在转录水平上。     

GL-7ACA酰化酶基因工程菌(Escherichia coli MMR204/pZC1)批式补糖发酵条件优化

戊二酰 7 氨基头孢烷酸 (GL-7-ACA)酰化酶 (3.1.5.11)可有效催化GL-7ACA分子中戊二酰基侧链水解 ,形成7-氨基头孢烷酸 (7-ACA)而成为两步酶法生产7-ACA的重要工业用酶之一。在已构建的GL-7ACA酰化酶基因(acy)重组质粒pZC1基础上 ,进一步对酰化酶基因工程菌EscherichiacoliMMR204pZC1的产酶发酵条件进行了考察。研究表明 ,工程菌的最佳发酵温度为 33℃ ,pH7.5～ 8.5的微碱条件有利于酶的生成。LB培养基补加适量葡萄糖 (1～ 5g/L) ,可提高发酵生物量和产酶水平 ,但葡萄糖的过量补加 (6g/L以上 ) ,则导致发酵液偏酸 (低至pH4.0 )而完全抑制酰化酶生成 ,并证明工程菌生长和产酶对乙酸的抑制效应较为敏感。同时通过5L自控发酵罐的批式补糖试验 ,对恒速流加、pH反馈控制和指数流加等三种补糖模式的发酵产酶进程进行了比较。结果发现 ,三种方式的补糖条件下 ,acy基因在tac启动子控制下 ,呈组成型表达 ,细胞生长与产酶同步 ,无需诱导 ;其中 ,以指数流加方式得到的生物量和产酶水平最高。而从acy基因的表达效率,即比酶活看,pH反馈的补料方法略高于恒速或指数流加模式。     

微生物脂肪酶在正庚烷中合成短链芳香酯的研究

用微生物脂肪酶在溶剂相中合成短链芳香酯的研究表明：脂肪酶在正庚烷中催化合成芳香酯转化率比水相中有明显的优势。在10个不同来源的商品脂肪酶中,选择了其中对己酸乙酯转化率在85％以上来自Mucor miehei,Candida rugosa和Porcine pancreas的脂肪酶对7个短链脂肪酸酯进行合成,其中Mucor miehei脂肪酶对这几个芳香酯48 h合成转化率均在90％以上,乙酸乙酯21．28g^-1．丙酸乙酯19．6gL^-1,丁酸乙酯26．61gL^-1,戊酸乙酯24．95gL^-1,己酸乙酯产量34.60gL^-1,丁酸异戊酯30.76gL^-1,乙酸异戊酯12．76gL^-1。同时．Mucor miehei脂肪酶在正庚烷批式反应中稳定性也较好,己酸乙酯半衰期为120h,最少的乙酸乙酯也在140h。并且还发现脂肪酶在正庚烷中分解酶活降低很多,但对其酯合成转化率没有直接的影响。     

酿酒酵母(Saccharomyces cerevisiae)半乳糖可诱导表达系统特性的研究

把大肠杆菌β一半乳糖苷酶基因克隆到带有酵母半乳糖可诱导启动子GALl的穿梭表达质粒pYES2中,并把得到的重组质粒分别转化到两种不同遗传性状的宿主菌中,其中一株菌为蛋白酶活性缺失90％以上的pep4-3突变菌株。通过比较两株重组菌产生的β一半乳糖苷酶活性水平发现在所述实验条件下,蛋白酶缺失突变菌株中产生的β一半乳糖苷酶活水平不仅均要高于另一对照菌株,并且pep4-3突变菌株表现出受葡萄糖阻遏的严紧程度高及对诱导反应迅速等特点。此外,带有重组质粒的pep4-3突变菌株在葡萄糖阻遏培养基中最大生长量和重组对照菌株基本相同,但β一半乳糖苷酶在pep4-3突变菌株中的表达对细胞生长的影响明显小于对照菌株。     

纳豆激酶产生菌——纳豆菌对木糖和葡萄糖的利用

在纳豆激酶 (Nattokinase,简称NK)发酵条件研究中 ,我们发现木糖是较葡萄糖更佳的产酶碳源。进一步的试验证明 ,NK的发酵菌种———Bacillussubtilisvar.natto在混合碳源中没有二次生长现象 ,对木糖和葡萄糖的吸收是同时的 ,且互不干扰 ,葡萄糖对木糖的吸收利用没有分解代谢阻遏。     

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。     

生淀粉糖化酶催化位点氨基酸及酶合成调控的初步研究

通过对Rhizopus OR-1UVN菌种所产生淀粉糖化酶在不同底物不同缓冲溶液条件下酶最适pH的测定,推测出该生淀粉糖化酶活力中心催化位点氨基酸是天冬氨酸(Asp)和谷氨酸(Glu)。实验证明5～50mg/mL浓度葡萄糖对生淀粉糖化酶没有抑制作用。分别以浓度<5mg/mL葡萄糖和淀粉为碳源的培养基进行不同碳源发酵实验,发现以淀粉为碳源的培养基Ⅰ发酵15h开始产生淀粉糖化酶,以葡萄糖为碳源的培养基Ⅱ发酵35h开始产酶(葡萄糖浓度<8mg/mL),而且前者菌体较后者少,由此可知葡萄糖对产酶有阻遏作用。实验还发现解阻遏熟淀粉糖化酶的葡萄糖浓度(15mg/mL)比生淀粉糖化酶的要高。由于葡萄糖的阻遏作用不发生在翻译水平,而发生在转录水平上,而且生淀粉糖化酶(G1)与熟淀粉糖化酶(G2)来自同一条DNA链,可以推测存在mRNA的拼接。通过以生淀粉为碳源的比较实验,发现生淀粉对生淀粉糖化酶形成的诱导作用可能主要是通过mRNA拼接的调节来实现的。     

微水体系中荧光假单胞菌脂肪酶催化合成单甘酯

研究了无溶剂微水体系中荧光假单胞菌脂肪酶 (PFL)催化油脂甘油解合成单甘酯的反应因素以及多温程非均相固液反应对单甘酯产率的影响。以初始体系最低共熔点 (PFL)取代临界温度学说中的油脂初熔点 ,通过考察不同IEP体系的甘油解 ,发现PFL酶促油脂甘油解时存在碳链基质特异性的函数关系 ,即反应物油脂中饱和碳残基的质量百分含量 (C₁₆+C₁₈)与单甘酯产率间符合以下多项式:Y =- 0.0006X³ +0.0592X²-0.8909X+26.753(13%<X<76.5%),式中X为C₁₆+C₁₈,Y为40℃时等温反应条件下的单甘酯产率。IEP为40℃时,最适等温反应条件如下：加水量3%～4.5%,加酶量为500μ/g油酯摩尔比1：2.5-5.0(油酯：甘油)反应温度40℃.实验条件下多步等程序降温反应48h后单甘酯最高产率为81.4%.     

生物法合成维生素C棕榈酸酯

研究了不同的脂肪酶在有机溶剂体系中催化合成L-维生素C棕榈酸酯的反应。针对维生素C在有机溶剂中溶解度较低这一问题,对催化合成维生素C棕榈酸酯反应的脂肪酶和反应介质进行比较,同时对影响合成维生素C棕榈酸酯反应的因素（温度、底物浓度、底物摩尔比、反应时间和酶量等）进行探讨,优化了反应条件：在10mL的丙酮中,1.094g棕榈酸与0.107g维生素C在酶量为20％（W/W, 固定化酶/维生素C）的固定化脂肪酶催化下,初始含0.4nm分子筛20%,温度为60℃,转速为200r/min,反应48h转化率可以达到80%,产物维生素C棕榈酸酯的浓度可达20g/L。     

合成己酸乙酯脂肪酶产生菌的筛选及产酶条件

从２７株脂肪酶产生菌中筛选到能由乙醇和己酸合成己酸乙酯的菌株８株。其中Ｒｈｉｚｏｐｕｓｓｐ．Ｈ－３菌株脂肪酶活力为５０－６０ｕ／ｍｌ,全细胞在有机溶剂中的酯化率可达己酸的９１％。Ｈ３产酶的最适碳源为淀粉或葡萄糖。６％黄豆饼粉加４％蛋白陈复合氮源有利于酶活力的增加。     

Lipase Production and Activity as a Function of Incubation Time, pH and Temperature of Four Lipolytic Micro-organisms

S ummary . The lipolytic activity in supernatant fractions of cultures of Saccharomycopsis lipolytica, Micrococcus caseolyticus, Bacillus licheniformis , and a Staphylococcus sp. was studied. Nutrient broth with and without emulsified olive oil was used as substrate. Optimal pH values and temperatures for the lipase produced by the 4 different micro-organisms were determined. The lipolytic activity generally reached a maximum after incubation for 2–6 days. The subsequent decrease in the lipolytic activity was associated with a high proteolytic activity only for Micrococcus caseolyticus . The lipolytic activity was decreased by the presence of olive oil in the medium. Determination of the lipolytic activity after a certain time of incubation, the maximal lipolytic activity and a time-integrated lipolytic activity are compared as estimators for the potential hydrolytic capacity of micro-organisms.     

Lipase Induction in Mucor hiemalis

The influence on lipase induction in Mucor hiemalis of different types of triglycerides containing mainly oleic acid (olive oil), erucic acid (mustard oil), or saturated fatty acids of 8 to 16 carbons (coconut oil) was studied. The fungus was grown in shake flasks in a fermentation medium containing peptone, minerals, and glucose or one of the oils as the carbon source. Maximum lipase was produced when the initial pH of the fermentation medium was kept at 4.0. Addition of Ca²⁺ to the medium did not increase lipase production. The optimum pH for activity of both the mycelial and extracellular lipases was found to be 7.0. The fungus produced a significant amount of lipase in the presence of glucose, but the lipase activity increased markedly when olive oil was added to the medium at the beginning of the fermentation. Addition of olive oil at a later stage did not induce as much enzyme. Studies with washed mycelia showed that a greater amount of lipase was released when olive oil was present than when glucose was present. Among the various types of triglycerides used as the carbon source, olive oil was found to be most effective in inducing the lipase. Olive oil and mustard oil fatty acids inhibited the lipase more than those of coconut oil. The lipase induced by a particular type of triglyceride did not seem to be specific for the same triglyceride, nor was it inhibited specifically by it. Irrespective of the triglyceride used in the fermentation medium, the lipase produced was most active against coconut oil triglyceride, and this specificity, as shown by lipase activities in an n-heptane system, was not found to be due to a better emulsification of this oil. The lipase of M. hiemalis can be considered to be both constitutive and inducible.     

The enzymatic acyl exchange of phospholipids with lipases

The acyl exchange of phospholipids with lipases was investigated. The lipase from Rhizopus delemar catalyzed the acyl exchange reaction between various phospholipids and fatty acids. When we incubated 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) and oleic acid with lipase from R. delemar, the yield of diacyl phosphatidyl choline (PC) was 25% and the fatty acid composition of the converted PC was an oleic acid content of 25% and a palmitic acid content of 75%. This reaction exhibited 1-positional specificity. Three industrial lipases from Rhizopus sp., Mucor javanicus, and Candida cylindracea had the activity of the acyl exchange of phosphatidyl choline. The lipase from R. sp. gave the best result.     

Optimization of extracellular lipase production by Geotrichum sp. using factorial design

Response surface methodology was employed to study the effects of carbon source (soy oil, olive oil and glucose) and nitrogen source concentrations (corn steep liquor and NH(4)NO(3)) on the lipase production by Geotrichum sp. The experiment included a 2(4) central composite rotatable design (CCRD) and four others 2(3) CCRD. According to the responses from the experimental designs, the effects of each variable were calculated and the interactions between them were determined. The response surface methodology was applied for the optimization of the nutrient concentrations in the culture medium for the enzyme production, at 30 degrees C. The optimum medium composition for lipase production by Geotrichum sp. was ammonium nitrate 2.1-2.5%, corn steep liquor 13-15% and soy oil 0.6% as carbon source, which lead to a lipase activity of about 20 U/ml. Using olive oil as carbon source, the optimum composition was ammonium nitrate 0.8-1%, corn steep liquor 13-15% and olive oil 0.6%, leading to an activity of 17 U/ml.     

Investigation of lipase production by a new isolate of Aspergillus sp.

Fungi isolated from soil were screened for exogenous lipolytic activity. The highest lipase activity was found in a new soil isolate of Aspergillus sp. Some optimal cultural parameters influencing the growth and production of extracellular lipase from this Aspergillus sp. were investigated. The lipase yield was maximum on day 4 of incubation of the culture at pH 5.5 and 30 °C. When the medium was prepared using olive oil as carbon source and peptone as a nitrogen source, better lipase yields were obtained. Aeration enhanced growth and lipase production.     

Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2.

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5, 35.5 degrees C, at a dilution rate of 0.04 h-1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-1 and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-1 h-1 (1 LU equalled 1 mumol fatty acid released min-1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.     

Biocatalytic acylation of carbohydrates with fatty acids from palm fatty acid distillates

Palm fatty acid distillates (PFAD) are by-products of the palm oil refining process. Their use as the source of fatty acids, mainly palmitate, for the biocatalytic synthesis of carbohydrate fatty acid esters was investigated. Esters could be prepared in high yields from unmodified acyl donors and non-activated free fatty acids obtained from PFAD with an immobilized Candida antarctica lipase preparation. Acetone was found as a compatible non-toxic solvent, which gave the highest conversion yields in a heterogeneous reaction system without the complete solubilization of the sugars. Glucose, fructose, and other acyl acceptors could be employed for an ester synthesis with PFAD. The synthesis of glucose palmitate was optimized with regard to the water activity of the reaction mixture, the reaction temperature, and the enzyme concentration. The ester was obtained with 76% yield from glucose and PFAD after reaction for 74 h with 150 U ml⁻¹ immobilized lipase at 40°C in acetone.     

Enzymatic properties of lipase and characteristics production byLactobacillus delbrueckii subsp.bulgaricus

Some properties of an extracellular lipase produced byLactobacillus delbrueckii subsp.bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl₂ to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40° C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35° C gave the highest lipolytic activity.     

Quantitative study of lipase secretion, extracellular lipolysis, and lipid storage in the yeast Yarrowia lipolytica grown in the presence of olive oil: analogies with lipolysis in humans

Lipase secretion, extracellular lipolysis, and fatty acid uptake were quantified in the yeast Yarrowia lipolytica grown in the presence of olive oil and/or glucose. Specific lipase assays, Western blot analysis, and ELISA indicated that most of the lipase activity measured in Y. lipolytica cultures resulted from the YLLIP2 lipase. Lipase production was triggered by olive oil and, during the first hours of culture, most of the lipase activity and YLLIP2 immunodetection remained associated with the yeast cells. YLLIP2 was then released in the culture medium before it was totally degraded by proteases. Olive oil triglycerides were largely degraded when the lipase was still attached to the cell wall. The fate of lipolysis products in the culture medium and inside the yeast cell, as well as lipid storage, was investigated simultaneously by quantitative TLC–FID and GC analysis. The intracellular levels of free fatty acids (FFA) and triglycerides increased transiently and were dependent on the carbon sources. A maximum fat storage of 37.8% w/w of yeast dry mass was observed with olive oil alone. A transient accumulation of saturated FFA was observed whereas intracellular triglycerides became enriched in unsaturated fatty acids. So far, yeasts have been mainly used for studying the intracellular synthesis, storage, and mobilization of neutral lipids. The present study shows that yeasts are also interesting models for studying extracellular lipolysis and fat uptake by the cell. The quantitative data obtained here allow for the first time to establish interesting analogies with gastrointestinal and vascular lipolysis in humans.     

Immobilization of Burkholderia sp. lipase on a ferric silica nanocomposite for biodiesel production

In this work, lipase produced from an isolated strain Burkholderia sp. C20 was immobilized on magnetic nanoparticles to catalyze biodiesel synthesis. Core-shell nanoparticles were synthesized by coating Fe(3)O(4) core with silica shell. The nanoparticles treated with dimethyl octadecyl [3-(trimethoxysilyl) propyl] ammonium chloride were used as immobilization supporters. The Burkholderia lipase was then bound to the synthesized nanoparticles for immobilization. The protein binding efficiency on alkyl-functionalized Fe(3)O(4)-SiO(2) was estimated as 97%, while the efficiency was only 76% on non-modified Fe(3)O(4)-SiO(2). Maximum adsorption capacity of lipase on alkyl-functionalized Fe(3)O(4)-SiO(2) was estimated as 29.45 mg g(-1) based on Langmuir isotherm. The hydrolytic kinetics (using olive oil as substrate) of the lipase immobilized on alkyl-grafted Fe(3)O(4)-SiO(2) followed Michaelis-Menten model with a maximum reaction rate and a Michaelis constant of 6251 Ug(-1) and 3.65 mM, respectively. Physical and chemical properties of the nanoparticles and the immobilized lipase were characterized by Brunauer-Emmett-Teller (BET) analysis, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FT-IR). Moreover, the immobilized lipase was used to catalyze the transesterification of olive oil with methanol to produce fatty acid methyl esters (FAMEs), attaining a FAMEs conversion of over 90% within 30 h in batch operation when 11 wt% immobilized lipase was employed. The immobilized lipase could be used for ten cycles without significant loss in its transesterification activity.