费希尔曲霉脂肪酶在毕赤酵母中的优化表达及高密度发酵

【背景】脂肪酶广泛应用于纺织、食品、药品、皮革等工业领域,其在微生物中的异源表达研究进一步促进了脂肪酶产品的生产和应用。【目的】实现来源于费希尔曲霉的脂肪酶在毕赤酵母中的高效异源表达,探究其合适的表达及发酵条件,提高产量,降低成本。【方法】对费希尔曲霉的脂肪酶编码基因进行密码子优化后,应用pPIC9k质粒整合到毕赤酵母GS115基因组上,构建高产脂肪酶Lip605的毕赤酵母工程菌;并通过响应面发酵条件优化、筛选最适伴侣蛋白和高密度发酵相结合的方法,综合提高脂肪酶表达量。【结果】确定高产脂肪酶毕赤酵母工程菌的最优摇瓶发酵产酶条件为:甲醇3.103%(体积比),生物素0.4 mg/L,酵母粉11.5 g/L,酵母基础氮源培养基(yeast nitrogen base,YNB) 13.4 g/L,初始pH 6.4,装液量50 mL/250 mL,转速220 r/min,温度24°C,培养时间40 h。优化后的胞外脂肪酶酶活达到72.34 U/mL,较优化前提高了5.8倍;进一步选择12个伴侣蛋白分别与脂肪酶Lip605进行共表达,其中共表达伴侣蛋白Rpl10(pPICZA-RPL10)效果最佳,可使Lip605表达量进一步提高46.8%;在此基础上,经过10 L发酵罐分批补料的高密度发酵,工程菌株发酵142 h,胞外脂肪酶酶活最高达到680 U/mL,蛋白浓度为15.89 g/L。【结论】应用复合策略有效提高了脂肪酶Lip605在毕赤酵母中的发酵产量,为其进一步工业化生产奠定了良好的基础。     

The profile of enzymes relevant to solvent production during direct fermentation of sago starch by <Emphasis Type="Italic">Clostridium saccharobutylicum</Emphasis> P262 utilizing different pH control strategies

The profile of enzymes relevant to solvent production during direct fermentation of sago starch by Clostridium saccharobutylicum P262 in a 2 L stirred tank fermenter was determined utilizing different pH control strategies. During fermentation without pH control (initial pH of 6), the specific activity of crotonase, thiolase, and β-hydroxybutyryl-CoA dehydrogenase increased proportionally with solvent production. The highest crotonase (3,450.7 kat) and phosphotransbutyrylase activity (1,475.6 kat) was observed in fermentation where pH was maintained at 5 during the acidogenic phase and corresponded to a fairly high acid accumulation but low solvent production. During fermentation with a controlled pH of 5.25 during the sol-ventogenic phase, the highest thiolase specific activity (255.7 kat) was obtained and corresponded to the highest production of acetone. On the other hand, the highest specific activities of crotonase, β-hydroxybutyryl-CoA dehydrogenase, and phosphotransbutyrylase were observed at pH 5.5 and corresponded to the highest production of ethanol and butanol. Butyryl-CoA dehydrogenase had no significance role in solvent fermentation. These results suggested that pH control strategies were important for improvement of solvent production during direct fermentation of sago starch by C. saccharobutylicum.     

纳豆激酶液态发酵条件的研究

考察纳豆杆菌在7.5 L发酵罐中分批发酵产纳豆激酶的条件,纳豆激酶酶活采用四肽底物测定。结果表明:纳豆杆菌生长和产酶的适宜条件不一致。发酵过程中发酵罐搅拌转速控制为500 r/min不变,0～12 h时控制pH为8.0、温度为37℃;12～36 h调整pH为7.0、温度为30℃;16 h时补加外源C源,连续发酵36 h。该过程中发酵液中比酶活最高达到3 232 U/mL,与摇瓶发酵相比,比酶活提高了58%。     

Swine manure fermentation for hydrogen production

Biohydrogen fermentation using liquid swine manure as substrate supplemented with glucose was investigated in this project. Experiments were conducted using a semi-continuously-fed fermenter (8 L in total volume and 4 L in working volume) with varying pHs from 4.7 through 5.9 under controlled temperature (35 ± 1 °C). The hydraulic retention time (HRT) tested include 16, 20, and 24 h; however, in two pH conditions (5.0 and 5.3), an additional HRT of 12 h was also tried. The experimental design combining HRT and pH provided insight on the fermenter performance in terms of hydrogen generation. The results indicated that both HRT and pH had profound influences on fermentative hydrogen productivity. A rising HRT would lead to greater variation in hydrogen concentration in the offgas and the best HRT was found to be 16 h for the fermenter in this study. The best pH value in correspondence to the highest hydrogen generation was revealed to be 5.0 among all the pHs studied. There was no obvious inhibition on hydrogen production by methanogenesis when methane content in the offgas was lower than 2%. Otherwise, an inverse linear relationship between hydrogen and methane content was observed with a correlation coefficient of 0.9699. Therefore, to increase hydrogen content in the offgas, methane production has to be limited to below 2%.     

Pongamia pinnata seed cake: a promising and inexpensive substrate for production of protease and lipase from Bacillus pumilus SG2 on solid-state fermentation

The production of a protease and a lipase from Bacillus pumilus SG2 on solid-state fermentation using Pongamia pinnata seed cake as substrate was studied. The seed cake was proved to be a promising substrate for the bacterial growth and the enzyme production. The initial pH, incubation time and moisture content were optimized to achieve maximal enzyme production. Maximum protease production was observed at 72 h and that of the lipase at 96 h of incubation. The production of protease (9840 U/g DM) and lipase (1974 U/g DM) were maximum at pH 7.0 and at 60% moisture content. Triton X-100 (1%) was proved to be an effective extractant for the enzymes and their optimal activity was observed at alkaline pH and at 60 C. The molecular mass of the protease and lipase was 24 and 40 kDa, respectively. Both the enzymes were found to be stable detergent additives. The study demonstrated that inexpensive and easily available Pongamia seed cake could be used for production of industrially important enzymes, such as protease and lipase.     

Kinetics of kojic acid fermentation byAspergillus flavus link S44-1 using sucrose as a carbon source under different pH conditions

Kojic acid production byAspergillus flavus strain S44-1 using sucrose as a carbon source was carried out in a 250-mL shake flask and a 2-L stirred tank fermenter. For comparison, production of kojic acid using glucose, fructose and its mixture was also carried out. Kojic acid production in shake flask fermentation was 25.8 g/L using glucose as the sole carbon source, 23.6 g/L with sucrose, and 6.4 g/L from fructose. Reduced kojic acid production (13.5 g/L) was observed when a combination of glucose and fructose was used as a carbon source. The highest production of kojic acid (40.2 g/L) was obtained from 150 g/L sucrose in a 2 L fermenter, while the lowest kojic acid production (10.3 g/L) was seen in fermentation using fructose as the sole carbon source. The experimental data from batch fermentation and resuspended cell system was analysed in order to form the basis for a kinetic model of the process. An unstructured model based on logistic and Luedeking-Piret equations was found suitable to describe the growth, substrate consumption, and efficiency of kojic acid production byA. flavus in batch fermentation using sucrose. From this model, it was found that kojic acid production byA. flavus was not a growth-associated process. Fermentation without pH control (from an initial culture pH of 3.0) showed higher kojic acid production than single-phase pH-controlled fermentation (pH 2.5, 2.75, and 3.0).     

Fermentation of Detoxified Acid-Hydrolyzed Pyrolytic Anhydrosugars into Bioethanol with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 2.399

Pyrolysate obtained from the pyrolysis of waste cotton is a source of fermentable sugars that could be fermented into bioethanol fuel and other chemicals via microbial fermentation. However, pyrolysate is a complex mixture of fermentable and non-fermentable substrates causing inhibition of the microbial growth. The aim of this study was to detoxify the hydrolysate and then ferment it into bio-ethanol fuel in shake flasks and fermenter applying yeast strain Saccharomyces cerevisiae 2.399. Pyrolysate was hydrolyzed to glucose with 0.2 M sulfuric acid, neutralized with Ba(OH)₂ followed by treatment with ethyl acetate and activated carbon to remove fermentation inhibitors. The effect of various fermentation parameters such as inoculum concentration, pH and hydrolysate glucose was evaluated in shake flasks for optimum ethanol fermentation. With respect to inoculum concentration, 20% v/v inoculum i.e. 8.0 × 10⁸–1.2 × 10⁹ cells/mL was the optimum level for producing 8.62 ± 0.33 g/L ethanol at 9 h of fermentation with a maximum yield of 0.46 g ethanol/g glucose. The optimum pH for hydrolysate glucose fermentation was found to be 6.0 that produced 8.57 ± 0.66 g/L ethanol. Maximum ethanol concentration, 14.78 g/L was obtained for 4% hydrolysate glucose concentration after 16 h of fermentation. Scale-up studies in stirred fermenter produced much higher productivity (1.32 g/L/h^–1) compared to shake flask fermentation (0.92 g/L/h^–1). The yield of ethanol reached a maximum of 91% and 89% of the theoretical yield of ethanol in shake flasks and fermenter, respectively. The complex of integrated models of development was applied, that has been successfully tested previously for the mathematical analysis of the fermentation processes.     

Culture condition improvement for whole-cell lipase production in submerged fermentation by Rhizopus chinensis using statistical method

Rhizopus chinensis CCTCC M201021 was a versatile strain capable of producing whole-cell lipase with synthetic activity in submerged fermentation. In order to improve the production of whole-cell lipase and study the culture conditions systematically, the combination of taguchi method and response surface methodology was performed. Taguchi method was used for the initial optimization, and eight factors viz., maltose, olive oil, peptone, K2HPO4, agitation, inoculum size, fermentation volume and pH were selected for this study. The whole-cell lipase activity yield was two times higher than the control experiment under initial optimal conditions, and four significant factors (inoculum, olive oil, fermentation volume and peptone) were selected to test the effect on the lipase production using response surface methodology. The optimal fermentation parameters for enhanced whole-cell lipase yield were found to be: inoculum 4.25 x 10(8) spores/L, olive oil 2.367% (w/v), fermentation volume 18 mL/250 mL flask, peptone 4.06% (w/v). Subsequent experimental trails confirmed the validity of the model. These optimal culture conditions in the shake flask led to a lipase yield of 13875 U/L, which 120% increased compare with the non-optimized conditions.     

脂肪酶产生菌Candida rugosa产酶条件研究

脂肪酶(Lipase,EC3.1.1.3)是用来催化酯类化合物的分解、合成和酯交换的特殊酶,具有高度的化学选择性和立体异构性,它广泛应用于食品、轻纺、皮革、香料、化妆品、洗涤剂、有机合成、医药等领域.本世纪80年代,美国科学家发现酶在近无水的有机溶剂中不仅能保存其催化活力,而且还获得许多新的催化特征[1],此后,脂肪酶在非水相酶催化领域的研究和应用逐渐增多.     

Production of phytase (myo-inositolhexakisphosphate phosphohydrolase) by Aspergillus niger van Teighem in laboratory-scale fermenter

The growth and production pattern of phytase by a filamentous fungus, Aspergillus niger van Teighem, were studied in submerged culture at varying agitation rates and controlled and uncontrolled pH conditions. Allowing the initial culture to grow under neutral condition with subsequent decline in pH resulted in increased phytase productivity. A maximum of 141 nkat/mL phytase was obtained when the broth pH was maintained at pH 2.5 as compared to 17 nkat/mL units at controlled pH 5.5. The culture morphology and rheological properties of the fermentation broth significantly varied with the agitation rate. The volumetric oxygen transfer coefficient was determined at different phases of fungal growth during batch fermentation using static gassing out and dynamic gassing out methods. The oxygen transfer coefficient (k(L)a) of the fermenter was found to be 125 h(-)(1) at 500 rpm as compared to 38 h(-)(1) at 200 rpm. The oxygen transfer rates at different phases of growth were significantly affected by cell mass concentration and fungal morphology. During the course of fermentation there was a gradual decline of k(L)a from 97 h(-)(1) on day 2 to 63 h(-)(1) on day 6 of fermentation, after which no significant change was observed. The degree of agitation considerably influenced the culture morphology where shear thinning of filamentous fungus was observed with the increase in agitation.     

Production and properties of an alkaline,thermophilic lipase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> NS2W

Eighteen bacterial strains were isolated from soil samples and screened for alkaline, thermophilic lipase production. Pseudomonas fluorescens NS2W was selected and its production of lipase was optimized in shake flasks using a statistical experimental design. Cell growth and lipase production were studied in shake flasks and in a 1-l fermenter in the optimized medium. Maximum lipase yields were 69.7 and 68.7 U ml⁻¹, respectively. The optimized medium resulted in about a five-fold increase in the enzyme production, compared to that obtained in the basal medium. The lipase had an optimal activity at pH 9.0 and was stable over a wide pH range of 3–11 with more than 70% activity retention. The lipase had an optimal activity at 55°C and was stable up to 60°C with more than 70% activity retention for at least 2 h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 344–348 DOI: 10.1038/sj/jim/7000254 Received 06 September 2001/ Accepted in revised form 15 March 2002     

Enhanced production of Penicillium expansum PED-03 lipase through control of culture conditions and application of the crude enzyme in kinetic resolution of racemic Allethrolone

Alkaline lipase production was performed in submerged fermentation by Penicillium expansum PED-03. It was found that the suitable carbon source and nitrogen source for lipase production were 0.5% starch and 4.0% soybean meal, respectively. The maximal lipase activity (850 U/mL) of production was achieved at initial pH 5.5-6.0, 26 degrees C, 72 h. Tween-80 was an effective enhancer for lipase production. Agitation speed of the fermentor played an important role, and the suitable agitation speed for lipase production was 500 r/min. The lipase was stable within the range of pH 7.0-10.0 and 20-40 degrees C, and the optimum conditions for the enzymatic reaction were 35 degrees C and pH 9.5. The enzymatic resolution of racemic allethrolone (4-hydroxy-3-methyl-2-(2-propenyl)-2- cyclopenten-1-one) was carried out by the lipase from P. expansum PED-03, and the conversion reached 48% with excellent enantioselectivity (E > 100), which showed a good application potential in the production of optically pure allethrolone.     

Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738

AIMS: The objective of the present study was to determine the optimal culture conditions for mycelial biomass and exo-polysaccharide (EPS) by Cordyceps militaris C738 in submerged culture. METHODS AND RESULTS: The optimal temperatures for mycelial biomass and EPS production were 20 degrees C and 25 degrees C, respectively, and corresponding optimal initial pHs were found to be 9 and 6, respectively. The suggested medium composition for EPS production was as follows: 6% (w/v) sucrose, 1% (w/v) polypeptone, and 0.05% (w/v) K2HPO4. The influence of pH on the fermentation broth rheology, morphology and EPS production of C. militaris C738 was carried out in a 5-l stirred-tank fermenter. The morphological properties were comparatively characterized by pellet roughness and compactness by use of image analyser between the culture conditions with and without pH control. The roughness and compactness of the pellets indicated higher values at pH-stat culture (pH 6.0), suggesting that larger and more compact pellets were desirable for polysaccharide production (0.91 g g(-1) cell d(-1). CONCLUSIONS: Under the optimized culture conditions (with pH control at 6), the maximum concentration of biomass and EPS were 12.7 g l(-1) and 7.3 g l(-1), respectively, in a 5-l stirred-tank fermenter. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effect of pH on fungal morphology and rheology presented in this study can be widely applied to other mushroom fermentation processes.     

Dissolved-oxygen-stat fed-batch fermentation of 1,3-dihydroxyacetone from glycerol by Gluconobacter oxydans ZJB09112

This study investigated the effects of DO concentration on DHA fermentation and of DO-stat fed-batch fermentation using a pH control strategy, on 1,3-dihydroxyacetone (DHA) production. The results showed that DO-stat fed-batch fermentation with pH-shift control was the optimal bioprocess for DHA production. DO-stat fed-batch fermentation was carried out at 30% air saturation, and the culture pH was automatically maintained at pH 6.0 during the first 20 h and then shifted to pH 5.0 until the end of the fermentation. An optimal DHA concentration of 175.9 ± 6.7 g/L, with a production yield to glycerol of 0.87 ± 0.04 g/g, was obtained at 72 h of DO-stat fed-batch fermentation at 30°C in a 15 L fermenter.     

Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77x10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.     

Lycopene production from synthetic medium by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> NRRL 2895 (+) and 2896 (−) in a stirred-tank fermenter

The dissolved oxygen tension of 20% of air saturation, pH-shift from 4.0 to 5.5 on day 3, and a moderate shear stress (calculated as an impeller tip speed, V_\texttip = 0. 9 2 6- 2. 1 6 1  \textm/\texts V_{\text{tip}} = 0. 9 2 6- 2. 1 6 1 \, {\text{m}}/{\text{s}} ) were identified to be the key factors in scaling-up the mated fermentation of Blakeslea trispora NRRL 2895 (+) and 2896 (−) for lycopene production from a shake flask to a stirred-tank fermenter. The maximal lycopene production of 183.3 mg/L was obtained in 7.5-L stirred-tank fermenter, and then the mated fermentation process was successfully step-wise scaled-up from 7.5- to 200-L stirred-tank fermenter. The comparability of the fermentation process was well controlled and the lycopene production was maintained during the process scale-up. Furthermore, with the integrated addition of 150 μmol/L abscisic acid on day 3, 0.5 g/L leucine and 0.1 g/L penicillin on day 4, the highest lycopene production of 270.3 mg/L was achieved in the mated fermentation of B. trispora in stirred-tank fermenter.     

产琥珀酸工程菌株的发酵工艺条件优化

对实验室构建的产琥珀酸大肠杆菌工程菌株(E.coliQZ1111)进行发酵工艺条件研究。以AM1低盐培养基为基础,研究不同C、N源及其质量浓度,培养基初始pH和发酵温度等因素对琥珀酸的影响,并在5L发酵罐中进行了补料-分批发酵实验。优化后的发酵条件为葡萄糖20g/L,玉米浆10g/L,pH6.4,发酵温度37℃。在5L发酵罐中培养,琥珀酸产量达到47.9g/L。     

Production of lipase in a fermentor using a mutant strain of Corynebacterium species: its partial purification and immobilization

Extracellular Corynebacterium lipase was produced using a 2.5 L Chemap fermentor using 1300 ml fermentation medium at temperature 33 degrees C, agitator speed 50 rpm, aeration rate 1 VVM having KLa 16.21 hr(-1). Crude lipase was purified by salting out method followed by dialysis and immobilized using calcium alginate gel matrix followed by glutaraldehyde cross linking Purification process increased specific activity of enzyme from 2.76 to 114.7 IU/mg. Activity of immobilized enzyme was 107.31 IU/mg. Optimum temperature for purified and immobilized enzyme activity were 65 degrees and 50 degrees C respectively. Optimum pH was 8.0 in both the cases, Km and Vmax value for purified lipase were 111.1 micromol/min and 14.7% respectively. Ca2+ (5 mM) was found to be stimulator for enzyme activity. Immobilized lipase retained 68.18% of the original activity when stored for 40 days.     

Evaluation of Medium Components by Plackett-Burman Statistical Design for Lipase Production by Candida rugosa and Kinetic Modeling

Lipase production by Candida rugosa was carried out in submerged fermentation. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effect of twelve medium components was studied in sixteen experimental trials. Glucose, olive oil, peptone and FeCl3?6H2O were found to have more significance on lipase production by Candida rugosa. Maximum lipase activity of 3.8 u mL-1 was obtained at 50 h of fermentation period. The fermentation was carried out at optimized temperature of 30oC, initial pH of 6.8 and shaking speed of 120 r/min. Unstructured kinetic models were used to simulate the experimental data. Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were found suitable to efficiently predict the cell mass, lipase production and glucose consumption respectively with high determination coefficient(R2). From the estimated values of the Luedeking-Piret kinetic model parameters, α and β, it was found that the lipase production by Candida rugosa is growth associated.     

Production and regulation of different lipase activities from Rhizopus chinensis in submerged fermentation by lipids

The fungal Rhizopus chinensis could produce several types of lipase, which were mainly intracellular. During the whole-cell lipase production by this strain in submerged fermentation, it was observed that two catalytic characteristics (hydrolytic and synthetic activity) of lipases were different with addition of lipids. The hydrolytic activity of the lipase was not induced by lipids efficaciously and could be detected regardless of whether substrate-related compounds were present. However, it was found that the induction of lipids for the synthetic activity lipase was significant, and that nearly no synthetic activity was detected while the medium contained no lipids. When only a little lipid (1 g/L) was added to medium, the synthetic activity increased sharply in the initial process of fermentation. Analysis of crude membrane-bound lipase by SDS-PAGE confirmed this induction. De novo biosynthesis of lipases, especially the lipase with synthetic activity occurred only when lipids existed. Cell growth and maltose repress the lipase production with synthetic activity, but have little influence on the lipase production with hydrolytic activity. Since the production process of mycelium-bound lipase with hydrolytic activity was different, it was reasonable to consider hydrolytic activity and synthetic activity for different application purposes. Whole-cell lipase obtained from fermentation process with high synthetic activity showed excellent catalytic ability in solvent free system on synthesis of ethylcaprylate and ethyloleate, the conversion could reach more than 90% in 5 h.