Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation

Solid-state fermentation (SSF) is a bioprocess that doesn’t need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.     

Formation of lipolytic enzymes by Brevibacterium linens

When Brevibacterium linens ATCC 9172 was grown in shake flasks, it produced a cell-associated lipase with a specific activity of 152 to 188 U g^–1 cells depending on the composition of the growth medium. There was no growth in media containing tributyrine as the sole carbon source. The cell-associated lipase had maximum activity at pH 8.0 and 37 °C and was strongly inhibited by 3,4-dichloroisocoumarin, an inhibitor specific for serine esterases. Cell-associated activity was released from the cells by treatment with lysozyme. The kinetics of lipase formation was closely related to the amount of biomass formed during growth.     

Effect of cultivation conditions on cell-surface display of Flo1 fusion protein using sake yeast

The cell-surface display of the Flo1p anchor system with a flocculation functional domain was examined under various cultivation conditions. As a model system, lipase from Rhizopus oryzae with the pro sequence was genetically fused to the Flo1 short (FS) anchor (FSProROL) and displayed on the sake yeast cell-surface under the control of the SED800 promoter (pSED800). The nutrients and carbon source in the culture media affected the display of the fusion protein FSProROL on the sake yeast cell-surface. The lipase activity in whole cells cultivated in poor media, without peptone and/or yeast extracts, were higher than those cultivated in rich media. In addition, glucose and maltose were effective carbon sources for increasing the lipase activity in whole cells, and the addition of di- or tri-saccharide as the carbon source reduced the release of the lipase activity into the culture supernatants. The initial glucose concentration was found to influence the total lipase activity and it mainly affected the lipase activity in whole cells. Under the optimum condition, sake yeast was found to show high cell density and high lipase activity in short time cultivation.     

Improvement of lipase production by addition of catalase during fermentation

A batch fermentation process for lipase production with the recombinant strain Staphylococcus carnosus (pLipMut2) was studied in a bubble column. The rates of growth and lipase production in this type of fermentor were compared with results from shakeflasks. It was seen that cultivation in the bubble column resulted in a prolonged lag time and a reduced lipase activity in comparison to flask cultures. However, by addition of catalase during the fermentation in the bubble column this different behaviour could be avoided. Correspondence to: E. Wenzig     

EXTRACELLULAR LIPOLYTIC ENZYME ACTIVITY OF A NEWLY ISOLATED DEBARYOMYCES HANSENII

A strain isolated from waste of a milk products plant and exhibited extracellular lipolytic activity was identified as Debaryomyces hansenii by 5.8S rRNA and 28S rRNA gene sequence analyses. Lipolytic activity was assayed spectrophotometrically by using p-nitrophenylpalmitate. Higher specific lipolytic activities were obtained in the presence of tristearin (0.68 U/mg prot), oleic acid (0.56 U/mg prot), and soybean oil (0.36 U/mg prot) than other triglycerides, fatty acids, and vegetable oils considered as carbon sources. Cheese whey appeared to be a good alternative to lipidic substances for lipolytic activity. Among various organic and inorganic nitrogen sources, soy flour was found to attain the lipolytic activity similar to that provided by universal yeast medium components. This work is the first report on the discussion of lipolytic activity enhancement by D. hansenii through modulating the cultivation medium. It also proposes low cost medium nutrients that could be of industrial value and could serve as basal nutrients for further optimization studies on the lipase production by D. hansenii.     

Characterization of a Triacylglycerol Lipase That Liberates Arachidonic Acid from Bovine Chromaffin Cells During Secretion

Abstract: Primary cultures of chromaffin cells from bovine adrenal medullae were used as a model to study lipolytic events during stimulus-secretion coupling. It has been shown that chromaffin cells liberate arachidonic acid in addition to their main secretion product, the catecholamines. To understand more about the mechanism of arachidonic acid liberation, chromaffin cells were labeled with radioactive arachidonic acid, stimulated, and then analyzed for changes in lipid composition. After stimulation with 10^?4M acetylcholine, the radioactivity of triacylglycerols decreased to the same extent that the free arachidonic acid level rose. This finding suggests that in bovine chromaffin cells a stimulation-dependent triacylglycerol lipase (triacylglycerol hydrolase; EC 3.1.1.3) is involved in arachidonic acid liberation. Further work was performed on detection, characterization, and isolation of this enzyme. Triacylglycerol lipase activity was found in whole cell homogenates and in plasma membrane fractions isolated from adrenal medullary tissue. The plasma membrane lipase showed a pH optimum of 4.3. The apparent Michaelis constant was determined as 3.3 × 10^?4 mol/L. Ca²⁺ did not influence the enzymatic activity. To differentiate the plasma membrane triacylglycerol lipase from the previously described plasma membrane diacylglycerol lipase of chromaffin cells, the influence of RG 80267, a specific diacylglycerol lipase inhibitor, was examined. RG 80267 (50 μM) inhibited the triacylglycerol lipase by only 24%, although diacylglycerol lipase was totally inhibited with only 20 μM RG 80267. The pH optimum of homogenate lipase was broad, lying between 4 and 7. Starting from the soluble fraction of whole cell homogenates, the triacylglycerol lipase was partially purified by ultracentrifugation and size-exclusion chromatography. The molecular mass of the enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was found to be between 47 and 57 kDa.     

Effects of the Reaction Conditions on the Enantio-Selective Esterification Using Candiada Cylindracea Lipase

Parameters influencing the synthetic activity and enantioselectivlty of the Candida cylindracea lipase were investigated. The test reaction was presented through the enantioselective esterification of 2—substituted propionic acids in organic solvents. It was found that both the activity and the enantioselectivity of the lipase could be most effectively influenced by solvent engineering. Since the solvent has a different effect on activity and enantioselectivity of the lipase, selection of the most suitable solvent or solvent mixture is an important optimization question.     

Culture conditions for intracellular lipase production by Rhizopus chinensis and its immobilization within biomass support particles

Acetone-dried cells of Rhizopus chinensis (with a 1,3-positional specificity lipase) were investigated for the interestierification reaction of olive oil and methyl stearate. First, the culture conditions for intracellular lipase production were examined, and then the activities of dried cells obtained from immobilization in Biomass Support Particles (BSPs) were compared with those of freely suspended cells.It was clear from cultivation of freely suspended cells that intracellular lipase activity for the interesterification reaction was enhanced sifnificantly by the presence of oleic acid, oil, and tea oil, but that the presence of glucose reduced the activity.The specific activity of dried cells within BSPs increased 7-fold compared with that obrained from freely suspended cells.The process presented here, using immobilization within BSPs, can provide cells directly as a catalyst with high activity, where cells become immobilized simply during batch operation, and no special preparation of cells is necessary. Therefore, the reaction system using dried cells immobilized within BSPs is a promising interesterifcation process for industrial applications.     

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.     

Purification and characterization of an extracellular lipase from Clostridium tetanomorphum

Summary The strictly anaerobic bacterium Clostridium tetanomorphum formed an extracellular lipase when the growth medium contained glycerol in addition to fermentable substrates such as l-glutamate or glucose. The lipase was purified from the concentrated culture supernatant and exhibited a final specific activity of 900 U/mg. The purified lipase had a Stokes’ radius of 5.0 nm and a sedimentation coefficient of 5.7S. The native molecular mass calculated from these values was 118,000 Da, which is considerably higher than the molecular mass calculated by PAGE (70,000 Da). With p-nitrophenyl esters of different fatty acids as substrates enzyme activity was highest when the acyl chain was short (C₂). The purified lipase showed no protease or thioesterase activity.     

扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达

将编码扩展青霉碱性脂肪酶 (PEL)的cDNA克隆到酵母整合型质粒pPIC3.5K ,电转化His4缺陷型巴斯德毕赤酵母 (Pichiapastoris)GS115 ,通过橄榄油 MM平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDS PAGE分析、橄榄油检验板鉴定 ,表明扩展青霉碱性脂肪酶基因在巴斯德毕赤酵母中获得了高效表达。表达蛋白分泌至培养基中 ,分子量约 2 8kD ,与扩展青霉碱性脂肪酶大小一致 ,占分泌蛋白的 95 %。橄榄油检验板检验表明该表达蛋白可分解橄榄油 ,通过优化该表达菌的发酵条件 ,以橄榄油为底物进行酶活测定 ,其发酵液酶活可达 2 6 0u mL。     

A Novel Olive Oil Degrading Thermoactinomyces Species with a High Extremely Thermostable Lipase Activity

A filamentous, Gram‐positive, spore forming aerobic bacterium was isolated from olive oil contaminated soil (Al Koura, Lebanon) on rhodamine agar plates at 60 °C. The isolate, HRK‐1 produced large quantities of an extracellular thermostable lipase which degrades olive oil. It was primarily classified as a Thermoactinomyces sp. due to the filamentous structure of its cells that bear one spore each on an un‐branched sporophore, the resistance of its spores to boiling, utilisation of sucrose as a carbon source and production of dark pigments. The isolate grew optimally at a temperature of 60 °C, a pH of 7.3 and an orbital shaking of 250 rpm. It showed an efficient olive oil degrading ability. No traces of triolein were detected after a 36‐h cultivation. A concentration of 10 % [v/v] olive oil did not inhibit its growth. Lipase production was constitutive, and did not depend on the presence of olive oil. The optimum concentration of olive oil for lipase activity was 1 % [v/v], and the activity was not enhanced at higher concentrations, but on the contrary, a decrease in enzyme activity was recorded. The lipase of HRK‐1 was preliminarily characterised in the crude cell‐free supernatant with a specific activity of 0.14 U/mg. It has an optimum activity at 60 °C and a pH of 8.0. This lipolytic enzyme showed resistance to boiling and to a wide range of metallic ions and inhibitors. The formation of this heat‐stable lipase started in the early exponential growth phase, while a maximum extracellular enzyme activity was detected at the end of the decline phase, when most of the cells appeared as spherical spores. The exceptionally high activity of lipase (2.37 U/ml) produced by HRK‐1 measured in the cell free supernatant clearly indicated the commercial importance of this isolate, especially after it showed great stability at elevated temperatures.     

Isolation,Identification, and Characterization of a Novel,Oil-Degrading Bacterium, <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> T1

A novel, oil-degrading bacterium (strain T1) was isolated from a hot spring in Hokkaido, Japan. It efficiently degrades different types of fats and oils, including edible oil waste. When grown in a mineral salt medium containing 1% triacylglycerol (as salad oil), hydrolysis products were 1,3- and 1,2-diacylglycerols, monoacylglycerol, and free fatty acid. However, these products were almost completely consumed during cultivation at 30°C for 5 days, indicating that extracellular lipase acts randomly at different sn-positions of acylglycerols and that strain T1 has a high capacity to utilize free fatty acids. Secreted lipase activity was induced by salad oil and oleic acid. This strain was a Gram-negative straight rod shaped, aerobic, with a polar flagellum, capable of growing in temperature ranges between 15°C and 55°C. The 16S rRNA gene sequence analysis and DNA-DNA hybridization revealed it as a new strain of Pseudomonas aeruginosa. The type strain was T1.     

八角莲组织培养的器官发生途径研究

为探究小檗科植物八角莲组织培养的器官发生方式,该研究以八角莲离体叶片、叶柄在MS培养基上诱导产生的愈伤组织、不定芽、不定根为对象,用连续石蜡切片技术分析八角莲组织培养的器官发生途径。结果表明:八角莲愈伤组织形成的解剖学特征是靠近表皮的薄壁细胞经激素刺激恢复分裂能力,继续培养形成拟分生组织。拟分生组织可形成许多分化中心。通过对八角莲组织培养产生的不定芽细胞组织学观察发现芽原基起源于愈伤组织外侧的几层薄壁细胞,芽原基背离愈伤组织中央生长形成不定芽,故八角莲脱分化形成的芽起源方式为外起源。而八角莲的根原基起源于组织深处髓部薄壁细胞和部分维管形成层细胞,进而形成类似球形或楔形并朝韧皮部突起的根原基轮廓,根原基继续发育会突破表皮生成不定根,起源方式为内起源。八角莲离体再生途径为器官发生型,在组培苗生长过程中先诱导形成不定芽,再诱导形成不定根,在愈伤组织上形成维管组织将不定芽和不定根连接成完整植株。     

Digestive enzyme activity of the Japanese grenadier anchovy Coilia nasus during spawning migration: influence of the migration distance and the water temperature

In this study, we investigated the activity levels of two major digestive enzymes (pepsin and lipase) in the commercially important Japanese grenadier anchovy Coilia nasus during its upstream migration to analyse the digestive physiological responses to starvation and to analyse the influence of the water temperature on enzyme activity. Water temperature had a significant effect on pepsin activity, while long-term starvation resulted in a significant decrease in pepsin activity. As starvation continued, however, a slight increase in pepsin activity between the Wuhu (440 river km) and Anqing (620 river km) regions may indicate that C. nasus had refeeding behaviour due to its large expenditure of energy reserves. In contrast, lipase activity was not significantly affected by the water temperature but the effect of fasting increased as much as 13% of lipase activity from the Chongming region (20 river km) to Anqing region, suggesting that the stored lipids of grenadier anchovy were mobilised to meet energy requirements of upstream migration activity and gonad development. Lipid mobilisation activated lipoprotein lipase (LPL; proteins with lipase activity) to hydrolyse triacylglycerides (TAG), which is the first step of lipid assimilation and obtained energy from fatty acids under fasting conditions. Therefore, the increased lipase activity is attributed mainly to the lipase that is involved in endogenous lipid hydrolysis. Grenadier anchovy appears to adapt to long-term starvation during migration and the increased lipase activity may indicate a crucial effect on lipid metabolism. This study demonstrated that distinct alterations occur in pepsin and lipase activities during the spawning migration of grenadier anchovy due to exogenous nutrition and endogenous metabolism. Furthermore, it provides a basis for further research on the digestive physiology and energy metabolism in this species.     

Sequential application of waste whey as a medium component for Kluyveromyces lactis cultivation and a co-feeder for lipase immobilization by CLEA method

Currently, much attention is paid to technologies which can be drivers of the circular economy across different sectors, in particular, to develop technologies for utilization or reusability of biocompatible materials from industrial waste. One of such is the milk whey, which is a cheap biobased raw material, the disposal of which is a major problem for the dairy industry. Our proposed and investigated technology is based on a continuous exploitation of the whey combining microbiology and biotechnology. Primarily, whey was used as a nutrition source for the cultivation of Kluyveromyces lactis with the aim to produce the targeted biocatalyst—lipase. During cultivation, the whey was transformed into the hydrolyzed form, which was further successfully applied as a protein feeder (external linker) for immobilization of lipase by cross-linked enzyme aggregate (CLEA) method. The first time use of whey as a co-feeder for immobilization of enzymes by CLEA method has shown promising results and increased the stability of lipases for temperature and organic solvents. Hydrolysis of rapeseed oil catalyzed with immobilized derivatives was obtained with 45–96% efficiency at non-optimized conditions. Additionally, the determined kinetic parameters indicated that the rate of p-nitrophenyl palmitate hydrolysis was not changed drastically after immobilization.

     

Exploring the effects of oil inducer on whole cell-mediated methanolysis for biodiesel production

Whole cell-mediated methanolysis of renewable oils for biodiesel production has drawn much attention in recent years since it can avoid the complex preparation procedures of traditional immobilized lipase. During the cultivation of Rhizopus oryzae whole cell catalyst, plant oils are usually added into the medium as inducer for lipase synthesis. It was found that oil inducer not only influenced lipase production, but also led to varied whole cell's catalytic performance. In this paper, the related mechanisms were explored and it was found that the higher unsaturated fatty acid (UFA) was contained in oil inducer, the higher intracellular lipase could be obtained. Different oil inducers also resulted in varied compositions of cell membrane, which was further found to be responsible for the operational stability of the catalyst. Cells with membrane enriched with saturated fatty acid (SFA) exhibited better stability than those enriched with UFA. And further study showed that after glutaraldehyde cross-linking treatment, the operational stability of both UFA enriched cells and SFA enriched cells were enhanced greatly and no loss in cell's catalytic activity was detected after being repeatedly used for 15 batches.     

Yarrowia lipolytica lipase production enhanced by increased air pressure

Aims: To study the cellular growth and morphology of Yarrowia lipolytica W29 and its lipase and protease production under increased air pressures. Methods and Results: Batch cultures of the yeast were conducted in a pressurized bioreactor at 4 and 8 bar of air pressure and the cellular behaviour was compared with cultures at atmospheric pressure. No inhibition of cellular growth was observed by the increase of pressure. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization enhanced the extracellular lipase activity from 96·6 U l^?1 at 1 bar to 533·5 U l^?1 at 8 bar. The extracellular protease activity was reduced by the air pressure increase, thereby eliciting further lipase productivity. Cell morphology was slightly affected by pressure, particularly at 8 bar, where cells kept the predominant oval form but decreased in size. Conclusions: OTR improvement by total air pressure rise up to 8 bar in a bioreactor can be applied to the enhancement of lipase production by Y. lipolytica. Significance and Impact of the Study: Hyperbaric bioreactors can be successfully applied for yeast cells cultivation, particularly in high‐density cultures used for enzymes production, preventing oxygen limitation and consequently increasing overall productivity.     

Immunocytochemical identification and localization of lipase in cells of the mycelium of Penicillium cyclopium variety

The localization of lipase in cells of the fungus Penicillium cyclopium was investigated. It was shown by differential centrifugation of a homogenate of mycelial cells that the activity of the enzyme is associated with the cell wall. A study of ultrathin sections of mycelium fixed using the method of Zvyagintseva in an electron microscope showed that the final products of lipolytic activity of the enzyme is localized on the cell wall. Antibodies were raised against the purified A and B lipases from P. cyclopium and their specificity was assessed by enzyme-linked immunosorbent assay. The antibody preparation was used in cytochemical investigation by immunogold labelling. This study permits the localization of cell-bound lipase mainly in the cell wall and in the periplasmic space. The identity of the cell-bound lipase with one of the two extracellular lipases is also demonstrated.     

Centrifugal cell hybridization

Centrifugation of cell suspensions containing two or several cell types at forces up to 2 million g results in several basic events in succession: 1. Intracellular stratification. 2. Extrusion of the most dense cell parts (nuclei or cytoplasmic components) and their penetration into an adjacent cell in the compact sediment. Such introduction of protoplasmic elements from one cell into another is considered as centrifugal hybridization and fusion of cells. It differs from other methods of cell hybridization by its selectivity for cell components. 3. Further intermingling and mixing of cells into a fused protoplasmic mass. 4. With continuing increase of centrifugal force fractions of subcellular components are formed from the protoplasmic mass. These components are presumably viable since cells are not exposed to chemical treatment. Morphological demonstration of hybridization is based on centrifuging microscopy and on labeling donor cells or recipient cells by staining or fluorescence. Genetic evidence can be provided by cultivation of hybrid cellsin vitro and their cloningin vivo.