固液态发酵中橄榄油对Rhizopus chinensis全细胞脂肪酶的影响

主要对华根霉全细胞脂肪酶固态和液态两种发酵过程进行比较,并着重探讨不同培养方式下橄榄油对其合成活力和水解活力的影响。结果表明：液态培养较有利于菌体生长,对脂肪酶的生产也有一定的促进作用。橄榄油的加入不仅有利于菌体生长、提高脂肪酶水解活力,更可使脂肪酶的合成活力显著增加,液态发酵下的效果更为明显。橄榄油在整个发酵过程中可能既作为碳源又是脂肪酶的诱导物。另外,全细胞脂肪酶的水解活力和合成活力在固液态发酵条件下均存在不对应性,表明华根霉可能产性质不同的脂肪酶同功酶。     

Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice

Fluorescent triacylglycerols containing pyrenedecanoic (P10) and pyrenebutanoic (P4) acids were synthesized and their hydrolysis by lipases from human gastric juice and stomach homogenate was investigated. The existence in stomach homogenate of four different lipolytic enzymes hydrolyzing fluorescent triacylglycerols is suggested by the comparison of various enzymatic properties: acyl chain length specificity, heat inactivation and effect of detergents (Triton X-100 and taurocholate), serum albumin, diethyl-para-nitrophenyl phosphate (E600) and other inhibitors. (1) The acid pH4-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol and exhibited the characteristic properties of the lysosomal lipase: the maximal activating effect of detergents occurs at relatively high concentrations (the substrate/detergent optimal molar ratios were 1:5 and 1:25 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively); its activity was strongly inhibited by para-chloromercuribenzoate (2.5 mmol/l), but was not significantly affected by serum albumin and E600 (10(-2) mmol/l). (2) The neutral pH7-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol. It is resistant to E600 and heat-stable, similarly to the acid pH4-lipase, but it is well discriminated from the acid enzyme by its substrate/detergent optimal molar ratios (1:2 and 1:3 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively), whereas higher detergent concentrations, optimal for the acid lipase, are strongly inhibitory for the neutral enzyme. (3) The pH5-lipase present in gastric juice as well as in stomach homogenate exhibited properties obviously discriminating it from the other lipolytic enzymes from stomach homogenate: broad substrate specificity for P10- as well as P4-triacylglycerols, activation by low concentrations of amphiphiles (with optimal ratios triacylglycerols/taurocholate, triacylglycerols/taurocholate and triacylglycerols/phosphatidylcholine around 1:1, 1:3 and 1:0.1, respectively), heat-lability, strong activation by serum albumin and inhibition by E600 (10(-2) mmol/l). This pH5-lipase is the sole lipolytic enzyme present in gastric juice and is probably identical with the well-known 'gastric' lipase. (4) A pH7.5-enzyme is characterized by its specificity for P4-triacylglycerols, its heat-lability at 50 degrees C and its strong inhibition by E600 (10(-2) mmol/l).     

Characterization and solubilization of membrane bound diacylglycerol lipases from bovine brain

Bovine brain contains two diacylglycerol lipases. One is localized in purified microsomes and the other is found in the plasma membrane fraction. The microsomal enzyme is markedly stimulated by the non-ionic detergent, Triton X-100, and Ca2+, whereas the plasma membrane diacylglycerol lipase is strongly inhibited by Triton X-100 and Ca2+ has no effect on its enzymic activity. Both enzymes were solubilized using 0.25% Triton X-100. The solubilized enzymes followed Michaelis-Menten kinetics. The apparent Km values for microsomal and plasma membrane enzymes are 30.5 and 12.0 microM respectively. Both lipases are strongly inhibited by RHC 80267, with Ki values for microsomal and plasma membrane diacylglycerol lipases of 70 and 43 microM, respectively. The retention of microsomal diacylglycerol lipase on a concanavalin A-Sepharose column and its elution by methyl alpha-D-mannoside indicates the glycoprotein nature of this enzyme.     

Immobilization in the presence of Triton X-100: modifications in activity and thermostability of <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis> CCR11 lipase

A partially purified lipase produced by the thermophile Geobacillus thermoleovorans CCR11 was immobilized by adsorption on porous polypropylene (Accurel EP-100) in the presence and absence of 0.1% Triton X-100. Lipase production was induced in a 2.5% high oleic safflower oil medium and the enzyme was partially purified by diafiltration (co. 500,000 Da). Immobilization conditions were established at 25 °C, pH 6, and a protein concentration of 0.9 mg/mL in the presence and absence of 0.1% Triton X-100. Immobilization increased enzyme thermostability but there was no change in neither the optimum pH nor in pH resistance irrelevant to the presence of the detergent during immobilization. Immobilization with or without Triton X-100 allowed the reuse of the lipase preparation for 11 and 8 cycles, respectively. There was a significant difference between residual activity of immobilized and soluble enzyme after 36 days of storage at 4 °C (P < 0.05). With respect to chain length specificity, the immobilized lipase showed less activity over short chain esters than the soluble lipase. The immobilized lipase showed good resistance to desorption with phosphate buffer and NaCl; minor loses with detergents were observed (less than 50% with Triton X-100 and Tween-80), but activity was completely lost with SDS. Immobilization of G. thermoleovorans CCR11 lipase in porous polypropylene is a simple and easy method to obtain a biocatalyst with increased stability, improved performance, with the possibility for re-use, and therefore an interesting potential use in commercial conditions.     

Membrane-bound 'synthetic lipase' specifically cultured under solid-state fermentation and submerged fermentation by Rhizopus chinensis: a comparative investigation

Rhizopus chinensis was able to produce synthetic lipases under both solid-state and submerged fermentations. These lipases were extracted from cell membrane using Triton X-100, and purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction chromatography and gel filtration chromatography. Judging from SDS-PAGE, the specific synthetic lipases associated with SSF (named as SSL) and SmF (named as SML) were different in the apparent molecular mass (62 and 40kDa). In term of hydrolytic activity, both enzymes exhibited maximum values at pH 8.0 and 40 degrees C; SSL appeared to be more pH tolerant and thermostable than SML. PMSF negligibly affected SSL but strongly reduced the activity of SML. Both enzymes showed clear preference for long-chained p-nitrophenyl esters, yielding maximum activity towards p-nitrophenyl palmitate (with SSL) and p-nitrophenyl laurate (with SML). In term of synthetic activity, lyophilized enzymes gave the highest values both at 30 degrees C, but at different pH memories (7.5 for SSL and 6.5 for SML). Most of ethyl esters synthesized by the two enzymes achieved good yields (>90%), and tetradecanoic acid and laurate acid separately served as the best acyl donors.     

Partial purification and characterization of phosphatidic acid-specific phospholipase A(1) in porcine platelet membranes

We have shown previously that the phospholipase A (PLA) activity specific for phosphatidic acid (PA) in porcine platelet membranes is of the A(1) type (PA-PLA(1)) [J. Biol. Chem. 259 (1984) 5083]. In the present study, the PA-PLA(1) was solubilized in Triton X-100 from membranes pre-treated with 1 M NaCl, and purified 280-fold from platelet homogenates by sequential chromatography on blue-Toyopearl, red-Toyopearl, DEAE-Toyopearl, green-agarose, brown-agarose, polylysine-agarose, palmitoyl-CoA-agarose and blue-5PW columns. In the presence of 0.1% Triton X-100 in the assay mixture, the partially purified enzyme hydrolyzed the acyl group from the sn-1 position of PA independently of Ca(2+) and was highly specific for PA; phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) were poor substrates. The enzyme exhibited lysophospholipase activity for l-acyl-lysoPA at 7% of the activity for PA hydrolysis but no lipase activity was observed for triacylglycerol (TG) and diacylglycerol (DG). At 0.025% Triton X-100, the enzyme exhibited the highest activity, and PA was the best substrate, but PE was also hydrolyzed substantially. The partially purified PA-PLA(1) in porcine platelet membranes was shown to be different from previously purified and cloned phospholipases and lipases by comparing the sensitivities to a reducing agent, a serine-esterase inhibitor, a PLA(2) inhibitor, a Ca(2+)-independent phospholipase A(2) inhibitor, and a DG lipase inhibitor.     

Laccase production in solid‐state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×10⁵ U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.     

Effects of lipidic carbon sources on the extracellular lipolytic activity of a newly isolated strain of Bacillus subtilis

An isolate exhibiting high extracellular lipolytic activity was identified as Bacillus subtilis by 16S rRNA gene sequence analysis. The enzyme activity of the isolate was improved by using different concentrations of lipidic carbon sources such as vegetable oils, fatty acids and triglycerides. Lipolytic activity was assayed spectrophotometrically using p-nitrophenyl palmitate. One percent (v/v) of sesame oil provided the highest activity with 80 and 98% enhancements with respect to 1% (v/v) concentrations of linoleic acid and triolein as the favored fatty acid and triglyceride, respectively. Glucose presented a repressive effect on lipase production. Lipase secreted by B. subtilis was partially purified by ultrafiltration and anion exchange chromatography; and the purified enzyme was tested for its residual activity in the presence of EDTA, SDS, Triton X-100, Tween 20, Tween 80 and protease. The present work reports, for the first time, that the lipolytic activity of a B. subtilis strain can be improved by using inexpensive vegetable oils; and also that B. subtilis lipase is suitable for use in detergents.     

Partial purification and characterization of phosphatidic acid-specific phospholipase A1 in porcine platelet membranes

We have shown previously that the phospholipase A (PLA) activity specific for phosphatidic acid (PA) in porcine platelet membranes is of the A₁ type (PA-PLA₁) [J. Biol. Chem. 259 (1984) 5083]. In the present study, the PA-PLA₁ was solubilized in Triton X-100 from membranes pre-treated with 1 M NaCl, and purified 280-fold from platelet homogenates by sequential chromatography on blue-Toyopearl, red-Toyopearl, DEAE-Toyopearl, green-agarose, brown-agarose, polylysine-agarose, palmitoyl-CoA-agarose and blue-5PW columns. In the presence of 0.1% Triton X-100 in the assay mixture, the partially purified enzyme hydrolyzed the acyl group from the sn-1 position of PA independently of Ca²⁺ and was highly specific for PA; phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) were poor substrates. The enzyme exhibited lysophospholipase activity for l-acyl-lysoPA at 7% of the activity for PA hydrolysis but no lipase activity was observed for triacylglycerol (TG) and diacylglycerol (DG). At 0.025% Triton X-100, the enzyme exhibited the highest activity, and PA was the best substrate, but PE was also hydrolyzed substantially. The partially purified PA-PLA₁ in porcine platelet membranes was shown to be different from previously purified and cloned phospholipases and lipases by comparing the sensitivities to a reducing agent, a serine-esterase inhibitor, a PLA₂ inhibitor, a Ca²⁺-independent phospholipase A₂ inhibitor, and a DG lipase inhibitor.     

Effect of different cultural conditions for phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> CFR 335 in submerged and solid-state fermentations

The present article deals with the studies on the effect of media ingredients, such as carbon, nitrogen, inorganic phosphates, surfactants, and metal salts, on phytase enzyme production by Aspergillus niger CFR 335 in submerged (SmF) and solid-state fermentations (SSF). The results obtained showed a 1.5-fold higher enzyme yield in the presence of sucrose in both SmF and SSF, while peptone was found to be a favorable nitrogen source for SmF. Sodium dihydrogen phosphate (NaH₂PO₄) favored 34% higher enzyme yield than the control, which was followed by 19% higher activity in potassium dihydrogen phosphate (KH₂PO₄) in SSF at 0.015% w/v. The addition of Tween-20 in SmF showed a maximum yield of 12.6 U/mL while, SDS suppressed the growth of the fungus. None of the surfactants favored the enzyme yield in SSF. Calcium chloride (CaCl₂) was extensively efficient in stimulating more than 55% higher phytase production in SmF at 0.01% v/v. In SSF, none of the metal salts stimulated phytase production.     

Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid

Tannase production by Aspergillus niger Aa-20 was studied in submerged (SmF) and solid-state (SSF) fermentation systems with different tannic acid and glucose concentrations. Tannase activity and productivity were at least 2.5 times higher in SSF than in SmF. Addition of high tannic acid concentrations increased total tannase activity in SSF, while in SmF it was decreased. In SmF, total tannase activity increased from 0.57 to 1.03 IU/mL, when the initial glucose concentration increased from 6.25 to 25 g/L, but a strong catabolite repression of tannase synthesis was observed in SmF when an initial glucose concentration of 50 g/L was used. In SSF, maximal values of total tannase activity decreased from 7.79 to 2.51 IU when the initial glucose concentration was increased from 6.25 to 200 g/L. Kinetic results on tannase production indicate that low tannase activity titers in SmF could be associated to an enzyme degradation process which is not present in SSF. Tannase titers produced by A. niger Aa-20 are fermentation system-dependent, favoring SSF over SmF. Journal of Industrial Microbiology & Biotechnology (2001) 26, 296–302. Received 07 July 2000/ Accepted in revised form 15 February 2001     

Study on the potential of cold-active lipases from psychrotrophic fungi for detergent formulation

Lipases from psychrotrophic fungal isolates BPF4 and BPF6 identified as Penicilium canesense and Pseudogymnoascus roseus respectively were characterized for their compatibility towards laundry detergent. BPF4 and BPF6 lipases showed maximum activity at pH 11 and 9 respectively and at 40?°C. The residual activities at 20?°C and 4?°C of BPF4 lipase were 35% and 20% and of BPF6 lipase were 70% and 20?°C respectively. Both the enzymes were stable at 4?°C, 20?°C and 40?°C for 2?h losing at the most 20% of activities. Both the enzymes were metalloenzymes with activity enhancement by nearly threefold by Ca²⁺. Contrary to BPF6 lipase, BPF4 enzyme was not stimulated by EDTA nor inhibited, rather stimulated by SDS and Triton X-100 by 125% and 330% respectively. Both the lipases showed minor to moderate inhibition by NaClO₃ and H₂O₂, and exhibited nearly 90% residual activity after 1?h of incubation in selected detergent brands thus indicating potential for their inclusion in detergent formulation thereby facilitating cold-washing as a step towards mitigation of climate change.     

Evidence for Membrane-Associated Choline Kinase Activity in Rat Striatum

The distribution of choline kinase (EC 2.7.1.32) activity was investigated in subcellular fractions of rat striatum. Enzyme activity in the crude mitochondrial fraction, determined after dissolution in Triton X-100, was 5.90 mumol/g initial wet weight/h. When a crude mitochondrial preparation was hypoosmotically shocked and fractionated, followed by the addition of Triton X-100, choline kinase activity in the soluble and particulate fractions was 4.58 and 1.40 mumol/g initial wet weight/h, respectively. Enzyme activity in the particulate fraction was not detected in the absence of Triton X-100 or in the presence of NaCl (up to 1.5 M). Subcellular enzyme markers indicated that the membrane-associated activity was not attributable to mitochondrial or microsomal contamination. Kinetic analysis of the activity of soluble and membrane-solubilized choline kinase indicated Km values of 0.74 mM and 0.68 mM, respectively. Results indicate that choline kinase activity may be measured in both the soluble and the particulate fractions of rat striatum, the latter most likely involving enzyme associated with membrane through hydrophobic or covalent interactions. The specific function of the membrane-associated enzyme has not yet been determined.     

Comparative production of cellulases by mutants of Penicillium janthinellum NCIM 1171 and its application in hydrolysis of Avicel and cellulose

Mutants of Penicillium janthinellum NCIM 1171 were evaluated for cellulase production using both submerged fermentation (SmF) and solid state fermentation (SSF). Mutant EU2D-21 gave highest yields of cellulases in both SmF and SSF. Hydrolysis of Avicel and cellulose were compared using SmF and SSF derived enzyme preparations obtained from EU2D-21. Surprisingly, the use of SSF derived preparation gave less hydrolysis compared to SmF derived enzymes. This may be due to inactivation of β-glucosidase at 50 °C in SSF derived enzyme preparations. SmF derived enzyme preparations contained both thermostable and thermosensitive β-glucosidases where as SSF derived enzyme preparations contained predominantly thermosensitive β-glucosidase. This is the first report on less thermostability of SSF derived β-glucosidase which is the main reason for getting less hydrolysis.     

Properties and characterization of a highly purified sarcoplasmic reticulum Ca2+-ATPase from dog cardiac and rabbit skeletal muscle

Sarcoplasmic reticulum (SR) Ca2+-ATPase was purified from dog cardiac and rabbit skeletal muscle using Triton X-100 at optimal ratios of 0.5 for cardiac and 0.5 to 1.0 for skeletal SR. The yields of Ca2+-ATPase were 4 to 5 and 1 to 2.2 mg/100 mg of cardiac and skeletal SR protein, respectively. The enzyme activities were 547 +/- 67 mumol ADP/mg/h for cardiac and 1192 +/- 172 mumol ADP/mg/h for skeletal Ca2+-ATPase. Removal of excess Triton X-100 increased the enzyme activities to 719 +/- 70 and 1473 +/- 206 mumol ADP/mg/h, respectively. The residual content of Triton X-100 for cardiac and skeletal Ca2+-ATPase was 20 and 5 mol/mol of enzyme, respectively. Maximum levels of phosphoenzyme were 4.4 +/- 0.2 and 5.6 +/- 0.6 nmol/mg in each case. A single protein band of 100 kDa was obtained for each purified Ca2+-ATPase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparations were stable at -80 degrees C for 5 months in the presence of 1 mM Ca2+. The phospholipid content of the purified enzyme was 2-fold greater than that of native cardiac and skeletal SR microsomes. Repeated washing of the purified enzyme preparation did not alter the phospholipid content or the specific activities.     

Mixed substrate solid state fermentation for production and extraction of lipase from Aspergillus niger MTCC 2594

A novel mixed substrate solid-state fermentation (SSF) process has been developed for Aspergillus niger MTCC 2594 using wheat bran (WB) and gingelly oil cake (GOC) and the results showed that addition of GOC to WB (WB : GOC, 3 : 1, w/w) increased the lipase activity by 36.0% and the activity was 384.3+/-4.5 U/g dry substrate at 30 degrees C and 72 h. Scale up of lipase production to 100 g and 1 kg tray-level batch fermentation resulted in 95.0% and 84.0% of enzyme activities respectively at 72 h. A three-stage multiple contact counter-current extraction yielded 97% enzyme recovery with a contact time of 60 min. However, extraction by simple percolation and plug-flow methods resulted in decreased enzyme recoveries. The mixed substrate SSF process has resulted in a significant increase in specific activity (58.9%) when compared to a submerged fermentation (SmF) system. Furthermore, an efficient process of extraction has been standardized with this process. Use of GOC along with WB as potential raw materials for enzyme production could be of great commercial significance. This is the first report on the production and extraction of lipase from Aspergillus niger using mixed solid substrates, WB and GOC, which are potential raw materials for the production of enzymes and other value-added products.     

Exopectinases produced by Aspergillus niger in solid-state and submerged fermentation: a comparative study

Exopectinase production by Aspergillus niger was compared in submerged fermentation (SmF) and solid-state fermentation (SSF). SSF was carried out using polyurethane foam (PUF) as the solid support. The purpose was to study the effect of sucrose addition (0 or 40 g/l) and water activity level (A _w=0.99 or 0.96) on the level of enzyme activity induced by 15 g/l of pectin. Mycelial growth, as well as extracellular protease production, was also monitored. Sucrose addition in SmF resulted in catabolite repression of exopectinase activity. However, in SSF, an enhancement of enzyme activity was observed. Protease levels were minimal in SSF experiments with sucrose and maximal in SmF without sucrose. Exopectinase yields (IU/g X) were negligible in SmF with sucrose. The high levels of exopectinase with sucrose and high A _w in SSF can be explained by a much higher level of biomass production without catabolite repression and with lower protease contamination. Journal of Industrial Microbiology & Biotechnology (2001) 26, 271–275. Received 05 July 2000/ Accepted in revised form 27 January 2001     

Screening Botryosphaeria species for lipases: Production of lipase by Botryosphaeria ribis EC-01 grown on soybean oil and other carbon sources

Nine isolates of Botryosphaeria spp. were screened for lipases when cultivated on eight different plant seed oils and glycerol, and all produced lipases. Botryosphaeria ribis EC-01 produced highest lipase titres on soybean oil and glycerol, while eight isolates of Botryosphaeria rhodina produced significantly lower enzyme titres. B. ribis EC-01 produced lipase when grown on different fatty acids, surfactants, carbohydrates and triacylglycerols, with highest enzyme titres produced on Triton X-100-emulsified stearic (316.7 U/mL), palmitic (283.5 U/mL) and oleic (247.4 U/mg) acids, and soybean oil (105.6 U/mL), as well as castor oil (191.2 U/mg); an enhancement of 9-fold over soybean oil-grown cultures. Glycerol was also a good substrate for lipase production. The crude lipase extract was optimally active at pH 8.0 and 55 °C, stable between 30 and 55 °C and pH 1–10, and tolerant to 50% (v/v) glycerol, methanol and ethanol. The crude lipase showed affinity for substrates of short, average and long-chain fatty acids (different esters of p-nitrophenol and triacylglycerols). Zymograms developed with 4-methylumbelliferyl-butyrate showed two bands of lipolytic activity at 45 and 15 kDa. This is the first report on the production of lipases by B. ribis grown on these different carbon sources.     

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.