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1.
Recombinant Candida rugosa lipase 5 (LIP5) has been functionally expressed along with other isoforms in our laboratory. However, the characterization and codon optimization of LIP5 have not been done. In this work, we characterized, codon-optimized and compared LIP5 with commercial lipase. LIP5 activity on hydrolysis of p-nitrophenyl (p-NP) butyrate was optimal at 55 °C as compared with 37 °C of the commercial lipase. Several assays were also performed to determine the substrate specificity of LIP5. p-NP butyrate (C(4)), butyryl-CoA (C(4)), cholesteryl laurate (C(12)), and N-carbobenzoxy-l-tyrosine-p-nitrophenyl ester (l-NBTNPE) were found as preferred substrates of LIP5. Interestingly, LIP5 specificity on hydrolysis of amino acid-derivative substrates was shown to be the highest among any lipase isoforms, but it had very weak preference on hydrolyzing triacylglycerol substrates. LIP5 also displays a pH-dependent maximum activity of a lipase but an esterase substrate preference in general. The characterization of LIP5 along with that of LIP1-LIP4 previously identified shows that each lipase isoform has a distinct substrate preference and catalytic activity.  相似文献   

2.
Subtilisin was immobilized on polycaprolactam and used for food packaging applications to reduce the transference of microorganisms from the packaging material to the packaged food material. The optimized conditions for subtilisin immobilization was as follows: pH, 8; temperature, 4 °C; glutaraldehyde, 0.5%; incubation time, 25 h; and subtilisin concentration, 600 μL. The formation of -CH═N- at 1576 cm(-1) in the Fourier transform infrared (FTIR) spectrum confirmed the immobilization. Subtilisin-immobilized polycaprolactam (SIP) exhibited the highest residual activity of 106.67 ± 4.41% and 104.67 ± 0.88% at 40 °C and pH 8 and retained residual activity of 94% at the end of 56 days when compared to 21.33 ± 4.10% in the case of free subtilisin. SIP significantly (p < 0.05) lowered the colony forming units (CFU), dry weight, and protein and carbohydrate contents in bacterial and fungal biofilm. Practical application of the SIP on ham steaks at 4 and 20 °C showed a 2-3 times reduction of Staphylococcus aureus as well as Escherichia coli cells in the range of p < 0.05.  相似文献   

3.
Peanut shells, a major waste stream of food processing, served as a renewable substrate for inducing the production of laccases by basidiomycetes. Of 46 surface cultures examined, 29 showed laccase activity under the experimental conditions. The edible fungus Pleurotus sapidus was selected as the most active producer, immobilized on the shells, and cultivated in the fed-batch mode. A continuous rise in laccase activity was found, indicating the inducibility of laccase secretion by the peanut shells and the reusability of the mycelium. Two laccase isoenzymes were purified by decoupled 2-D electrophoresis, and amino acid sequence information was obtained by electrospray ionization tandem mass spectrometry. cDNAs of the corresponding gene and another laccase were cloned and sequenced using a PCR-based screening of a synthesized P. sapidus cDNA library. Data bank searches against public databases returned laccases of P. ostreatus and P. sajor-caju as the best hits. The potential use of laccases by the food industry is discussed.  相似文献   

4.
Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.  相似文献   

5.
Stability of dried and intermediate moisture tomato pulp during storage   总被引：1，自引：0，他引：1  
Commercial tomato pulp was air-dried to two final moisture contents in order to obtain intermediate moisture pulp (IMP, 23% moisture) and dried pulp (DP, 9% moisture). IMP and DP were stored at 4, 20, and 37 degrees C for approximately 5 months; periodically samples were analyzed to evaluate heat and oxidative damage by measurement of color changes, total phenolics, rutin, lycopene and furosine concentrations, and antioxidant activity of the lipophilic extract. IMP and DP, despite different drying degree, had similar antioxidant activity; in fact, whereas lycopene was stable to drying treatments, ascorbic acid was totally degraded in both products. During storage, IMP and DP showed different behavior: IMP was more sensitive to degradation than DP, especially with regard to lycopene, rutin, and antioxidant activity degradation and to nonenzymatic browning. Effects of storage temperature varied with regard to different parameters: variations in rutin, furosine, and color indices were higher in products stored at higher temperatures, while lycopene and antioxidant activity of the lipophilic fraction were maximally degraded in products stored at 4 degrees C.  相似文献   

6.
Ready to drink (RTD) teas are a growing segment in the beverage category, brought about by improvements in the flavor of these products and healthy market trends driven by consumers. The presented results evaluated the antioxidant phytochemical stability of RTD teas from aqueous infusions of traditional green tea (Camellia sinensis) and a botanical tea from yaupon holly (Ilex vomitoria) as influenced by packaging materials during cold storage. Two common packaging materials for RTD products are glass and polyethylene terephthalate (PET) and have been compared to a retortable pouch (RP), an emerging packaging material for various types of food since it is durable, inexpensive, lightweight, and easy to sterilize. Storage stability was then evaluated for each aqueous infusion prepared at 10 g/L at 90 °C for 10 min and evaluated at 3 °C in the absence of light over 12 weeks. Analyses included quantification and characterization of individual polyphenolics by high-performance liquid chromatography-photodiode array and liquid chromatography-electrospray ionization-mass spectrometry as well as changes in total antioxidant capacity. For green tea, concentrations of the three major flavan-3-ols, epigallocatechin gallate, epigallocatechin, and epicatechin gallate were better retained in glass bottles as compared to other packages over 12 weeks. In yaupon holly, chlorogenic acid and its isomers that were the predominant compounds were generally stable in each packaging material, and a 20.6-fold higher amount of saponin was found as compared to green tea, which caused higher stability of flavonol glycosides present in yaupon holly during storage. The antioxidant capacity of green tea was better retained in glass and PET versus RP, whereas no differences were again observed for yaupon holly. Results highlight the superiority of oxygen-impervious glass packaging, but viable alternatives may be utilizable for RTD teas with variable phytochemical compositions.  相似文献   

7.
Enzymatic transformation of humic acids (HA), fulvic acids (FA) and indole was examined using naphthalene 1,2-dioxygenase (NDO). NDO was used as a model for dioxygenase enzymes found in various microbial species. Indole was used as a model substrate for NDO-catalyzed reactions resulting in condensation products. Although NDO is not classified as a soil enzyme, all HA and FA tested were susceptible to NDO-induced transformation. The extent of NDO-specific NADH oxidation in solutions containing HA and FA paralleled the percent aromaticity of the HA and FA. Furthermore, the UV–Vis absorptive properties of NDO-treated HA and FA were altered in a manner suggesting condensation reactions similar to the formation of indigo from indole. Condensation reactions were enhanced in NDO-treated mixtures containing indole and an FA. NDO retained activity for 2 weeks under ambient conditions, and retained some enzymatic activity for 9 days based on detection of specific metabolites by HPLC, suggesting prolonged extracellular activity. Humic substances have not previously been known to be substrates for dioxygenases; even more significant was that dioxygenase enzymes can facilitate condensation reactions between indole-like functional groups well-known to be present in HA and FA. These results illustrate how dioxygenases can be potential humic-modifying enzymes when released into the environment upon microbial death and concurrent cell lysis which could alter the bioavailability of organic contaminants associated with dissolved organic matter through specific modulation of enzyme activity involving substrate competition.  相似文献   

8.
Human milk fat substitutes (HMFSs) were synthesized by lipozyme RM IM-catalyzed acidolysis of chemically interesterified palm stearin (mp = 58 °C) with mixed FAs from rapeseed oil, sunflower oil, palm kernel oil, stearic acid, and myristic acid in a solvent-free system. Response surface methodology (RSM) was used to model and optimize the reactions, and the factors chosen were reaction time, temperature, substrate molar ratio, and enzyme load. The optimal conditions generated from the models were as follows: reaction time, 3.4 h; temperature, 57 °C; substrate molar ratio, 14.6 mol/mol; and enzyme load, 10.7 wt % (by the weight of total substrates). Under these conditions, the contents of palmitic acid (PA) and PA at sn-2 position (sn-2 PA) were 29.7 and 62.8%, respectively, and other observed FAs were all within the range of FAs of HMF. The product was evaluated by the cited model, and a high score (85.8) was obtained, which indicated a high degree of similarity of the product to HMF.  相似文献   

9.
The effect was studied of drying and ageing leaves on the ability of their water extracts to dissolve hydrous ferric oxide. Extracts of freshly collected picked and autumn-fallen oak, beech, and larch leaves were compared with those of leaves from the same collections that were either freeze-dried or dried at 105°C. The iron-dissolving properties of the fallen leaves were not significantly affected by either method of drying, but drying at 105°C greatly increased the activities of picked larch and beech leaves. Freeze-drying had relatively little effect. Larch needles lost their activity very quickly when aged aerobically for 8 months, while the activity of beech leaves decreased slowly. The activity of picked and fallen oak leaves increased dramatically during the first 1–2 weeks, and then decreased, rapidly at first; after 8 months the activity was still comparable with the initial value. There was no direct relationship between the activities of the extracts and the pH or the concentrations of polyphenols.  相似文献   

10.
The kinetics of the thermal hydrolysis of the fructans of Agave salmiana were determined during the cooking step of mezcal production in a pilot autoclave. Thermal hydrolysis was achieved at different temperatures and cooking times, ranging from 96 to 116 °C and from 20 to 80 h. A simple kinetic model of the depolymerization of fructans to monomers and other reducing sugars and of the degradation of reducing sugars to furans [principally 5-(hydroxymethyl)furfural, HMF] was developed. From this model, the rate constants of the reactions were calculated, as well as the pre-exponential factors and activation energies of the Arrhenius equation. The model was found to fit the experimental data well. The tradeoff between a maximum fructan hydrolysis and a critical furan concentration in allowing for the best ethanol yield during fermentation was investigated. The results indicated that the thermal hydrolysis of agave was optimal, from the point of view of ethanol yield in the ensuing fermentation, in the temperature range of 106-116 °C and the cooking range time of 6-14 h. The optimal conditions corresponded to a fructan hydrolysis of 80%, producing syrups with furan and reducing sugar concentrations of 1 ± 0.1 and 110 ± 10 g/L, respectively.  相似文献   

11.
The effect of different heat treatments of camelina (Camelina sativa) seeds on the phenolic profile and antioxidant activity of their hydrolyzed extracts was investigated. The results showed that total phenol contents increased in thermally treated seeds. Heat treatment affected also the quantities of individual phenolic compounds in extracts. Phenolics in unheated camelina seeds existed in bound rather than in free form. A temperature of 160 °C was required for release of insoluble bound phenolics, whereas lower temperatures were found to be optimal to liberate those present as soluble conjugates. The best reducing power and alkyl peroxyl radical scavenging activity in the emulsion was expressed by phenolics which were bound to the cell wall, whereas the best iron chelators and 2,2-diphenyl-1-picrylhydrazyl (DPPH?) radical scavengers were found to be those present in free form. The heat treatment of seeds up to 120 °C increased the reducing power and DPPH? radical scavenging ability of extracts, but negatively affected iron chelating ability and their activity in an emulsion against alkyl peroxyl radicals.  相似文献   

12.
Previously-frozen stores of organic carbon (C) are now subject to decomposition due to a warming Arctic climate and associated permafrost thaw; however, estimates of the amount of greenhouse gases (GHG) that may be released are not well constrained. Knowing more about the functions of the extant permafrost microbial community will inform this knowledge gap. The exploration of microbial functional traits may be useful to elucidate the relationship between microbial diversity and ecosystem function. We characterized the community traits and functional diversity of the bacterial and Archaeal component of the microbial community from three depths of permafrost, as well as the organic and mineral horizons of the seasonally-thawed active layer, by assessing ‘substrate-use richness,’ ‘substrate preference,’ ‘growth rate,’ ‘and substrate specific growth rate.’ We measured the microbial community response to 31 substrates with an EcoPlate (Biolog, Inc.) assay at three incubation temperatures (1, 10, and 20 °C) using a kinetic approach, and modeled the microbial response to each substrate with a modified logistic growth function. We hypothesized that the permafrost communities would be selected for high functional potential and activity at cold temperatures. Rather, we found that the permafrost community did not have a higher functional diversity or activity at 1 °C than the organic active layer soils. In addition, permafrost communities increased their growth rates with increasing temperature, indicating that the highest incubation temperature (20 °C) was below their temperature optimum for growth. As predicted, the permafrost communities did exhibit temperature dependent substrate preferences. Thus, permafrost microbial communities did not appear to be selected for higher metabolism and the ability to use a broad suite of substrates at low temperatures, which suggests that they may have limited function immediately following thaw when temperatures are near 0 °C. However, changes in community composition or additional permafrost warming will increase the functional capabilities of permafrost microbes to decompose the C stored in those soils.  相似文献   

13.
Studies with surface samples of Iowa soils selected to obtain a wide range in properties showed that the following treatments of field-moist soils had no effect on urease activity: leaching with water ; drying for 24 h at temperatures ranging from 30 to 60°C ; storage for 6 months at temperatures ranging from ?20 to 40°C; incubation under aerobic or waterlogged conditions at 30 or 40°C for 6 months. No loss of urease activity could be detected when field-moist soils were air-dried and stored at 21–23°C for 2yr, but complete loss of urease activity was observed when they were dried at 105°C for 24 h or autoclaved (120°C) for 2h. Inactivation of urease in moist soils was detected at temperatures above 60°C.Treatment of field-moist soils with proteolytic enzymes which cause rapid destruction of jackbean urease did not decrease urease activity, but jackbean urease was destroyed or inactivated when added to sterilized or unsterilized soils.Although no decrease in urease activity could be detected when field-moist soils were air-dried, an appreciable (9–33%) decrease in urease activity was observed when air-dried soils were incubated under aerobic or waterlogged conditions. This decrease occurred within a few days, and prolonged incubation or repetition of the drying-incubation treatment did not lead to a further decrease in urease activity. Treatment of incubated air-dried soil with urease or glucose initially increased urease activity to a level exceeding that of the undried soil, but this activity decreased with time and eventually stabilized at the level observed for the undried soil.The work reported supports the conclusions from previous work that the native urease in Iowa soils is remarkably stable and that different soils have different levels of urease activity determined by the ability of their constituents to protect urease against microbial degradation and other processes leading to inactivation of enzymes.  相似文献   

14.
Characterization of the autolytic profile of arrowtooth flounder (ATF) muscle indicated the involvement of heat-activated proteinases active at both acidic and alkaline pH values. Further assay of fish extract exhibited the maximum activity at 60 degrees C against casein used as a substrate at both pH 5.5 and 8.0. The maximum activity shifted to lower temperatures by the addition of urea with two distinctive patterns: activity reduction at pH 5.5 and activity enhancement at pH 8.0. The highest inhibition by E-64 indicated the proteinase belongs to the cysteine proteinase class. At pH 5.5, the proteinase hydrolyzed Z-Phe-Arg-NMec and all types of protein substrates tested at higher rate than that at pH 8.0. Activity bands, observed on the activity-stained substrate gels, indicated similar proteinases are responsible for the proteolytic activity observed at both pH values. When proteins of fish extract were separated by HPLC-SEC, only one proteolytic peak was observed at the retention time of 26 min with an estimated molecular weight of 39800 Da. The results implied cathepsin L is a predominant proteinase responsible for autolysis of ATF muscle at elevated temperatures.  相似文献   

15.
The press-residue of black currants provides a good source of phenolic antioxidants. The purpose of this study was to optimize the extraction of phenolic compounds from the press-residue by use of extraction conditions compatible with food use. The effects of temperature, extraction duration, and use of ultrasound-assisted extraction on the juice yield, total phenolics (TP), and anthocyanin content of aqueous extracts were studied. Within the variables and response factors tested, the optimal conditions were a 15 min extraction at 90 °C. No significant effect from ultrasound-assisted extraction was found. The composition of anthocyanins and polyphenols was highly dependent on the extraction temperature. The percentage contribution of delphinidin- and cyanidin-3-rutinoside to TP had a negative linear correlation with temperature, while delphinidin- and cyanidin-3-glucoside had a positive linear correlation with temperature, with a maximum amount obtained at 80 °C and 55 °C, respectively. Furthermore, extracts obtained at higher temperatures showed a stronger inhibition of proliferation of Caco-2, HT-29, and HCT 116 cells than extracts obtained at lower temperatures. This may be due to the decomposition of complex polyphenols at higher temperatures, making them more accessible to the cells.  相似文献   

16.
Different drying methods were applied to fresh Canadian-grown Echinacea purpurea flowers to determine optimal drying procedures for preserving caffeic acid derivatives. Fresh flowers of E. purpurea were dried by freeze-drying (FD), vacuum microwave drying with full vacuum (VMD), and air-drying (AD) at 25, 40, and 70 degrees C. Using HPLC, chicoric acid and caftaric acid levels were quantitated in dried flowers. These acids were significantly affected by the drying method conditions used. Although significant (p < 0.05) loss of chicoric acid was observed when flowers were stored at high moisture, VMD flowers with a low moisture content retained the highest levels of chicoric acid and caftaric acid similar to FD flowers. Flowers that were AD at 25 degrees C retained about 50%, while those dried by AD at 70 degrees C resulted in the lowest retention of these acids. Although flowers dried by AD at 40 degrees C retained relatively high amounts of chicoric acid and caftaric acid, the time (55 h) required to reach optimal drying was considerably longer than that (47 min) for VMD.  相似文献   

17.
To obtain basic information about enzymatic deterioration of buckwheat flour, triacylglycerol lipase (LIP; EC 3.1.1.3) was purified from buckwheat seed. The LIP consisted of two isozymes, LIP I and LIP II, and they were purified with purification folds of 60 and 143 with final specific activities of 0.108 and 0.727 mumol of fatty acid released per minute per milligram of protein at 30 degrees C using triolein as a substrate. Molecular weights were estimated to be 150 (LIP I) and 28.4 kDa (LIP I) by gel filtration and 171 (LIP I) and 26.5 kDa (LIP II) by SDS-PAGE. Optimal pHs of LIP activities were 3.0 (LIP I) and 6.0 (Lip II) using triolein as a substrate. Both LIP I and II reacted in the acidic pH range. Optimal temperatures were 30 (LIP I) and 40 degrees C (LIP II), and both LIP I and II were stable below 30 degrees C when p-nitrophenyl-laurate was used as a substrate. However, they were inactivated above 60 degrees C. On the other hand, when triolein was used as a substrate, optimal temperatures were 30 degrees C for both LIP I and II, and they retained 40% of their activity after a 4 h incubation of enzymes at 70 degrees C. LIP I and II had higher activity against triolein than monoolein or tri/monopalmitin. Most of the LIP activity was distributed in the embryo.  相似文献   

18.
Hydroxyphenylureas are the first main metabolites formed in the environment from pesticide and biocide urea compounds. Because fungi release potent exocellular oxidases, we studied the ability of laccases produced by the white rot fungus, T. versicolor, to catalyze in vitro the transformation of five hydroxyphenylureas, to identify transformation pathways and mechanisms. Our results establish that the pH of the reaction has a strong influence on both the kinetics of the reaction and the nature of the transformation products. Structural characterization by spectroscopic methods (NMR, mass spectrometry) of eleven transformation products shows that laccase oxidizes the substrates to quinones or to polyaromatic oligomers. Slightly acidic conditions favor the formation of quinones as final transformation products. In contrast, at pH 5-6, the quinones further react with the remaining substrate in solution to give hetero-oligomers via carbon-carbon or carbon-oxygen bond formation. A reaction pathway is proposed for each of the identified products. These results demonstrate that fungal laccases could assist the transformation of hydroxyphenylureas.  相似文献   

19.
An enzyme having activity toward n-hexanol was purified from apple, and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephadex G-100 column. The obtained enzyme had a yield of 16.00% with a specific activity of 18879.20 U/mg protein and overall purification of 142.77-fold. The enzyme showed activity to isoamylol, 1-propanol, n-hexanol, and isobutanol but not toward methanol and ethanol. With n-hexanol as a substrate, the optimum conditions were pH 4.0 and 30 °C for enzyme activity and pH 3.0-4.0 and temperatures below 40 °C for enzyme stability. The enzyme activity was increased significantly by adding l-cysteine and Fe(2+) at all tested concentrations and slightly by Zn(2+) at a high concentration but decreased by additions of EDTA, Ga(2+), K(+), Mg(2+), sodium dodecyl sulfate (SDS), sodium aluminum sulfate (SAS), dithiothreitol (DTT), and glutathione (GSH). The enzyme activities toward n-hexanol and n-hexanal were increased by NADH but decreased by NAD(+), in contrast to a decrease toward n-hexane by addition of both NAD(+) and NADH.  相似文献   

20.
Our study showed that sorghum and millet followed a similar pattern of changes when they were malted under similar conditions. When the malt from these cereals was mashed, both cereal types produced wide spectra of substrates (sugars and amino acids) that are required for yeast fermentation when malted at either lower or higher temperatures. At the germination temperatures of 20, 25, and 30 °C used in malting both cereal types, production of reducing sugars and that of free amino nitrogen (FAN) were similar. This is an important quality attribute for both cereals because it implies that variation in temperature during the malting of sorghum and millet, especially when malting temperature is difficult to control, and also reflecting temperature variations, experienced in different countries, will not have an adverse effect on the production and release of amino acids and sugars required by yeast during fermentation. Such consistency in the availability of yeast food (substrates) for metabolism during fermentation when sorghum and millet are malted at various temperatures is likely to reduce processing issues when their malts are used for brewing. Although sorghum has gained wide application in the brewing industry, and has been used extensively in brewing gluten-free beer on industrial scale, this is not the case with millet. The work described here provides novel information regarding the potential of millet for brewing. When both cereals were malted, the results obtained for millet in this study followed patterns similar to those of sorghum. This suggests that millet, in terms of sugars and amino acids, can play a role similar to that of sorghum in the brewing industry. This further suggests that millet, like sorghum, would be a good raw material for brewing gluten-free beer. Inclusion of millet as a brewing raw material will increase the availability of suitable materials (raw material sustainability) for use in the production of gluten-free beer, beverages, and other products. The availability of wider range of raw materials will not only help to reduce costs of beer production, but by extension, the benefit of reduced cost of production can be gained by consumers of gluten-free beer as the product would be cheaper and more widely available.  相似文献   

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