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1.
κ-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by κ-carrageenase is safe and controllable. Therefore, κ-carrageenases have captured more and more attentions. In this study, a κ-carrageenase encoding gene, cgk X, was cloned from Pseudoalteromonas sp. QY203 with degenerate and inverse PCR. It comprised an ORF of 1194 bp in length, encoding a protein with 397 amino acid residues. Cgk X is a new member of glycoside hydrolase family 16. The deduced amino acid sequence shared a high similarity with Cgk X of Pseudoalteromonas κ-carrageenase; however, the recombinant Cgk X showed different biochemical characteristics. The recombinant enzyme was most active at p H 7.0 and 55℃ in the presence of 300 mmol L~(~(-1))Na Cl. It was stable in a broad range of acidity ranging from p H 3.0 to p H 10.0 when temperature was below 40℃. More than 80% of its activity was maintained after being incubated at p H 3.6–10.0 and 4℃ for 24 h. Cgk X retained more than 90% of activity after being incubated at 40℃ for 1 h. EDTA and SDS(1 mmol L~(-1)) did not inhibit its activity. Cgk X hydrolyzed κ-carrageenan into disaccharide and tetrasaccharide as an endo-cleaver. All these characteristics demonstrated that Cgk X is applicable to both κ-carrageenan oligosaccharide production and κ-carrageenase structure-function research.  相似文献   

2.
A new κ-carrageenase gene cgk S was cloned from marine bacterium Shewanella sp. Kz7 by using degenerate and site-finding PCR. The gene was comprised of an open reading frame of 1224 bp, encoding 407 amino acid residues, with a signal peptide of 24 residues. Based on the deduced amino acid sequence, the κ-carrageenase Cgk S was classified into the Glycoside Hydrolase family 16. The cgk S gene was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity with a specific activity of 716.8 U mg-1 and a yield of 69%. Recombinant Cgk S was most active at 45℃ and p H 8.0. It was stable at p H 6.0–9.0 and below 30℃. The enzyme did not require Na Cl for activity, although its activity was enhanced by Na Cl. Cgk S degraded κ-carrageenan in an endo-fashion releasing tetrasaccharides and disaccharides as main hydrolysis products.  相似文献   

3.
Absract A lipase gene, lip1233, isolated from Pseudoalteromonas lipolytica SCSIO 04301, was cloned and expressed in E. coli. The enzyme comprised 810 amino acid residues with a deduced molecular weight of 80 k Da. Lip1233 was grouped into the lipase family X because it contained a highly conserved motif GHSLG. The recombinant enzyme was purified with Ni-NTA affinity chromatography. The optimal temperature and p H value of Lip1233 were 45℃ and 8.0, respectively. It retained more than 70% of original activity after being incubated in p H ranging from 6.0 to 9.5 for 30 min. It was stable when the temperature was below 45℃, but was unstable when the temperature was above 55℃. Most metal ions tested had no significant effect on the activity of Lip1233. Lip1233 remained more than original activity in some organic solvents at the concentration of 30%(v/v). It retained more than 30% activity after incubated in pure organic solvents for 12 h, while in hexane the activity was nearly 100%. Additionally, Lip1233 exhibited typical halotolerant characteristic as it was active under 4M Na Cl. Lip1233 powder could catalyze efficiently the synthesis of fructose esters in hexane at 40℃. These characteristics demonstrated that Lip1233 is applicable to elaborate food processing and organic synthesis.  相似文献   

4.
A total of 69 strains of thermophilic bacteria were isolated from water, soil and sediment samples from three Indonesia’s hot spring areas (Pantai cermin, Kalianda and Banyu wedang) by using Minimal Synthetic Medium (MSM). The extreme thermophile Brevibacillus sp. PLI-1 was found to produce extracellular thermophilic alkaline protease with optimal activity at 70℃ and pH 8.0-9.0. The molecular weight of the protease was estimated to be around 56 kD by SDS-PAGE. The maximum activity of the protease was 26.54 U mL-1. The protease activity did not decrease after 30 min and still retained more than 70% of relative activity after 60 min at 70℃ and pH 8.0. The ion Mg2+ was found to promote protease activity at both low and high concentrations, whereas Cu2+ and Zn2+ could almost completely inhibit the activity. Divalent cation chelator EDTA inhibited the enzyme activity by 55.06% ± 0.27%, while the inhibition caused by PMSF, Leupeptin, Pepstain A and Benzamidine were 66.78% ± 3.25%, 52.37% ± 0.25%, 62.47% ± 2.96% and 50.99% ± 0.24%, respectively. Based on these observations, the enzyme activity was conspicuously sensitive to the serine and cysteine protease inhibitors. All these results indicated that the protease isolated from the strain PLI-1 was a thermophilic protease and had a high-temperature stability and a pH stability.  相似文献   

5.
Small heat shock proteins encompass a widespread but diverse class of proteins,which play key roles in protecting organisms from various stressors.In the present study,the full-length cDNAs of two small heat shock proteins(MgsHSP22 and MgsHSP24.1)were cloned from Mytilus galloprovincialis,which encoded peptides of 181 and 247 amino acids,respectively.Both MgsHSP22 and MgsHSP24.1 were detected in all tissues examined by real-time PCR,with the highest expression being observed in muscle and gonad tissues.The real-time PCR results revealed that Cd signifi cantly inhibited MgsHSP22 expression at 24 h and MgsHSP24.1 at 24 and 48 h under 5 μg/L Cd 2 + exposure.MgsHSP24.1 expression was also signifi cantly inhibited after 50 μg/L Cd 2+ exposure for 48 h.With regard to antioxidant enzymes,increased GPx and CAT activity were detected under Cd 2+ stress(5 and 50 μg/L),while no signifi cant difference in SOD activity was observed throughout the experiment.Overall,both MgsHsps and antioxidant enzymes revealed their potential as Cd stress biomarkers in M.galloprovincialis.  相似文献   

6.
In the present research, the strain SLYY-3 was isolated from sediments of Jiaozhou Bay, Qingdao, China. The strain SLYY-3, which produced a bacteriocin-like substance(BLS), was characterized to be a strain of Bacillus subtillis by biochemical profiling and 16 S r DNA sequence analysis. It is the first time to report that Bacillus subtilis from Jiaozhou Bay sediments could produce a BLS. The BLS of B. subtillis SLYY-3 exhibited strong inhibitory activity against gram-positive bacteria(including Staphylococcus aureus and B. subtillis) and some fungi(including Penicillium glaucum, Aspergillus niger and Aspergillus flavus). The antimicrobial activity was detected from culture in the exponential growth phase and reached its maximum when culture entered into stationary growth phase. It was thermo-tolerant even when being kept at 100℃ for 60 min without losing any activity and stable over a wide p H range from 1.0 to 12.0 while being inactivated by proteolytic enzyme and trypsin, indicating the proteinaceous nature of the BLS. The BLS was purified by precipitation with hydrochloric acid(HCl) and gel filteration(Sephadex G-100). SDS-PAGE analysis of the extracellular peptides of SLYY-3 revealed a bacteriocin-like protein with a molecular mass of 66 k Da. Altogether, these characteristics indicate the potential of the BLS for food industry as a protection against pathogenic and spoilage microorganisms.  相似文献   

7.
Protease is wildly used in various fields,such as food,medicine,washing,leather,cosmetics and other industrial fields.In this study,an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized.The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30 th hour and the enzyme activity reached the maximum value at the 36 th hour.The protease was purified with 3 steps involving ammonium sulfate precipitation,ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery.The molecular mass of the protease was estimated to be 25 k Da by SDS-PAGE analysis.The optimum temperature and p H for the protease activity were 50℃ and pH 10.0,respectively.The protease showed a strong stability in a wide range of pH values ranging from 6.0–11.0,and maintained 90% enzyme activity in strong alkaline environment with p H 11.0.Inhibitor trials indicated that the protease might be serine protease.But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn~(2+).Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS(MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.  相似文献   

8.
The effects of salinity on hemolymph osmotic pressure, Na^＋ concentration and Na^＋-K^＋-ATPase activity of gill of Chinese crab Eriocheir sinensis were studied. The results showed that hemolymph osmotic pressure and Na^＋ concentration increased significantly （P〈0.05）, and the Na^＋-K^＋-ATPase activity of gills decreased significantly （P〈0.05） when salinity increased from 0 to 16. The hemolymph osmotic pressure and Na^＋ concentration in each treatment group rose remarkably at 0.125 d or 0.25 d, while the Na^＋-K^＋-ATPase activity of gill reduced gradually with increased experiment time in 3 d. Then the three parameters remained at a constant level after 0.25 d, 0.125 d and 3 d, respectively, and higher hemolymph osmotic pressure, higher Na^＋ concentration and lower Na^＋-K^＋-ATPase activity of gill occurred at higher salinity. The effect of salinity change on protein concentration of hemolymph was indistinct （P〉0.05）; However, the protein concentration decreased gradually with the increase of salinity from 0.25 d to 1d, and then tended to be stable from day 1 to day 15.  相似文献   

9.
Carrageenan is a highly sulfated polysaccharide alternating of 1,4-O-α-D-galactopyranose-2,6-sulfate(D2S,6S) and 1,3-O-β-D-galactopyranose-2-sulfate(G2S). λ-Carrageenases are desirable tools for λ-carrageenan degradation. Based on the genome mining, a novel λ-carrageenase Cgl150A＿Wa was cloned from the bacterium Wenyingzhuangia aestuarii and expressed in Escherichia coli. Cgl150A＿Wa was an endo-acting enzyme and exhibited its maximum activity at 30℃ and pH 8.0. By employing a glycomics strategy ...  相似文献   

10.
The protease activity in digestive tract of young turbot Scophthalmus maximum was studied, and the optimal pH, temperature and NaCl concentration were determined for different portions of the fish＇s internal organs. The optimal activity in the fish＇s stomach was at pH of 2.2, while that in the intestinal extracts was within the alkaline range from 9.5 to 10.0. In hepatopancreas, the optimal pH was in low alkalinity at 8.5. The optimal reaction temperature was above 40℃ in stomach, intestine and hepatopancreas. With increasing temperature, the pH value increased in stomach, while in the intestine, an opposite tendency was observed due to combined effect of pH and temperature. NaCl concentration showed inhibitory impact on protein digestion in hepatopancreas. The main protease for protein digestion in turbot seemed to be pepsin. Moreover, the maximum protease activity in different segments of intestine existed in the hindgut.  相似文献   

11.
Accurate information on the spatial distribution and temporal change of wetlands is vital to devise effective measures for their protection. This study uses satellite images in 1994 and 2001 to assess the effects of topography and proximity to channels on wetland change in Maduo County on the Qinghai-Tibet Plateau, western China. In 1994 wetlands in the study area extended over 6,780.0 km2. They were distributed widely throughout the county, with a higher concentration in the south, and were especially prominent close to streams. The pattern of wetlands demonstrated a bell-shaped distribution curve with elevation, ranging over hill slopes with gradients from 0-19°, the commonest gradient being around 3°. Although the aspects of these hill slopes range over all directions, there is a lower concentration of wetlands facing east and southeast. The extent of wetlands in 2001 decreased to 6,181.1 km2. Marked spatial differentiation in the pattern of wetlands is evident, as their area increased by 1,193.3 km2 at lower elevations but decreased by 1,792.2 km2 at higher ground, resulting in a net decrease of 598.8 km2. In areas with a gradient <2° or >9° the area of wetlands remained approximately consistent from 1994-2001. Newly retained wetlands are situated in relatively flat lowland areas, with no evident preference in terms of aspect. Wetlands on north-, east- and northeast-facing hillslopes with a bearing of 1-86° were more prone to loss of area than other orientations. The altered pattern of wetland distribution from higher to lower elevation on north-facing slopes coincided with the doubling of annual temperature during the same period, suggesting that climate warming could be an important cause.  相似文献   

12.
Anadara broughtonii is one of the main commercially important species of shellfish in northern China.A.broughtonii lives in relatively stable subtidal zone where the clam could re spond to environmental changes with minimum energy.Therefore,slight fluctuations in water environment may have a great impact on physiological processes such as feeding and metabolism.Large-scale mortality often occurs during the intermediate rearing processes,and high temperatures in summer are considered the leading cause of mortality.To understand the physiological and molecular response patterns of A.broughtonii at high temperatures and to estimate the appropriate metabolism temperature for A.broughtonii,the effects of temperature on the respiratory metabolism and food intake at different growth stages were studied.The response patterns of heat shock protein genes to heat stress and post-stress recovery were also explored.Results show that the temperature and growth stage of A.broughtonii were two important factors affecting metabolism and feeding.The optimum temperature for feeding and physiological activities in each shelllength group was 24℃.The temperature at which the peak metabolic rate occurred in each shell-length group increased with the increase in shell length.With the increase in heat stress,the expression of three heat shock protein genes(Abhsp60,Abhsp70,and Abhsp90) in the tissues of two size groups of A.broughtonii increased significantly when temperature was above 24℃ and reached a peak at 30℃.After heat shock at30℃ for 2 h,the expression of the three heat shock protein genes rapidly increased,peaked at 2 h after the heat shock,and then gradually decreased to the level of the control group at 48 h after the heat shock.The three heat shock protein genes respond rapidly to heat stress and can be used as indicators to the cellular stre ss response in A.broughtonii under an environmental stre ss.  相似文献   

13.
EmpA is an extracellular metalloprotease secreted by Vibrio anguillarum.For better understanding its role in the patho-genicity of V.anguillarum strain M3,empA insertion mutant was constructed.In the mutant it decreased in extracellular proteolytic activity,swarming motility,hemolytic activity and virulence on turbot(Scophthalmus maximus).Significant decline(by 5-fold)of extracellular proteolytic activity and similar growth curve between mutant and wild type strains indicated that EmpA was the major extracellular protease of M3.LD50 of mutant increased by 38-fold compared with wild type.No pro-EmpA was detected in the su-pernatant of culture,indicating that EmpA autolyzed to mature protein after 24 h.Secretion of EmpA in M3 was similar to that in NB10 strain.Attenuated virulence of mutant was similar to that of M93Sm strain.It was demonstrated that specific operation of EmpA was different from that in previous studies and EmpA contributed to the swarming motility and hemolytic activity in V.an-guillarum strain M3.The results provides insight into understanding the function of EmpA and its potential application in vaccine development.  相似文献   

14.
A rapid method of microwave assisted acid hydrolysis was established to prepare κ-carra-oligosaccharides. The optimal hydrolysis condition was determined by an orthogonal test. The degree of polymerization(DP) of oligosaccharides was detected by high performance thin layer chromatography(HPTLC) and polyacrylamide gel electrophoresis(PAGE). Considering the results of HPTLC and PAGE, the optimum condition of microwave assisted acid hydrolysis was determined. The concentration of κ-carrageenan was 5 mg m L-1; the reaction solution was adjusted to p H 3 with diluted hydrochloric acid; the solution was hydrolyzed under microwave irradiation at 100 for 15℃ min. Oligosaccharides were separated by a Superdex 30 column(2.6 cm × 90 cm) using AKTA Purifier UPC100 and detected with an online refractive index detector. Each fraction was characterized by electrospray ionization mass spectrometry(ESI-MS). The data showed that odd-numbered κ-carra-oligosaccharides with DP ranging from 3 to 21 could be obtained with this method, and the structures of the oligosaccharides were consistent with those obtained by traditional mild acid hydrolysis. The new method was more convenient, efficient and environment-friendly than traditional mild acid hydrolysis. Our results provided a useful reference for the preparation of oligosaccharides from other polysaccharides.  相似文献   

15.
Preparation of Fe^2＋ chelate of fish protein hydrolysate （Fe-FPH） obtained from low value fish proteins was introduced and its bioactivity was studied by compound enzymolysis. The optimum conditions for hydrolysate chelating Fe^2＋ are DH （degree of hydrolysis） at 5%, pH 7.0, 20℃ and 15 min chelating time for FM （material not being defatted）. Four types of Fe-FPH including CA （deposit after chelating）, CB （deposit in 50% of absolute ethanol solution）, CC （suspended deposit in 80% of absolute ethanol solution）, and CD （bottom deposit in 80% of absolute ethanol solution） were fractionated with absolute ethanol from FM. Structural analysis through infra-red spectrum revealed that Fe^2＋ was combined strongly with amino-group and carboxyl-group in each chelate and each Fe^2＋ could form two five-member ring structures. All of the four chelates were shown more significant antioxidative activity and can be used as natural hydrophobic and hydrophilic antioxidant. Among all the chelates, the CB possesses the most effective antioxidative activity at 92% as high as that of a-tocopherol. Among all Fe-FPHs, only CD showed the most effective antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella typhi, and Bacillus subtilis and can be used as natural antibacterial. It provides a more effective way for utilization of low value fish proteins and key information of Fe-FPH as additive in food industry.  相似文献   

16.
A novel continuous ovary cell line from barfin flounder(Verasper moseri)(BFO cell line) was established with its primitive application in transgenic expression demonstrated in this study. Primarily cultured cells grew well at 22℃ in Dulbecco's modified Eagle medium/F12 medium(DMEM/F12, 1:1; p H 7.2) supplemented with 20% fetal bovine serum(FBS), carboxymethyl chitooligosaccharide, basic fibroblast growth factor(b FGF) and insulin-like growth factor-I(IGF-I). The primary BFO cells in fibroblastic morphology proliferated into a confluent monolayer about 2 weeks later, and were able to be subcultured. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 22℃. The BFO cells can be continuously subcultured to Passage 120 steadily with a population doubling time of 32.7 h at Passage 60. Chromosome analysis revealed that 72% of BFO cells at Passage 60 maintained the normal diploid chromosome number(46) with a normal karyotype of 2st+44t. The results of gene transformation indicated that green fluorescence protein(GFP) positively expressed in these cells after being transformed with pc DNA3.1-GFP. Therefore, a continuous and transformable BFO cell line was successfully established, which may serve as a useful tool for cytotechnological manipulation and transgenic modification of this fish.  相似文献   

17.
The densities of 36 water samples from the Huanghe River estuary and Bohai Bay were determinedby a magnetic float densimcter under three temperatures from 15℃ to 25℃.All the measured densities ofsamples were greater than that of the values calculated from the International Equation of State of Seawater.The differences between the measured and calculated densities increased with the decrease of salinities.The dif-ferences appeared exponentially correlated with[Ca~(2+)]/s,[Mg~(2+)]/s and[SO_4~(2-)]/s,and had"s"type curverelationship with the alkalinity in all salinity range.But in the salinity ranging from 25.72 to 31.57,therelationships were all linear.The density difference can be estimated from the equation △ρ（10~3kg·m~(-3)）=（-2.79+236.5([Ca~(2+)]/s)/(-9.7464×10~（-3）+[Ca~(2+)]/s).It was the high alkalinity and[Ca~(2+)]/s that resulted in the measured densi-ties of seawaters being higher than the calculated densities in the Huanghe estuary and Bohai Bay.  相似文献   

18.
We evaluated the ability of juvenile Amur sturgeon (Acipenser schrenckii) to osmoregulate and grow in saltwater. Hatchery-reared juveniles (mean weight 106.8 g, 5-month old) were transferred from freshwater to 10, 20, and 25 salinity saltwater over a period of 20 d. We measured the growth, serum osmolality, ion concentrations, and Na+/K+-ATPase activity. In addition, we prepared samples of gill tissue to quantify morphological changes in gill ultrastructure. Rearing in up to 25 saltwater for 30 d had no significant effect on growth. Similarly, serum osmolality and ion concentrations were similar to levels reported in other teleosts following acclimation to saltwater. Serum osmolality and Na+, Cl- concentrations increased significantly with the initial increase in salinity. Afterwards, levels tended to stabilize and then decrease. Serum K+ levels did not change during acclimation to saltwater. Gill Na+/K+-ATPase activity increased initially as salinity was increased. However, the activity later decreased and, finally stabilized at 3.7±0.1 μmol Pi/mg·prot·h in 25 saltwater (1.6 times higher than the level in those in freshwater). In fish that were held only in freshwater, the chloride cells were located in the interlamellar regions of the filament and at the base of the lamella. Following acclimation to 25 saltwater for 30 d, the number and size of chloride cells increased significantly. Our results suggest that juvenile Amur sturgeon is able to tolerate, and grow in, relatively high concentrations of saltwater.  相似文献   

19.
Myosin subfragment-1 was prepared from the myofibrils of bighead carp (Aristichthys nobilis). The myosin subfrag- ment-1 was proved to have the activity of tripolyphosphatase (TPPase) responding to the hydrolysis of sodium tripolyphosphate (STPP). The optimum temperature and pH for the TPPase of myosin subfragment-1 were 30℃ and pH 5.0, and at pH 8.0 the TPPase also showed a high activity. Mg2 was necessary to TPPase. The TPPase activity of myosin subfragment-1 was activated by Mg2 under low concentrations, but was inhibited when the concentration was over 17 mmolL-1. The TPPase activity was also affected by KCl. The optimum concentration of KCl for TPPase was 0.3 molL-1 under the condition of 17 mmolL-1 Mg2 . The TPPase activity was significantly inhibited by EDTA-Na2. Reagents such as KBr, KI and KIO3 could inhibit the TPPase effectively. K2Cr2O7 as well as KMnO7 and KNO3 exhibited weak inhibiting effects. The TPPase converted STPP to pyrophosphate (PP) and orthophosphate (Pi) stoichiometrically with a KM of 3.2 mmolL-1.  相似文献   

20.
An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phospholipase C activity at the optimal values of temperature 50℃ and pH 8.0.The enzymatic activity decreased when the temperature increased to 40℃.The N-terminal sequence of the purified protein was determined as ADVPAGTKLA,which is similar to that of OppA.The OppA pre-cursor gene was cloned from th...  相似文献   

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