In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amin...In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6(PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity(258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃ for 30 min. Al163 activity was stimulated by Cd^(2+), Ca^(2+), Fe^(3+), and Mn^(2+), but inhibited by Cu^(2+), Si^(2+), Fe^(2+), and Ni^(2+). Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, poly M, and poly G, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides.展开更多
Twenty-seven antarctic bacteria producing extracellular polysaccharide （EPS） were selected from 57 strains by staining technology. The effects of major environmental factors on the growth and EPS production of Pseud...Twenty-seven antarctic bacteria producing extracellular polysaccharide （EPS） were selected from 57 strains by staining technology. The effects of major environmental factors on the growth and EPS production of Pseudoalteromonas sp. S - 15 - 13 were investigated, and the EPS was separated and purified for characterization analysis. The results showed that the optimal conditions for the EPS production were culture period, 56 h; growth temperature, 8 ℃ ; carbon source, 1.0% glucose; NaCI concentration, 3.0% ; pH 6.0 - 7.0. The EPS was purified by cold ethanol precipitation, proteins removal, ion exchange chromatography and gel chromatography technology. The molecular mass of EPS - H was 62 kDa as determined by the high performance gel permeation chromatography. Its sugar composition was a homopolymer of marmose analyzed by gas chromatograph spectroscopy. After repeated freezing and thawing of the bacteria hiomass in the presence of EPS, the bacterial growth was much higher than that observed after freezing in the absence of EPS and the difference augmented with the increase of freeze-thaw cycles. It is hypothesized that the adaptation of Pseudoalteromonas sp. S- 15 - 13 to the antarctic marine conditions, characterized by low temperature, high NaCl concentration and repeated freeze-thaw cycles, might be related to the EPS production ability.展开更多
Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession ...Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.展开更多
A new extracelluar polysaccharide （EPS） was isolated and purified from Antarctic bacterium S-15-13, identified as Pseudoalteromonas sp. After being separated and purified by DEAE-Sephadex A-50 ionexchange and Sephad...A new extracelluar polysaccharide （EPS） was isolated and purified from Antarctic bacterium S-15-13, identified as Pseudoalteromonas sp. After being separated and purified by DEAE-Sephadex A-50 ionexchange and Sephadex G-100 gel chromatography, two mains fractions （EPS I and EPS Ⅱ ） were ob-tained. EPS I was composed of mannose, glucose and galactose with a molecular weight of 23kDa and EPS Ⅱ was composed of mannose only with a molecular weight of 62kDa. The effect of the polysaccharide EPS I on the cellular immune response of mice was investigated. Results demonstrated that EPS I could markedly facilitate lymphocyte proliferation, and might be a strong immunomodulator.展开更多
A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus ...A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus japonicus. Sea cucumber individuals were fed with the diets containing 0(control), 105, 107 and 109 CFU g-1 diet of BC228 for 45 days. Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107 and 109 CFU g-1 diet of BC228 in comparison with control(P < 0.01). The phagocytic activity in the coelomocytes of sea cucumber fed the diet supplemented with 107 CFU g-1 diet of BC228 was significantly higher than that of those fed control diet(P < 0.05). In addition, 105 and 107 CFU g-1 diet of BC228 significantly enhanced lysozyme and phenoloxidase activities in the coelomic fluid of sea cucumber, respectively, in comparison with other diets(P < 0.01). Sea cucumbers, 10 each diet, were challenged with Vibrio splendidus NB13 after 45 days of feeding. It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet. Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber, stimulated its immune response and enhanced its resistance to the infection of V. splendidus.展开更多
Agar is an essential polysaccharide that has been utilized in numerous fields.Many kinds of literature have been published regarding agarolytic microorganisms’isolation and agarases biochemical studies.In this search...Agar is an essential polysaccharide that has been utilized in numerous fields.Many kinds of literature have been published regarding agarolytic microorganisms’isolation and agarases biochemical studies.In this search,a local marine agarolytic bacterium associated with marine alga Ulva lactuca surface was isolated and identified as Pseudoalteromonas sp.MHS.The agarase production was parallel to the growth of Pseudoalteromonas sp.MHS as cells displayed a lag phase(2 h),subsequently an exponential growth that prolonged till 10 h where maximum growth(OD550nm=3.9)was achieved.The enzyme activity increased rapidly as cells increased exponentially where the maximum activity of 0.22 U/mL was achieved after 8h and remained constant till 12 h during the stationary phase of growth.Agarase production was optimized using Plackett-Burman statistical design by measuring enzyme activity as a response and the design was validated using a verification experiment;the activity of the enzyme increased from 0.22 U/mL to 0.29 U/mL.Pseudoalteromonas sp.MHS agarase was partially purified and its molecular weight(MW)was determined by SDS-PAGE(15-25 kDa).Agarase showed approximately 94% of its activity at 40℃.The enzyme stability decreased as the temperature increased;the enzyme could retain about 98,90,80,75,and 60% of its activity at 20,30,40,50,and 60℃,respectively.Biomass of the red alga Pterocladia capillacea proved to be a suitable substrate for agarase production using Pseudoalteromonas sp.MHS;the enzyme activity recorded after 24 h of incubation was 0.35 U/mL compared to 0.29 U/mL from the optimized medium.展开更多
kappa-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by kappa-carrageenase is safe and controllable. Therefore, kappa-carrageenases have captured more and more attentions. In...kappa-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by kappa-carrageenase is safe and controllable. Therefore, kappa-carrageenases have captured more and more attentions. In this study, a kappa-carrageenase encoding gene, cgkX, 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. CgkX is a new member of glycoside hydrolase family 16. The deduced amino acid sequence shared a high similarity with CgkX of Pseudoalteromonas kappa-carrageenase; however, the recombinant CgkX showed different biochemical characteristics. The recombinant enzyme was most active at pH 7.0 and 55A degrees C in the presence of 300 mmol L-1 NaCl. It was stable in a broad range of acidity ranging from pH 3.0 to pH 10.0 when temperature was below 40A degrees C. More than 80% of its activity was maintained after being incubated at pH 3.6-10.0 and 4A degrees C for 24 h. CgkX retained more than 90% of activity after being incubated at 40A degrees C for 1 h. EDTA and SDS (1 mmol L-1) did not inhibit its activity. CgkX hydrolyzed kappa-carrageenan into disaccharide and tetrasaccharide as an endo-cleaver. All these characteristics demonstrated that CgkX is applicable to both kappa-carrageenan oligosaccharide production and kappa-carrageenase structure-function research.展开更多
A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperat...A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃ to 50℃ with the highest activity at 45℃ and pH7.2. Sodium chloride increased its activity markedly, and KC1 increased its activity slightly. The divalent and trivalent metal ions including Cu^2＋, Ni^2＋, Zn^2＋, Mn^2＋, Al^3＋ and Fe^3＋ significantly inhibited its activity, while Mg^2＋ did not. CgkP remained 70% of original activity after being incubated at 40℃ for 48h, and remained 80% of the activity after being incubated at 45℃ for 1 h. It exhibited endo-κ-carrageenase activity, mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide. CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.展开更多
An agar-degrading bacterium, designated as Pseudoalteromonas sp. NJ21, was isolated from an Antarctic sediment sample. The agarase gene aga1161 from Pseudoalteromonas sp. NJ21 consisting of a 2 382-bp coding region wa...An agar-degrading bacterium, designated as Pseudoalteromonas sp. NJ21, was isolated from an Antarctic sediment sample. The agarase gene aga1161 from Pseudoalteromonas sp. NJ21 consisting of a 2 382-bp coding region was cloned. The gene encodes a 793-amino acids protein and was found to possess characteristic features of the Glyco_hydro 42 family. The recombinant agarase （rAgal 161） was overexpressed in Escherichia coli and purified as a fusion protein. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were 30--40℃ and 8.0, respectively. rAga 1161 was found to maintain as much as 80% of its maximum activity at 10℃, which is typical of a cold- adapted enzyme. The pattern of agar hydrolysis demonstrated that the enzyme is an β-agarase, producing neoagarobiose （NA2） as the final main product. Furthermore, this work is the first proof of an agarolytic activity in Antarctic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.展开更多
The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genom...The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains： a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/（Arg＋Lys） ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 （DE3） by pColdlII was also successfully observed in this study.展开更多
基金Supported by the Public Science and Technology Research Funds Project of Ocean(No.201505026-4)the Chinese Polar Environment Comprehensive Investigation&Assessment Programs(No.CHINARE2016-01-05)+1 种基金the Basic Scientific Research Funds of First Institute of Oceanography,State Oceanic Administration(SOA)(No.2014T09)the Qingdao Applied Basic Research Project(No.14-2-4-14-jch)
文摘In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6(PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity(258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃ for 30 min. Al163 activity was stimulated by Cd^(2+), Ca^(2+), Fe^(3+), and Mn^(2+), but inhibited by Cu^(2+), Si^(2+), Fe^(2+), and Ni^(2+). Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, poly M, and poly G, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides.
文摘Twenty-seven antarctic bacteria producing extracellular polysaccharide （EPS） were selected from 57 strains by staining technology. The effects of major environmental factors on the growth and EPS production of Pseudoalteromonas sp. S - 15 - 13 were investigated, and the EPS was separated and purified for characterization analysis. The results showed that the optimal conditions for the EPS production were culture period, 56 h; growth temperature, 8 ℃ ; carbon source, 1.0% glucose; NaCI concentration, 3.0% ; pH 6.0 - 7.0. The EPS was purified by cold ethanol precipitation, proteins removal, ion exchange chromatography and gel chromatography technology. The molecular mass of EPS - H was 62 kDa as determined by the high performance gel permeation chromatography. Its sugar composition was a homopolymer of marmose analyzed by gas chromatograph spectroscopy. After repeated freezing and thawing of the bacteria hiomass in the presence of EPS, the bacterial growth was much higher than that observed after freezing in the absence of EPS and the difference augmented with the increase of freeze-thaw cycles. It is hypothesized that the adaptation of Pseudoalteromonas sp. S- 15 - 13 to the antarctic marine conditions, characterized by low temperature, high NaCl concentration and repeated freeze-thaw cycles, might be related to the EPS production ability.
基金The work was supported by the Hi-Tech Research and Development Program of China under contract Nos 2006AA09Z414 and 2007AA091903；the China Ocean Mineral Resources R ＆ D Association under contract No. DYXM - 115 - 02 - 2 - 6；the National Natural Science Foundation of China under contract No. Z2004D02；the Natural Science Foundation of Shandong Province of China under contract No. Z2004D02；the Foundation for Young Excellent Scientists in Shandong Province of China under contract No. 2006BS02002；the Program for New Century Excellent Talents in University under contract No. NCET - 06 - 0578.
文摘Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.
文摘A new extracelluar polysaccharide （EPS） was isolated and purified from Antarctic bacterium S-15-13, identified as Pseudoalteromonas sp. After being separated and purified by DEAE-Sephadex A-50 ionexchange and Sephadex G-100 gel chromatography, two mains fractions （EPS I and EPS Ⅱ ） were ob-tained. EPS I was composed of mannose, glucose and galactose with a molecular weight of 23kDa and EPS Ⅱ was composed of mannose only with a molecular weight of 62kDa. The effect of the polysaccharide EPS I on the cellular immune response of mice was investigated. Results demonstrated that EPS I could markedly facilitate lymphocyte proliferation, and might be a strong immunomodulator.
基金financially supported by the National High Technology Research and Development Program of China (863 Project) of China (2012AA10A412)
文摘A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus japonicus. Sea cucumber individuals were fed with the diets containing 0(control), 105, 107 and 109 CFU g-1 diet of BC228 for 45 days. Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107 and 109 CFU g-1 diet of BC228 in comparison with control(P < 0.01). The phagocytic activity in the coelomocytes of sea cucumber fed the diet supplemented with 107 CFU g-1 diet of BC228 was significantly higher than that of those fed control diet(P < 0.05). In addition, 105 and 107 CFU g-1 diet of BC228 significantly enhanced lysozyme and phenoloxidase activities in the coelomic fluid of sea cucumber, respectively, in comparison with other diets(P < 0.01). Sea cucumbers, 10 each diet, were challenged with Vibrio splendidus NB13 after 45 days of feeding. It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet. Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber, stimulated its immune response and enhanced its resistance to the infection of V. splendidus.
文摘Agar is an essential polysaccharide that has been utilized in numerous fields.Many kinds of literature have been published regarding agarolytic microorganisms’isolation and agarases biochemical studies.In this search,a local marine agarolytic bacterium associated with marine alga Ulva lactuca surface was isolated and identified as Pseudoalteromonas sp.MHS.The agarase production was parallel to the growth of Pseudoalteromonas sp.MHS as cells displayed a lag phase(2 h),subsequently an exponential growth that prolonged till 10 h where maximum growth(OD550nm=3.9)was achieved.The enzyme activity increased rapidly as cells increased exponentially where the maximum activity of 0.22 U/mL was achieved after 8h and remained constant till 12 h during the stationary phase of growth.Agarase production was optimized using Plackett-Burman statistical design by measuring enzyme activity as a response and the design was validated using a verification experiment;the activity of the enzyme increased from 0.22 U/mL to 0.29 U/mL.Pseudoalteromonas sp.MHS agarase was partially purified and its molecular weight(MW)was determined by SDS-PAGE(15-25 kDa).Agarase showed approximately 94% of its activity at 40℃.The enzyme stability decreased as the temperature increased;the enzyme could retain about 98,90,80,75,and 60% of its activity at 20,30,40,50,and 60℃,respectively.Biomass of the red alga Pterocladia capillacea proved to be a suitable substrate for agarase production using Pseudoalteromonas sp.MHS;the enzyme activity recorded after 24 h of incubation was 0.35 U/mL compared to 0.29 U/mL from the optimized medium.
基金supported by the National High-Tech R&D Program(No.2011AA090703)the National Natural Science Foundation of China(No.31070712)the Special Fund for Marine Scientific Research in the Public Interest(Nos.201105027 and 201005024)
文摘kappa-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by kappa-carrageenase is safe and controllable. Therefore, kappa-carrageenases have captured more and more attentions. In this study, a kappa-carrageenase encoding gene, cgkX, 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. CgkX is a new member of glycoside hydrolase family 16. The deduced amino acid sequence shared a high similarity with CgkX of Pseudoalteromonas kappa-carrageenase; however, the recombinant CgkX showed different biochemical characteristics. The recombinant enzyme was most active at pH 7.0 and 55A degrees C in the presence of 300 mmol L-1 NaCl. It was stable in a broad range of acidity ranging from pH 3.0 to pH 10.0 when temperature was below 40A degrees C. More than 80% of its activity was maintained after being incubated at pH 3.6-10.0 and 4A degrees C for 24 h. CgkX retained more than 90% of activity after being incubated at 40A degrees C for 1 h. EDTA and SDS (1 mmol L-1) did not inhibit its activity. CgkX hydrolyzed kappa-carrageenan into disaccharide and tetrasaccharide as an endo-cleaver. All these characteristics demonstrated that CgkX is applicable to both kappa-carrageenan oligosaccharide production and kappa-carrageenase structure-function research.
基金supported by National Science Foundation of China (31000361 and 31070712)Program for Changjiang Scholars and Innovative Research Team in University (IRT0944)+1 种基金Special Fund for Marine Scientific Research in the Public Interest (201005024)the Fundamental Research Funds for the Central Universities(201013008)
文摘A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃ to 50℃ with the highest activity at 45℃ and pH7.2. Sodium chloride increased its activity markedly, and KC1 increased its activity slightly. The divalent and trivalent metal ions including Cu^2＋, Ni^2＋, Zn^2＋, Mn^2＋, Al^3＋ and Fe^3＋ significantly inhibited its activity, while Mg^2＋ did not. CgkP remained 70% of original activity after being incubated at 40℃ for 48h, and remained 80% of the activity after being incubated at 45℃ for 1 h. It exhibited endo-κ-carrageenase activity, mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide. CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.
基金Supported by the Public Science and Technology Research Funds Project of Ocean(No.201105027)the Shandong Province Young and the Middle-Aged Scientists Research Awards Fund(No.DS2010HZ001)the Basic Scientific Research Funds of First Institute of Oceanography,State Oceanic Administration(No.GY02-2011G17)
文摘An agar-degrading bacterium, designated as Pseudoalteromonas sp. NJ21, was isolated from an Antarctic sediment sample. The agarase gene aga1161 from Pseudoalteromonas sp. NJ21 consisting of a 2 382-bp coding region was cloned. The gene encodes a 793-amino acids protein and was found to possess characteristic features of the Glyco_hydro 42 family. The recombinant agarase （rAgal 161） was overexpressed in Escherichia coli and purified as a fusion protein. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were 30--40℃ and 8.0, respectively. rAga 1161 was found to maintain as much as 80% of its maximum activity at 10℃, which is typical of a cold- adapted enzyme. The pattern of agar hydrolysis demonstrated that the enzyme is an β-agarase, producing neoagarobiose （NA2） as the final main product. Furthermore, this work is the first proof of an agarolytic activity in Antarctic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.
基金supported by the National High Technology Research and Development Program of China (Grant no.2007AA091905)the Basic Scientific Research Operation Fund of China (Grant no. GY02-2007-T11)
文摘The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains： a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/（Arg＋Lys） ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 （DE3） by pColdlII was also successfully observed in this study.
