海洋微生物多糖水解酶的基因克隆和高效表达
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摘要
地球上的生命在海洋中诞生，至今已有数亿年的历史。海洋面积广阔，并拥有一系列复杂的生境和最多样的生命形式。海洋环境中，微生物之间空间和营养的竞争的选择压力导致了微生物的进化，这种进化促使微生物产生多样性的酶系统来适应竞争性的海洋环境。因此海洋微生物酶凭借其独特的特性，可以广泛应用于生物催化反应中。其中有关海洋微生物碳源利用的多糖水解酶多样性十分丰富，而且在发酵生产和生物质能源利用中有十分重要的应用价值。本论文就着重研究海洋微生物多糖水解酶中的褐藻胶裂解酶、κ-卡拉胶酶和菊糖酶，并对其进行特性分析、基因克隆和高效表达。
褐藻胶裂解酶基因ALY扩增自海洋病原菌Vibrio sp. QY101，连接到酵母表面展示质粒pINA1317-YlCWP110，在Yarrowia lipolytica Po1h中进行表面展示表达。展示有褐藻胶裂解酶的转化子菌落够在褐藻胶平板上形成透明圈，酶活力最高的转化子His7褐藻胶裂解酶展示酶活力为208.0±5.3U/g细胞干重。展示有褐藻胶裂解酶的酵母菌体可以水解β-D-甘露聚糖（M）、α-L-古罗聚糖（G）和褐藻胶，产生不同聚合度的具生物学活性的褐藻胶寡糖。
从中国黄海腐烂海藻表面分离到一株产κ-卡拉胶酶的海洋细菌LL1鉴定为Pseudoalteromonas porphyrae。其胞外分泌的κ-卡拉胶酶经超滤浓缩、阳离子交换层析和分子筛纯化，得到经SDS蛋白电泳检测为40.0kDa的单一条带。纯化κ-卡拉胶酶的最适温度55℃，最适pH8.0，最适底物κ-卡拉胶，Km值为4.4mg/mL，Vmax值为0.1mg/(min·mL)。纯化κ-卡拉胶酶水解κ-卡拉胶的最终产物经电喷雾离子化质谱（ESI-MS）分析为硫酸新κ-卡拉二糖和硫酸新κ-卡拉四糖。
采用反向PCR和基因组步移的方法克隆了P. porphyrae LL1的κ-卡拉胶酶基因和其转录调节因子基因。κ-卡拉胶酶基因（登录号：GU386342）ORF框1224bp，推导407个氨基酸，蛋白预测分子量42.5kDa。保守区具有糖水解酶家族16的特征序列E(162)xD(164)x(x)E(167)。κ-卡拉胶酶转录调节因子ORF框318bp，推导蛋白105个氨基酸，预测分子量12.1kDa，属于AraC家族，具有两个转角-螺旋-转角结构的DNA结合域。随后构建κ-卡拉胶酶基因双拷贝片段转化Y.lipolytica Po1h，所表达的酶嵌壁，酶活力4.9±1.2U/mL，比活力为210.3±3.2U/g细胞干重。重组κ-卡拉胶酶最适作用温度和pH较原始酶没有变化，温度稳定性提高。转化子细胞可以将κ-卡拉胶水解成硫酸新κ-卡拉二糖和少量硫酸新κ-卡拉四糖。在κ-卡拉胶2.0mg/mL，重组κ-卡拉胶酶量7.5U/mg κ-卡拉胶，反应温度55℃，反应时间40min的最优水解条件下，κ-卡拉胶的水解率为71.5±0.2%。因此重组κ-卡拉胶酶在制备具生物活性κ-卡拉胶寡糖方面有潜在应用价值。
菊糖和含菊糖作物是用于发酵生产生物酒精及其他有价值代谢产物的重要碳源物质。为了构建在Pichia guilliermondii酵母稳定遗传的重组菊粉酶表达质粒，本研究通过重叠延伸PCR和酶切连接构建了新的rDNA插入型表达菊糖外切酶基因质粒pMD-rDNA-HPT-INU，该质粒转化对潮霉素B敏感的天然P.guilliermondii菌株。经潮霉素平板和PCR验证证明菊糖酶基因重组到染色体中，酶活力最高的转化子R3经过72h培养，菊糖酶活力达到53.2±2.1U/mL，较天然菌株的菊糖酶活力32.5±0.7U/mL提高65.6%。将转化子R3进行1.5L发酵罐分批补料发酵，酶活力到达84.1±0.9U/mL。
为了进一步提高菊糖酶活力，以P. guilliermondii经定点突变改造的菊糖酶基因INUM为基础，用易错PCR的方法进行体外分子进化。通过转入Ycplac33进行转化子筛选测序，得到高酶活力菊糖酶基因INUDV82，将其与原基因INUM进行比对分析发现共有5个碱基、4个氨基酸发生变化，并进一步分析酶三级结构和催化位点的变化，发现其中突变氨基酸S97改善了催化中心内的电荷环境，间接增强了酶与底物之间的相互作用。将INUDV82基因连到rDNA重组表达质粒pMIRSC11，转入耐酒精的尿嘧啶缺陷型酵母S. cerevisiae W12d，获得的酶活力最高的转化子INUDV5，菊糖酶活力为25.0±1.8U/mL，较原INUM基因转化子菊糖酶活力16.75±1.5U/mL提高49%。
The ocean has been regarded as the origin of life on Earth. The ocean includesthe largest range of habitats and the most life-forms. Competition amongstmicroorganisms for space and nutrients in the marine environment is a powerfulselective pressure which has led to evolution. The evolution prompts the marinemicroorganisms to generate multifarious enzyme systems to adapt to the complicatedmarine environments. Therefore, marine microbial enzymes can offer novelbiocatalysts with extraordinary properties. Marine microbialpolysaccharide-degrading enzymes are also multifarious, and have importantapplications in fermentation and exploitation of biomass energy. In this study, marinemicrobial polysaccharide-degrading enzymes such as alginate lyases, κ-carrageenasesand inulinase were characterized, cloned and overexpressed.
The alginate lyase gene was amplified from the marine bacterium Vibriosp.QY101which was a pathogen of Laminaria sp. The alginate lyase gene was clonedinto the multiple cloning site of the surface display vector pINA1317-YlCWP110andexpressed in cells of Yarrowia lipolytica Po1h. The cells displaying the alginate lyasecould form clear zone on the plate containing sodium alginate, and the alginate lyaseactivity of transformant His7was208.0±5.3U/g. The cells displaying alginate lyasecould be used to hydrolyze poly-β-D-mannuronate and poly-α-L-guluronate andsodium alginate to produce different lengths of oligosaccharides. This was the firstreport that the yeast cells displaying alginate lyasewere used to produce differentlengths of oligosaccharidesfrom alginate.
A bacterial strain LL1producing κ-carrageenase was isolated from the decayedseaweed collected from Yellow Sea, China and identified as Pseudoalteromonasporphyrae. The extracellular κ-carrageenase in the supernatant of cell culture of the marine bacterium P. porphyrae LL1was purified to homogeneity with a202.6-foldincrease in specific κ-carrageenase activity as compared to that in the supernatantbyultrafiltration, anion-exchange chromatography, and gel filtration chromatography.According to the data from sodium dodecyl sulfatepolyacrylamide gel electrophoresis,the molecular mass of the purified enzyme was estimated to be40.0kDa. The optimalpH and temperature of the purified enzyme were8.0and55℃. The purified enzymecould hydrolyze κ-carrageenan into κ-neocarrabiose (DP2) and κ-neocarratetraose(DP4) sulfate，which were characterized by ESI-MS.
After that, the gene encoding κ-carrageenase and its transcriptional regulator in P.porphyae were cloned by reverse PCR and genome walking, and then characterized.The κ-carrageenase gene (CGK gene, accession number: GU386342) had an openreading frame of1224bp encoding a407amino acids protein with calculatedmolecular weight of42.5kDa. The deduced protein belonged to the family GH16.The catalytic residues of the deduced protein were constituted by the characteristicsequence pattern E(162)xD(164)x(x)E(167). Another gene encoding κ-carrageenasetranscriptional regulator had an open reading frame of318bp encoding a105aminoacids protein with calculated molecular weight of12.1kDa. The deduced proteinbelonged to the family AraC with two helix-turn-helix DNA binding domain. TheCGK gene was over-expressed in Y.lipolytica Po1h by constructing double CGK genecopies vector, and all the recombinant CGK was retained by the recombinant Y.lipolytica Po1h. The results of western blotting show that the molecular weight of therCGK was60.0kDa. The optimal pH and temperature of the rCGK were8.0and54°C, respectively. The rCGK had high κ-carrageenase activity and κ-carrageenan washydrolyzed into κ-neocarrabiose and κ-neocarratetraose sulfate. The optimalconditions for hydrolysis of κ-carrageenan were that the amount of rCGK was7.5U/mg of κ-carrageenan, κ-carrageenan concentration was2.0mg/mL, reaction timewas40min and temperature was55℃. Under such conditions,71.5±0.2%of addedκ-carrageenan was hydrolyzed. Therefore, the recombinant κ-carrageenase may have highly potential applications in biotechnology.
Inulin and inulin-containing materials are good substrates for biofuel production.In order to construct genetically stable recombinant Pichia guilliermondii yeast cellscarrying the additional inulinase gene for inulinase production, a new rDNAintegration vector pMD-rDNA-HPT-INU was constructed in this study. After the INUgene encoding exo-inulinase was ligated into the rDNA integration vector, the vectorwas transformed into P. guilliermondii which was susceptible to hygromycin B andintegrated into its chromosomes. The transformant R3obtained could produce53.2±2.1U/mL of inulinase activity within72h，while the inulinase activity of native strainwas32.5±0.7U/mL. In the1.5L Fed-batch fermentation，the inulinase activity ofthe cultures was84.1±0.9U/mL.
In order to improve the inulinase activity, error prone PCR was used to generaterandom point mutations of the native inulinase gene with the template INUM whichwas the directed mutagenesis inulinase gene from P. guilliermondii. Then the errorprone PCR products were ligased to expression vector Ycplac33and expreesed in S.cerevisiae W12d in order to screen and obtain high inulinase activity transformantswith mutated gene. The transformant carrying mutated gene INUDV82was indicatedas the highest inulinase activity, and the mutated gene INUDV82was found that fivebases and four amino acids were changed. Finally, expression vectorpMIRSC11-INUDV82was constructed and transformed into S. cerevisiae W12d,which was integrated into its chromosomes. The transformant INUDV5obtainedcould produce25.0±1.8U/mL of inulinase activity within72h, which increased49%than the transformant carrying unmutated gene INUM.
褐藻胶裂解酶基因ALY扩增自海洋病原菌Vibrio sp. QY101，连接到酵母表面展示质粒pINA1317-YlCWP110，在Yarrowia lipolytica Po1h中进行表面展示表达。展示有褐藻胶裂解酶的转化子菌落够在褐藻胶平板上形成透明圈，酶活力最高的转化子His7褐藻胶裂解酶展示酶活力为208.0±5.3U/g细胞干重。展示有褐藻胶裂解酶的酵母菌体可以水解β-D-甘露聚糖（M）、α-L-古罗聚糖（G）和褐藻胶，产生不同聚合度的具生物学活性的褐藻胶寡糖。
从中国黄海腐烂海藻表面分离到一株产κ-卡拉胶酶的海洋细菌LL1鉴定为Pseudoalteromonas porphyrae。其胞外分泌的κ-卡拉胶酶经超滤浓缩、阳离子交换层析和分子筛纯化，得到经SDS蛋白电泳检测为40.0kDa的单一条带。纯化κ-卡拉胶酶的最适温度55℃，最适pH8.0，最适底物κ-卡拉胶，Km值为4.4mg/mL，Vmax值为0.1mg/(min·mL)。纯化κ-卡拉胶酶水解κ-卡拉胶的最终产物经电喷雾离子化质谱（ESI-MS）分析为硫酸新κ-卡拉二糖和硫酸新κ-卡拉四糖。
采用反向PCR和基因组步移的方法克隆了P. porphyrae LL1的κ-卡拉胶酶基因和其转录调节因子基因。κ-卡拉胶酶基因（登录号：GU386342）ORF框1224bp，推导407个氨基酸，蛋白预测分子量42.5kDa。保守区具有糖水解酶家族16的特征序列E(162)xD(164)x(x)E(167)。κ-卡拉胶酶转录调节因子ORF框318bp，推导蛋白105个氨基酸，预测分子量12.1kDa，属于AraC家族，具有两个转角-螺旋-转角结构的DNA结合域。随后构建κ-卡拉胶酶基因双拷贝片段转化Y.lipolytica Po1h，所表达的酶嵌壁，酶活力4.9±1.2U/mL，比活力为210.3±3.2U/g细胞干重。重组κ-卡拉胶酶最适作用温度和pH较原始酶没有变化，温度稳定性提高。转化子细胞可以将κ-卡拉胶水解成硫酸新κ-卡拉二糖和少量硫酸新κ-卡拉四糖。在κ-卡拉胶2.0mg/mL，重组κ-卡拉胶酶量7.5U/mg κ-卡拉胶，反应温度55℃，反应时间40min的最优水解条件下，κ-卡拉胶的水解率为71.5±0.2%。因此重组κ-卡拉胶酶在制备具生物活性κ-卡拉胶寡糖方面有潜在应用价值。
菊糖和含菊糖作物是用于发酵生产生物酒精及其他有价值代谢产物的重要碳源物质。为了构建在Pichia guilliermondii酵母稳定遗传的重组菊粉酶表达质粒，本研究通过重叠延伸PCR和酶切连接构建了新的rDNA插入型表达菊糖外切酶基因质粒pMD-rDNA-HPT-INU，该质粒转化对潮霉素B敏感的天然P.guilliermondii菌株。经潮霉素平板和PCR验证证明菊糖酶基因重组到染色体中，酶活力最高的转化子R3经过72h培养，菊糖酶活力达到53.2±2.1U/mL，较天然菌株的菊糖酶活力32.5±0.7U/mL提高65.6%。将转化子R3进行1.5L发酵罐分批补料发酵，酶活力到达84.1±0.9U/mL。
为了进一步提高菊糖酶活力，以P. guilliermondii经定点突变改造的菊糖酶基因INUM为基础，用易错PCR的方法进行体外分子进化。通过转入Ycplac33进行转化子筛选测序，得到高酶活力菊糖酶基因INUDV82，将其与原基因INUM进行比对分析发现共有5个碱基、4个氨基酸发生变化，并进一步分析酶三级结构和催化位点的变化，发现其中突变氨基酸S97改善了催化中心内的电荷环境，间接增强了酶与底物之间的相互作用。将INUDV82基因连到rDNA重组表达质粒pMIRSC11，转入耐酒精的尿嘧啶缺陷型酵母S. cerevisiae W12d，获得的酶活力最高的转化子INUDV5，菊糖酶活力为25.0±1.8U/mL，较原INUM基因转化子菊糖酶活力16.75±1.5U/mL提高49%。
The ocean has been regarded as the origin of life on Earth. The ocean includesthe largest range of habitats and the most life-forms. Competition amongstmicroorganisms for space and nutrients in the marine environment is a powerfulselective pressure which has led to evolution. The evolution prompts the marinemicroorganisms to generate multifarious enzyme systems to adapt to the complicatedmarine environments. Therefore, marine microbial enzymes can offer novelbiocatalysts with extraordinary properties. Marine microbialpolysaccharide-degrading enzymes are also multifarious, and have importantapplications in fermentation and exploitation of biomass energy. In this study, marinemicrobial polysaccharide-degrading enzymes such as alginate lyases, κ-carrageenasesand inulinase were characterized, cloned and overexpressed.
The alginate lyase gene was amplified from the marine bacterium Vibriosp.QY101which was a pathogen of Laminaria sp. The alginate lyase gene was clonedinto the multiple cloning site of the surface display vector pINA1317-YlCWP110andexpressed in cells of Yarrowia lipolytica Po1h. The cells displaying the alginate lyasecould form clear zone on the plate containing sodium alginate, and the alginate lyaseactivity of transformant His7was208.0±5.3U/g. The cells displaying alginate lyasecould be used to hydrolyze poly-β-D-mannuronate and poly-α-L-guluronate andsodium alginate to produce different lengths of oligosaccharides. This was the firstreport that the yeast cells displaying alginate lyasewere used to produce differentlengths of oligosaccharidesfrom alginate.
A bacterial strain LL1producing κ-carrageenase was isolated from the decayedseaweed collected from Yellow Sea, China and identified as Pseudoalteromonasporphyrae. The extracellular κ-carrageenase in the supernatant of cell culture of the marine bacterium P. porphyrae LL1was purified to homogeneity with a202.6-foldincrease in specific κ-carrageenase activity as compared to that in the supernatantbyultrafiltration, anion-exchange chromatography, and gel filtration chromatography.According to the data from sodium dodecyl sulfatepolyacrylamide gel electrophoresis,the molecular mass of the purified enzyme was estimated to be40.0kDa. The optimalpH and temperature of the purified enzyme were8.0and55℃. The purified enzymecould hydrolyze κ-carrageenan into κ-neocarrabiose (DP2) and κ-neocarratetraose(DP4) sulfate，which were characterized by ESI-MS.
After that, the gene encoding κ-carrageenase and its transcriptional regulator in P.porphyae were cloned by reverse PCR and genome walking, and then characterized.The κ-carrageenase gene (CGK gene, accession number: GU386342) had an openreading frame of1224bp encoding a407amino acids protein with calculatedmolecular weight of42.5kDa. The deduced protein belonged to the family GH16.The catalytic residues of the deduced protein were constituted by the characteristicsequence pattern E(162)xD(164)x(x)E(167). Another gene encoding κ-carrageenasetranscriptional regulator had an open reading frame of318bp encoding a105aminoacids protein with calculated molecular weight of12.1kDa. The deduced proteinbelonged to the family AraC with two helix-turn-helix DNA binding domain. TheCGK gene was over-expressed in Y.lipolytica Po1h by constructing double CGK genecopies vector, and all the recombinant CGK was retained by the recombinant Y.lipolytica Po1h. The results of western blotting show that the molecular weight of therCGK was60.0kDa. The optimal pH and temperature of the rCGK were8.0and54°C, respectively. The rCGK had high κ-carrageenase activity and κ-carrageenan washydrolyzed into κ-neocarrabiose and κ-neocarratetraose sulfate. The optimalconditions for hydrolysis of κ-carrageenan were that the amount of rCGK was7.5U/mg of κ-carrageenan, κ-carrageenan concentration was2.0mg/mL, reaction timewas40min and temperature was55℃. Under such conditions,71.5±0.2%of addedκ-carrageenan was hydrolyzed. Therefore, the recombinant κ-carrageenase may have highly potential applications in biotechnology.
Inulin and inulin-containing materials are good substrates for biofuel production.In order to construct genetically stable recombinant Pichia guilliermondii yeast cellscarrying the additional inulinase gene for inulinase production, a new rDNAintegration vector pMD-rDNA-HPT-INU was constructed in this study. After the INUgene encoding exo-inulinase was ligated into the rDNA integration vector, the vectorwas transformed into P. guilliermondii which was susceptible to hygromycin B andintegrated into its chromosomes. The transformant R3obtained could produce53.2±2.1U/mL of inulinase activity within72h，while the inulinase activity of native strainwas32.5±0.7U/mL. In the1.5L Fed-batch fermentation，the inulinase activity ofthe cultures was84.1±0.9U/mL.
In order to improve the inulinase activity, error prone PCR was used to generaterandom point mutations of the native inulinase gene with the template INUM whichwas the directed mutagenesis inulinase gene from P. guilliermondii. Then the errorprone PCR products were ligased to expression vector Ycplac33and expreesed in S.cerevisiae W12d in order to screen and obtain high inulinase activity transformantswith mutated gene. The transformant carrying mutated gene INUDV82was indicatedas the highest inulinase activity, and the mutated gene INUDV82was found that fivebases and four amino acids were changed. Finally, expression vectorpMIRSC11-INUDV82was constructed and transformed into S. cerevisiae W12d,which was integrated into its chromosomes. The transformant INUDV5obtainedcould produce25.0±1.8U/mL of inulinase activity within72h, which increased49%than the transformant carrying unmutated gene INUM.
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Bergkamp RJM., Bootsman TC., Toschka HY., Mooren ATA., Kox L., Verbakel JMA., Geerse RH.,Planta RJ. Expression of an-galactosidase gene under control of homologous inulinase promoterin Kluyveromyces marxianus. Appl Microbiol Biotechnol1993,40:309–417.
Boyd A, Chakrabarty AM. Role of alginate lyase in cell detachment of Pseudomonas aeruginosa.Appl Environ Microbiol,1994,60(7):2355-2359.
Boyen C, Bertheau Y, Barbeyron T, KloaregB. Preparation of guluronate lyase from Pseudomonasalginovora for protoplast isolation in Laminaria. Enzyme Microb. Technol.,1990,12:885–90
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Brown BJ, Preston JF III, et al. Lguluronan-specific alginate lyase from a marine bacteriumassociated with Sargassum. Carbohydr. Res.,1991,211:91–102
Cadwell R C,Joyce G F,. Randomization of genes by PCR mutagenesis. Genome Res.1992,2:28-33.
Catana R, Eloy M., Stability evaluation of an immobilized enzyme system for inulin hydrolysis.Food Chemistry,2005,91:517–520.
Chi ZM., Chi Z, Zhang T, Liu GL, Yue L. Inulinase expressing microorganisms and applicationsof inulinases. Appl Microbiol Biotechnol2009,82:211–220.
Divne, C., et al., and Jones, T.A. The three-dimensional crystal structure of the catalytic core ofcellobiohydrolase I from Trichoderma reesei. Science,1994,265,524–528.
Frederic C., Jean W., et al. Cloning, sequencing and overexpression in Escherichia coli of thealginatelyase-encoding aly gene of Pseudomonas alginovora: identification of three classes ofalginate lyases. Biochem. J.,1996,319:575-583
Gacesa P. et al. Alginate-modifying enzymes:a proposed unified mechanis action for the lyasesand epimerases.FEBS Lett,1987,212:199-202.
Gacesa P. et al. Enzymic degradation of alginates. Int J Biochem,1992,24(4):545-552.
Gary F., Walter G., David K., et al. Inulin and Oligofructose as Dietary Fiber: A Review of theEvidence.2001,41:253-362.
Gl ckner FO, Kube M, et al. Complete genome sequence of the marine planctomycete Pirellulasp. Strain1. Proc Natl Acad Sci U S A.,2003,100(14):8298-303.
Gong F, Chi ZM, Sheng J, Li J, Wang XH. Purification and characterization of extracellularinulinase from a marine yeastn Pichia guilliermondii and Inulin hydrolysis by the purifiedinulinase. Biotechnol Bioproc Eng2008,13:533–539.
Grasdalen H.， et al. Spectroscopy of alginate: sequential structure and linkage conformations.Carbohydr.Res.,1983,118:255-260.
Guibet M, Colin S, Barbeyron T, et al. Degradation of lambda-carrageenan by Pseudoalteromonascarrageenovora lambda-carrageenase: a new family of glycoside hydrolases unrelated tokappa-and iota-carrageenases. Biochem J.2007,404(1):105-14.
Hashimoto W, Miyake O, Momma K, Kawai S, Murata K， Molecular identification ofoligoalginate lyase of Sphingomonas sp. Strain A1one of the enzymes required forcompletedepolymerization of alginate. J Bacteriol,2000,182:4572-4577.
Hatada Y, Mizuno M, Li Z, Ohta Y. Hyper-production and characterization of the ι-carrageenaseuseful for ι-carrageenan oligosaccharide production from a deep-sea bacterium, Microbulbiferthermotolerans JAMB-A94T, and insight into the unusual catalytic mechanism. Mar Biotechnol(NY).2011,13(3):411-22.
Haug A，Larsen B，Smidsrod O., et al. Studies on the sequence of uronic acid residues in algnicacid.ActaChem. Scand.,1967,21:691-704.
Khambhaty Y, Mody K, Jha B, Gohel V. Statistical optimization of medium components forκ-carrageenase production by Pseudomonas longate. Enzy Microb Technol,2007,40:813–22.
Kim C.H., Rhee S.K. Fructose production from Jerusalem artichoke by inulinase immobilized onchitin.,1989,):201-206
Kim DE, Lee EY, Kim HS. Cloning and characterization of alginate lyase from a marinebacterium Streptomyces sp. ALG-5. Mar Biotechnol,2008doi:10.1007/s10126-008-9114-9
Kim KY, Alessandro S.Nascimento et al. Catalytic mechanism of inulinase from Arthrobacter sp.S37.2008,371:600–605.
Kuzuwa S., Yokoi K., Kondo M., Kimoto H., Yamakawa A., Taketo A., Kodaira K. Properties ofthe inulinase gene levH1of Lactobacillus casei IAM1045; cloning, mutational and biochemicalcharacterization. Gene2012, doi:10.1016/j.gene.2011.12.004.
Kwon HJ., Jeon SJ.,You DJ., Kim KH., Jeong YK., Kim YH., Kim YM., Kim BW. Cloning andcharacterization of an exoinulinase from Bacillus polymyxa. Biotechnol Lett2003,25:155–159.
Kwon YM., Kim HY., Choi YJ. Cloning and characterization of Pseudomonas mucidolensexo-inulinase. J Microbiol Biotechnol2000,10:238-243.
Liu GL, Li Y， Chi Z，Chi ZM. Purification and characterization of κ-carrageenase from themarine bacterium Pseudoalteromonas porphyrae for hydrolysis of κ-carrageenan. ProcessBiochemistry,2011,46:265–271.
Liu XY, Chi Z, Liu GL,et al. Inulin hydrolysis and citric acid production from inulin using thesurface-engineered Yarrowia lipolytica displaying inulinase. Metabolic Engineering,12(5):469–476
LOPES T. S., DE WIJS1. J.,et al. Factors Affecting the Mitotic Stability of High-copy-numberIntegration into the Ribosomal DNA of Saccharomyces cerevisiae.1996,12:467-477
Madzak, C., Gaillardin, C., Beckerich, J.M.,. Heterologous protein expression and secretion in thenon-conventional yeast Yarrowia lipolytica:a review. J. Biotechnol.2004,109:63-81.
Matsushima R, Danno H, Uchida M, Analysis of extracellular alginate lyase and its gene from amarine bacterial strain, Pseudoalteromonas atlantica AR06. Appl Microbiol Biotechnol.2010,86(2):567-576
Mavromatis K, Abt B, et al. Complete genome sequence of Coraliomargarita akajimensis typestrain (04OKA010-24). Stand Genomic Sci.2010,2(3):290-9.
May TB, Chakrabarty AM. Pseudomonas aeruginosa:genes and enzymes of alginate synthesis.Trends Microbiol,1994,2(5):151-157.
Michel G, Chantalat L， et al. The κ-carrageenase of P. carrageenovora features a tunnel-shapedactive site: A Novel Insight in the Evolution of Clan-B Glycoside Hydrolases. Structure,2001,9:513–525.
Michel G, Nyval-Collen P, Barbeyron T, Czjzek M, Helbert W. Bioconversion of red seaweedgalactans: a focus on bacterial agarases and carrageenases. Appl Microbiol Biotechnol,2006,71:23-33.
Michel G,Chantalat L,Fanchon,et al. The ι-Carrageenase of Alteromonas fortis. Biochem. J.2001,276(43):40202–40209.
Mou H,Jiang X,Guan H. The κ-carrageenan derived oligosaccharide prepared by enzymaticdegradation containing anti-tumor activity. J Appl Phychol,2003,15:297～303.
Nagem P., Rojas L., Golubev. M. et al. Crystal Structure of Exo-inulinase from Aspergillusawamori: The Enzyme Fold and Structural Determinants of Substrate Recognition. J. Mol. Biol.2004,344:471–480
Ni XM, Yue LX, Chi ZM, Li J, Wang XH, Madzak C. Alkaline protease gene cloning from themarine yeast Aureobasidium pullulans HN2-3and the protease surface display on Yarrowialipolytica for Bioactive Peptide Production. Mar Biotechnol,2009,11:81–89.
Onodera S., Murakami T., Ito H., Mori H., Matrui H., Honda M., Chiba S., Shiomi N., Molecularcloning and nucleotide sequence of cDNA and gene encoding endo-inulinase from Penicilliumpurpurgenum. Biosci Biotechn Biochem1996,60:1780-1785.
Potin P， Richard C， Barbeyron T， et a1． Processing and hydrolytic mechanism ofthecgkA-encoded κ-carrageenase of Alteromonas carrageenovora．Eur J Biochem，1995，228：971—975
Potin P., Bouarab K., Kupper F., Oligosaccharide recognition signals and defence reactions inmarine plant microbe inter. Actions[J].Ecology and Microbiology,1999,2:276-283.
PreissJ， AshwellG., et al. Alginic acid metabolism in bacteria.I. Enymatic formation ofunsaturated oligosaceharides and4-deoxy-L-erythro-5-hexoseulose uronie acid. Jbiol.Chem.,1962,237:309-316.
Preston LA, Wong TY, Bender CL, Schiller NL. Characterization of alginate lyase fromPseudomonas syringae pv. Syringae. J Bacteriol,2000,182(21):6268-71.
R.A. Muzzarelli, M. Mattioli-Belmonte, A. Pugnaloni, G. Biagini, Biochemistry, histology andclinical uses of chitins and chitosans in wound healing, in: P. Jolles, R.A.A. Muzzarelli (Eds.),Chitin and Chitinases, Birkhauser, Basel,1999.
Rebuffet E, Barbeyron T, Czjzek M,et al. Identification of Catalytic Residues and MechanisticAnalysis of Family GH82ι-Carrageenases. Biochemistry2010,49,7590–7599.
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