【作者】 付晓婷；

【导师】 林洪；

【作者基本信息】 中国海洋大学 ， 食品科学， 2008， 博士

【摘要】 从韩国东海岸的蝾螺(Turbinidae batillus cornutus)肠道中筛选出的菌株YKW-34,能够有效降解一些褐藻和红藻的细胞壁多糖。通过分析其16S rRNA基因序列,将该菌株鉴定为Agarivorans albus,命名为Agarivorans albus YKW-34 (中文名暂定为白色琼胶贪食菌YKW-34)。A. albus YKW-34在其生长培养基marine broth中不产生多糖降解酶;但在以多糖作为碳源的培养基中可产生1种褐藻胶裂解酶和2种琼胶酶。本研究旨在对A. albus YKW-34产生的褐藻胶裂解酶和琼胶酶进行生产优化、分离纯化、性质研究及基因克隆,以便为两种酶的工业化应用提供理论依据和实践基础。通过单因子和正交实验,对A. albus YKW-34产褐藻胶裂解酶的培养条件进行了优化。结果表明,其最适碳源为海带粉0.5% (w/v),氮源为KNO3 0.1% (w/v),最佳种龄为12h,接种量为10%,最适培养基初始pH值为8.0,培养温度为25°C。在上述条件下,110rpm震荡培养48h,褐藻胶裂解酶产量达到最大值5U/ml。在发酵培养基中添加过量的多糖或单糖可抑制产酶;增加氮源的浓度或种类有利于菌株的生长,但也会抑制产酶。通过聚醚酰亚胺沉淀,阴离子交换、疏水及凝胶过滤色谱,进行了A. albus YKW-34产褐藻胶裂解酶的纯化。其回收率为7%,纯化倍数为25倍。SDS-PAGE和凝胶过滤色谱表明此酶由单一肽链组成,分子量为60kDa。等电聚焦实验表明其等电点为5.5-5.7。此酶的最适pH为7.0,在pH 7.0?10.0稳定;最适温度为30?40°C,低于50°C时稳定。此酶具有钠/钾离子依赖性。透析除去反应体系中钠/钾离子后,酶活力丧失;添加钠/钾离子后,酶活力可完全恢复。此酶对还原剂如β-巯基乙醇和DTT、金属离子络合剂如EDTA和EGTA具有耐受性;变性剂如SDS和尿素可使酶活力提高30%。底物专一性实验表明,此酶能同时降解甘露糖醛酸和古罗糖醛酸片断。通过单因子实验、Plackett-Burman设计及响应面设计实验,对A. albus YKW-34发酵产琼胶酶的条件进行了优化。单因子实验表明,最佳碳源和氮源分别为琼胶和酵母浸膏,最适培养温度为25°C。在此条件下,确定最适培养时间为12h。Plackett-Burman设计表明琼胶、酵母浸膏及初始pH三个因素对琼胶酶产量具有显著影响(P<0.05)。响应面设计实验确定此三个因素的最适水平为:琼胶0.23%,酵母浸膏0.27%,初始pH为7.81。经优化后,琼胶酶产量由0.23U/ml提高至0.87U/ml。用活性染色法对琼胶酶进行了原位测定,结果表明,琼胶酶为培养液中的主要蛋白质组分,其分子量约为50kDa。离子交换色谱表明,A. albus YKW-34能产生两种琼胶酶,将其分别命名为AgaA34和AgaB34,其中AgaA34产量为747U/l,AgaB34产量为83U/l。AgaA34进一步通过凝胶过滤色谱得以纯化。SDS-PAGE和凝胶过滤色谱表明此酶由单一肽链组成,分子量为50kDa。纯化的回收率为7%,纯化倍数为25倍。经测定,AgaA34的N端氨基酸序列为ASLVTSFEEA,与糖基水解酶家族50的琼胶酶相似;AgaB34的N端氨基酸序列为ADWDNIPIPAELDAG,与糖基水解酶家族16的琼胶酶相似。AgaA34的酶学性质分析表明,此酶的最适pH为8.0,在pH 6.0-11.0稳定;最适温度为40°C,在温度≤50°C时稳定。MALDI-TOF MS和13C NMR分析表明AgaA34的琼胶酶解产物为75mol%新琼二糖和25mol%新琼四糖。薄层色谱表明此酶能将新琼四糖降解为新琼二糖。以琼脂糖和新琼四糖为底物,此酶的催化效率分别为4.04×103和8.1×102s–1M–1。海水中的常见金属离子对AgaA34的酶活力无显著影响。此酶对SDS和尿素具有耐受作用;β-巯基乙醇和DTT可提高酶活力。本研究建立了A. albus YKW-34的基因组文库,并从此文库中克隆出琼胶酶基因(agaB34)。此基因长1362bp,编码453个氨基酸。根据同源性分析,判断其氨基酸序列1-23aa为信号肽,25-297aa为糖基水解酶家族16催化组件,316-452aa为碳水化合物结合组件。成熟蛋白的分子量预测为48,637Da,等电点预测为6.34。通过构建N端包含信号肽序列及C端镶嵌6个组氨酸的琼胶酶重组质粒,以大肠杆菌DH5α为宿主对该酶进行了重组表达。结果表明,重组琼胶酶(rAgaB34)可分泌至胞外,最高产量为1670U/l。rAgaB34可经亲和色谱进行一步纯化,回收率为80%,纯化倍数为15倍。rAgaB34分子量为49kDa,其终产物为新琼四糖。其结构域及终产物表明其属于糖基水解酶家族16成员。此酶的最适pH为7.0,在pH 5.0-9.0稳定;最适温度为30°C,在温度≤50°C时稳定。以琼脂糖为底物,此酶的比活力和催化效率分别为242U/mg和1.7×106s–1M–1。此酶对EDTA和EGTA、SDS和尿素及β-巯基乙醇和DTT具有耐受性。更多还原

【Abstract】 A marine bacterium strain YKW-34 that degrades the cell wall of some seaweed including Laminaria japonica and Gelidium amansii was isolated from the gut of a turban shell. This strain was identified as Agarivorans albus based on 16S rRNA gene sequence analysis. This paper describes the optimization of production, purification, and characterization of an alginate lyase and an agarase, as well as gene cloning and expression of an agarase from this strain. The effects of medium composition and culture condition on the production of alginate lyase by A. albus YKW-34 were investigated using batch shake flasks. No alginate lyase was produced in the marine broth medium. After optimization, the activity of the alginate lyase reached 5 U/ml. The optimal production conditions for alginate lyase by A. albus YKW-34 were: inoculum volume, 10%; inoculum age, 12 h; initial pH of the medium, 8.0; culture temperature, 25°C; carbon source, 0.5% Laminaria powder; nitrogen source, 0.1% KNO3. Alginate could induce alginate lyase production, but not as efficient as did Laminaria powder. The addition of fucoidan, cellulose, and glucose had negative effect on the production of alginate lyase. Other kinds of nitrogen sources such as yeast extract, beef extract, and peptone affected positively the growth of the microorganism, but negatively the alginate lyase production. In addition, the optimal harvest time was 48 h based on the time course of alginate lyase production.An alginate lyase with high specific enzyme activity was purified from A. albus YKW-34 by in order of ion exchange, hydrophobic, and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. It was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for the activity of the alginate lyase were pH 7.0 and 40°C, respectively. It was stable over pH 7.0–10.0 and at temperature below 50°C. The enzyme had substrate specificity for both poly-guluronate and poly-mannuronate units. The kcat/Km value for alginate (heterotype) was 1.7×106 s-1M-1. The enzyme activity was completely lost by dialysis and restored by addition of Na+ or K+. The optimal activity exhibited in 0.1 M of Na+ or K+. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT), and chelating reagents (EGTA and EDTA).Effects of medium composition and culture conditions on agarase production by A. albus YKW-34 were investigated in shake flasks. Effects of carbon and nitrogen sources and culture temperature on agarase production were evaluated by one-factor-at-a-time design. Agar, yeast extract, and 25°C were found to be most suitable for agarase production. The most important nutritional components and culture conditions influencing agarase production were selected by Plackett-Burman design. Among the nine factors studied, agar, yeast extract, and initial pH had significant effect on agarase production. The optimum levels of these variables were further determined using a central composite design. The highest agarase production was obtained in the medium consisting of 0.23% agar and 0.27% yeast extract at initial pH 7.81. The whole optimization strategy resulted in the enhancement of agarase production from 0.23 U/ml to 0.87 U/ml. The activity staining of crude agarase preparation after electrophoresis revealed the presence of an agarase with molecular mass of 50 kDa.An extracellularβ-agarase, AgaA34, was purified from A. albus YKW-34 by ion exchange and gel filtration chromatographies to homogeneity with a recovery of 30% and a fold of 10. AgaA34 was composed of a single polypeptide chain with the molecular mass of 50 kDa. N-terminal amino acid sequencing revealed a sequence of ASLVTSFEEA, which exhibited a high similarity (90%) with those of agarases from glycoside hydrolase family 50. The pH and temperature optima of AgaA34 were pH 8.0 and 40°C, respectively. It was stable over pH 6.0–11.0 and at temperature below 50°C. Hydrolysis of agarose by AgaA34 produced neoagarobiose (75 mol%) and neoagarotetraose (25 mol%), whose structures were identified by MALDI-TOF MS and 13C NMR. AgaA34 cleaved both neoagarohexaose and neoagarotetraose into neoagarobiose. The kcat/Km values for agarose and neoagarotetraose were 4.04×103 and 8.1×102 s–1M–1, respectively. AgaA34 was resistant to denaturing reagents (SDS and urea). Metal ions were not required for its activity, while reducing reagents (β-Me and DTT) increased its activity by 30%.Aβ-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium Agarivorans albus YKW-34. The nucleotide sequence of agaB34 consisted of 1362 bp and encoded a protein of 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a glycoside hydrolase family 16 (GH-16) domain and a carbohydrate-binding module (CBM), showed 37–86% identity to those of known agarases in glycoside hydrolase family 16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli DH5αas a host. The purified rAgaB34 was aβ-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, while it could not further degrade NA4. The maximal activity of rAgaB34 was observed at 30°C and pH 7.0. It was stable over pH 5.0–9.0 and at temperature up to 50°C. Its specific activity and kcat/Km value for agarose were 242 U/mg and 1.7×106 s-1M-1, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT,β-Me), and denaturing reagents (SDS and urea).更多还原