南极菌产琼胶酶aga3311的表达、性质及其降解特性

【目的】本文通过对具有琼胶降解能力的南极菌Pseudoalteromonas sp.NJ21全基因组进行生物信息学分析,筛选获得琼胶酶疑似序列aga3311,采用基因工程手段对该基因的功能和性质进行了验证和分析。【方法】首先对aga3311进行克隆和表达;采用Ni-NTA对重组酶进行纯化;DNS-还原糖法测定重组酶的酶学性质;用薄层层析(TLC)和质谱(MS)技术对Aga3311的酶解产物进行分析。【结果】构建的重组表达质粒p ET-30(a)+aga3311能够在工程菌E.coli BL21(DE3)中实现高效表达,其中可溶性表达为30%左右;纯化的重组酶Aga3311分子量为87 k Da,其最适作用温度为35°C,30–45°C的范围内稳定性较高,50°C则迅速失活,具有热不稳定的特征;最适p H为7.0,在p H 4.0–10.0的范围内仍能保持50%以上的活性;金属离子Fe~(3+)、Be~(2+)、Zn~(2+)和Ca~(2+)均能显著提高Aga3311的活性,特别是Ca~(2+)使其酶活提高1倍。该酶的酶解终产物经TLC和质谱分析主要为新琼二糖。【结论】重组酶Aga3311为Glyco_hydro_42家族的外切型β-琼胶酶,能够特异性降解琼脂糖生成新琼二糖。     

Extracellular β-agarase LSL-1 producing neoagarobiose from a newly isolated agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1

Summary An agar-liquefying Acinetobacter species capable of utilizing agar as sole source of carbon and energy was isolated from soil samples and the culture conditions were standardized for the maximal production of extracellular agarase. The bacterium was capable of liquefying an agar-plate within 3 days of incubation and produced extracellular agarase within a short period of time (16–18 h) when grown in defined mineral salts medium. Bacterium grew in the pH range 4.0–9.0, optimal at pH 7.0; temperature 25–40 °C and optimal at 37 °C. The agarase secreted by the Acinetobacter strain was inducible by agar and not repressed by other simple sugars when supplemented along with agar in the medium. The bacterium did not require NaCl for growth or production of agarase. The bacterium did not utilize other polysaccharides like κ-carrageenan, alginate, cellulose, and CMC. The activity staining of partially purified agarase preparations after native-PAGE and SDS PAGE revealed the presence of a single zone of clearance corresponding to the molecular weight 100 kDa, suggesting that it is a monomer. Neoagarobiose was the end product of agarose hydrolysis by this enzyme. The agarase was an endo-type glycosidase and belongs to Group-III β-agarase family.     

深海太平洋火色杆菌琼胶酶Aga2660的克隆表达及发酵优化

【背景】琼胶寡糖已成为化妆品、食品、医药等领域的研究热点,而生物酶法被认为是制备琼胶寡糖的高效方法。【目的】从深海太平洋火色杆菌(Flammeovirga pacifica WPAGA1)筛选获得琼胶酶基因aga2660,对基因aga2660进行克隆转化,使其在大肠杆菌中进行表达,分析重组酶的性质以及酶解产物。【方法】采用克隆表达和镍柱纯化方法获得aga2660基因表达的纯化产物;采用薄层层析和离子色谱法分析酶解产物;5L发酵罐采用补料阶段指数流加、诱导阶段乳糖连续流加的策略进行产酶发酵条件的优化。【结果】基因aga2660是具有GH50家族典型特征的基因,酶解产物为单一的新琼二糖。最适温度为30°C,最适pH为7.0,Mn2+、Ca2+和Mg2+能促进琼胶酶Aga2660的酶活。采用发酵优化策略后的酶活达到11.81U/mL,比优化前提高了13.2倍。【结论】琼胶酶Aga2660具有良好的热、酸、碱稳定性,其酶解产物为单一的新琼二糖,这为特定聚合度琼胶寡糖的制备奠定了良好基础。