褐藻表面可培养褐藻酸降解菌的原位筛选

In situ screening of cultivable alginate-degrading microorganism on surface of brown seaweed

【背景】褐藻酸经酶解后生成的褐藻寡糖具有抗氧化、抗肿瘤、诱导免疫调节、调节植物生长等多元化的生物学功能,在食品、医药领域应用前景广阔。大量筛选褐藻酸降解菌有利于获得新结构、新功能的褐藻寡糖,有利于积极推动寡糖产业进程。【目的】高效筛选褐藻酸降解菌株,发掘具有开发前景的海藻原位微生物资源。【方法】利用褐藻酸唯一碳源培养基对天然铜藻表面微生物进行筛选;革兰氏碘液显色反应指示微生物降解褐藻酸的特性;牛津杯法初步测定产褐藻酸裂解酶菌株的酶活大小。【结果】从铜藻表面共获得81个菌落,经透明圈显色筛选出28株菌,通过16S rRNA基因测序分析得到7株褐藻酸降解菌,分别属于芽孢杆菌属、节杆菌属、德库菌属、短杆菌属和链孢子囊菌属。除芽孢杆菌属外,其余菌属的菌种此前均未被报道过有产褐藻酸裂解酶的能力。进一步分析表明,T-1菌株的产酶能力最强、酶活力最高。【结论】利用革兰氏碘液显色结合牛津杯法筛选到7株产酶菌株,并比较了各菌株的酶活大小,表明该筛选方法简便高效,适合大规模筛选褐藻酸降解菌株。

[Background] Alginate oligosaccharides produced by enzymatic hydrolysis of alginate have diverse biological functions such as anti-oxidation, anti-tumor, induction of immune regulation, and regulation of plant growth, and exhibit broad application prospects in the fields of food and medicine.Mass screening of alginate-degrading bacteria is beneficial to obtain new structures and functions of alginate oligosaccharides, which is conducive to actively promoting the industrial progress of oligosaccharides. [Objective] High-efficiency screening was carried out for alginate-degrading strains and exploring the in situ microbial resources of seaweeds with development prospects. [Methods] The only carbon source of sodium alginate was used to select the microorganisms on the surface of brown seaweed. The chromogenic reaction of gram iodine solution indicated the characteristics of microorganisms to degrade alginate. The activity of alginate lyase was determined by Oxford cup method. [Results] A total of 81 colonies were obtained from the surface of the algae samples. Twenty-eight strains were screened by clear circle staining. Seven strains of alginate-degrading bacteria were identified by 16S rRNA gene sequencing analysis. They belonged to the genus Bacillus （3/7）, Arthrobacter （1/7）, Desemzia （1/7）, Brachybacterium （1/7） and Streptosporangium （1/7）. The genera Arthrobacter, Desemzia, Brachybacterium and Streptosporangium have not been previously reported to produce alginate lyase. Further analysis showed that T-1 strain （Bacillus） had maximum enzyme production and highest enzyme activity. [Conclusion] Seven alginate lyase-excreting strains were isolated, and the enzyme activities of the strains were compared by Gram＇s iodine coloration combined with the Oxford cup method. This indicates that the screening method is simple, efficient and suitable for large-scale screening of alginate lyase-excreting microorganisms.