海藻酸裂解酶高产菌株Microbulbifer sp.SH-1的分离、鉴定及其产酶条件优化

Isolation, identification and culture optimization of an alginate lyase-producing strain Microbulbifer sp. SH-1

摘要:

[目的] 筛选一株海藻酸裂解酶高产菌株，并通过优化产酶条件提高海藻酸裂解酶活性。[方法] 以海藻酸钠为唯一碳源的培养基，对福建漳州滨海土壤中的微生物进行筛选和分离，获得海藻酸裂解酶高产菌株；依据形态、生理生化特征及16S rDNA序列分析对目的菌株进行鉴定；然后通过单因素和正交试验对其产酶条件进行优化。[结果] 十六烷基吡啶（CPC）染色得到4株透明圈与菌落直径比值（D/d）>3的菌株；DNS法测定4菌株发酵液中海藻酸裂解酶活力，其中菌株SH-1的海藻酸裂解酶活性最高，达到315.52 U/mL；经形态、生理生化和16S rDNA测序鉴定，将其命名为Microbulbifer sp.SH-1；通过单因素和正交试验优化，确定该菌株最适产酶培养基为：海藻酸钠10 g/L，NaCl 5 g/L，（NH₄）₂SO₄ 5 g/L，MgSO₄ 0.2 g/L，K₂HPO₄ 1 g/L，FeSO₄ 0.02 g/L。对培养条件的进一步优化结果发现，在初始pH 7.5、温度32℃条件下，以1%的接种量将SH-1菌株接入50 mL优化培养基中，240 r/min转速下振荡培养24 h，SH-1菌株产酶最大活性可达757.90 U/mL，比优化前提高了2.4倍。[结论] SH-1最佳产酶条件的建立，为海藻酸裂解酶的大规模制备以及更深层次研究提供了试验基础和理论依据

Abstract:

[Objective] The aim of this study was to screen a strain with high alginate lyase activity and optimize the culture conditions of the strain. [Methods] Using sodium alginate as the sole carbon source, we screened and isolated microorganisms from the coastal soil of Zhangzhou, Fujian province, and a high-yield strain of alginate lyase was obtained. According to the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, the target strain was identified. Then the enzyme production conditions were optimized by single factor and orthogonal test. [Results] Four strains with transparent circle to colony diameter ratio (D/d)>3 were obtained by cetylpyridine staining. DNS method was used to determine the activity of alginate lyase in the fermentation broth of four strains. The enzyme activity of strain SH-1 was the highest, reaching 315.5 U/mL. Based on morphological, physiological, biochemical and 16S rDNA sequencing identification, it was named Microbulbifer sp. SH-1. Through single factor and orthogonal test optimization, the optimum enzyme production medium was determined as follows (g/L):sodium alginate 10, NaCl 5, (NH₄)₂SO₄ 5, MgSO₄ 0.2, K₂HPO₄ 1 and FeSO₄ 0.02. Further optimization of culture conditions showed that SH-1 strain could be inoculated into 50 mL medium at initial pH 7.5 and 32℃ with 1% inoculation. The maximum enzyme activity of SH-1 strain could reach 757.9 U/mL under 240 r/min for 24-h shaking, 2.4 times higher than that before optimization. [Conclusion] The optimized conditions for SH-1 production provides reference for large-scale preparation of alginate lyase and further research.