产褐藻胶裂解酶菌株筛选及其发酵与酶解工艺优化研究

Screening of Alginate Lyase-Producing Strains and Optimization of Fermentation and Enzymatic Hydrolysis Processes

  • 摘要: 褐藻胶裂解酶作为一种可以高效降解褐藻胶的工具酶,对于工业化生产褐藻寡糖具有重要作用。本研究以海藻酸钠为唯一碳源,从腐烂的南非树皮藻(Ecklonia maxima)中筛选纯化出了能够高效降解海藻酸钠的菌株,根据菌株形态、生理生化特征和16S rRNA基因测序结果将该菌鉴定为肺炎克雷伯菌(Klebsiella pneumoniae),将其命名为SW06。通过优化产酶和酶解条件,菌株的酶活力显著提高。菌株SW06的最优培养基成分为:海藻酸钠5 g/L,(NH4)2SO4 5 g/L、MgSO4·7H2O 6 mmol/L、NaCl 120 mmol/L、K2HPO4 15 mmol/L以及FeSO4·7H2O 0.04 mmol/L。经过优化后最佳培养条件为:接种量1%、装液量90 mL(250 mL锥形瓶内)、转速180 r/min和pH 6.0初始培养基。优化后褐藻胶裂解酶的最大酶活力达到了311 U/mL,与优化前酶活力相比提高了395%。酶学性质实验表明该酶的最适酶解温度为40 °C,在该温度下pH稳定性较好,在pH6.0~9.0环境下可以稳定存在;β-巯基乙醇和NaCl的添加可以显著提高酶活力(酶活力提高≥50%)。酶解产物分析实验表明,该菌的酶解产物为褐藻二糖、褐藻三糖、褐藻四糖。本研究成功从南非树皮藻中筛选出一株酶活力较高的产酶菌株,具有潜在的应用前景,为褐藻寡糖的工业化生产提供了新的选择。

     

    Abstract: Alginate lyase, as a tool enzyme capable of efficiently degrading alginate, plays a significant role in the industrial production of alginate oligosaccharides. In this study, using sodium alginate as the sole carbon source, a bacterial strain with high sodium alginate degradation efficiency was isolated and purified from decaying Ecklonia maxima. Based on morphological, physiological, biochemical characteristics, and 16S rRNA gene sequencing, the strain was identified as Klebsiella pneumoniae and designated as SW06. Through optimization of enzyme production and enzymatic hydrolysis conditions, the enzyme activity of the strain was significantly enhanced. The optimal medium composition for strain SW06 was as follows: sodium alginate 5 g/L, (NH4)2SO4 5 g/L, MgSO4·7H2O 6 mmol/L, NaCl 120 mmol/L, K2HPO4 15 mmol/L, and FeSO4·7H2O 0.04 mmol/L. The optimized culture conditions were as follows: inoculum size 1%, medium volume 90 mL (in a 250 mL conical flask), shaking speed 180 r/min, and initial medium pH 6.0. After optimization, the maximum activity of alginate lyase reached 311 U/mL, representing a 395% increase compared to the pre-optimization level. Enzymatic property experiments revealed that the optimal temperature for enzymatic hydrolysis was 40 °C, with good pH stability at this temperature; the enzyme remained stable within the pH range of 6.0–9.0. The addition of β-mercaptoethanol and NaCl significantly enhanced enzyme activity (≥50% increase). Analysis of enzymatic hydrolysis products indicated that the main products were alginate disaccharide, trisaccharide, and tetrasaccharide. This study successfully isolated a high-activity alginate lyase-producing strain from Ecklonia maxima, demonstrating potential application prospects and providing a new alternative for the industrial production of alginate oligosaccharides.

     

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