海洋细菌DT-7产琼胶酶的培养基组分优化

Optimization of culture medium components for agarase-producing marine bacterial strain Brevundimonas sp. DT-7

摘要:

培养基是微生物生长的重要条件之一。为研究微生物发酵法获取高活性琼胶酶并为今后酶法生产琼胶寡聚糖奠定一定基础，利用筛选实验设计法，首次以海洋细菌Brevundimonas sp. DT-7为实验菌株，以琼胶酶活性为评价指标，对该菌株培养基进行筛选，所筛选的8个相关因子为X₁（琼胶粉）、X₂（NaCl）、X₃（FePO₄）、X₄（CaCl₂）、X₅（K₂HPO₄）、X₆（FeSO₄·7H₂O）、X₇（酵母膏）、X₈（蛋白胨），其中X₁(琼胶粉)、X₄(CaCl₂)、X₈(蛋白胨)对产琼胶酶活性有显著影响(P<0.05)，然后采用BoxBehnken设计法进行实验设计，再应用SAS 8.2软件的二次响应面回归程序，对实验点的响应值进行回归分析，最后建立了二次响应面回归模型Y₁=499.6+19.45X₁+13.99X₄－1.213X₈－33.16X₁²－5.3X₁X₄－3.45X₁X₈－39.64X₄²－10.23X₄X₈－15.79X₈²。优化后培养基组分(w/v)：琼胶粉0.536%，NaCl 2.5%，FePO₄ 0.1%，CaCl₂ 0.052%，K₂HPO₄ 0.1%，FeSO₄·7H₂O 0.03%，酵母膏1%，蛋白胨0.484%；经培养基组分优化后，在250 mL三角瓶装液量为50 mL、初始pH 7.5、摇床转速为120 r/min、培养温度为(23±1) ℃,培养28 h的条件下，菌株产酶活力从优化前的372.0 U/mL提高到503.3 U/mL，酶活力为前者的1.35倍。上述研究结果表明：SAS软件可快速、有效地优化培养基组分配方；优化后的DT7培养基配方的组分简单，这为今后低成本生产高活性琼胶酶提供有利条件；对菌株Brevundimonas sp. DT-7的选育及其发酵特性的深入研究有望解决琼胶酶的商品化。

Abstract:

The activity of agarase depended on the bacterial growth conditions such as growth medium formula. In this study, we used marine bacterial strain Brevundimonas sp. DT-7 as an extracellular agarase producer and optimized the growth medium formula to produce maximum agarase that can be used for producing oligosaccharides from agar on a commercial scale in the future. Eight components in the bacterial culture medium (X₁ agar powder，X₂ NaCl, X₃ FePO₄，X₄ CaCl₂，X₅ K₂HPO₄，X₆ FeSO₄·7H₂O，X₇ yeast extract， X₈ peptone) were selected for the optimization. PlackettBurman design was used in the experiments and the significance of each component (X₁-X₈) contributing to activity of agarase (Y₁) was determined. The significant levels were defined at P<0.05. The data show that among the eight components, three medium components (X₁ agar powder, X₄ CaCl₂ and X₈ peptone) are significant factors for production of agarase from DT7. The three significant factors (X₁ agar powder, X₄ CaCl₂ and X₈ peptone) were selected for further analysis using BoxBehnken design. A regression model was obtained by response surface regression method (SAS 8.2) as follows: Y₁=499.6+19.45X₁+13.99X₄－1.213X₈－33.16X₁²－5.3X₁X₄－3.45X₁X₈－39.64X₄²－10.23X₄X₈－15.79X₈². The results demonstrate the optimum culture medium components (w/v): 0.536 % agar powder, 2.5% NaCl, 0.1% FePO₄, 0.052% CaCl₂, 0.1% K₂HPO₄，0.03% FeSO₄·7H₂O, 1% yeast extract, and 0.484% peptone, respectively. Using those optimized culture components and combined with other culture conditions ［50 mL culture medium in 250 mL flask, initial pH 7.5, shaken at 120 r/min, incubated at （23±1）℃ for 28 h］, the agarase activity of 503.3 U/mL was obtained, which was significantly higher than that of 372.0 U/mL when the culture medium was not optimized. We conclude that (1) using SAS software is a very effective and quick way to optimize a bacterial growth culture medium; (2) the culture medium optimized in this study is simply formulated and can be used for production of high activity of agarase at low cost; (3) with further modification and improvement, Brevundimonas sp. DT7 can be a source bacterium for production of agarase on a commercial scale. This is a preliminary study. Further research will be required to evaluate Brevundimonas sp. DT-7 in commercial application.