Enhanced production of thermostable laccases from a native strain of Pycnoporus sanguineus using central composite design

The production of thermostable laccases from a native strain of the white-rot fungus Pycnoporus sanguineus isolated in Mexico was enhanced by testing different media and a combination of inducers including copper sulfate (CuSO4). The best conditions obtained from screening experiments in shaken flasks using tomato juice, CuSO4, and soybean oil were integrated in an experimental design. Enhanced levels of tomato juice as the medium, CuSO4 and soybean oil as inducers (36.8% (v/v), 3 mmol/L, and 1% (v/v), respectively) were determined for 10 L stirred tank bioreactor runs. This combination resulted in laccase titer of 143 000 IU/L (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), pH 3.0), which represents the highest activity so far reported for P. sanguineus in a 10-L fermentor. Other interesting media resulting from the screening included glucose-bactopeptone which increased laccase activity up to 20 000 IU/L, whereas the inducers Acid Blue 62 and Reactive Blue 19 enhanced enzyme production in this medium 10 times. Based on a partial characterization, the laccases of this strain are especially promising in terms of thermostability (half-life of 6.1 h at 60 °C) and activity titers.概要研究目的优化获得血红密孔菌(P. sanguineus)的最佳培养基组成, 提高耐热漆酶的产量。创新要点获得了目前文献报道的最高水平的漆酶活力。研究方法通过单因素试验研究了不同培养基(番茄汁、 麦麸、 麦芽提取物和葡萄糖细菌蛋白胨培养基) 和不同组合诱导剂(大豆油、 阿魏酸、 没食子酸、 二甲基苯胺、 酸性蓝62 和活性蓝19 分别与硫酸铜组合诱导剂)对P. sanguineus 产耐热漆酶的影响。 在此基础上采用中心组合试验设计, 进一步研究了番茄汁培养基结合硫酸铜和大豆油组合诱导剂对P. sanguineus 产耐热漆酶的影响。 利用SAS10.0 和响应面分析方法对试验结果进行了统计分析和建立回归模型。重要结论通过中心组合设计优化得出P. sanguineus 产耐热漆酶的最优培养基条件: 以36.8%番茄汁为培养基, 以3 mmol/L 硫酸铜和1%大豆油作为组合诱导剂。 该条件下在10 L 搅拌槽生物反应器中漆酶活力达到了143 000 IU/L(2,2′-联氮双(3-乙基苯并噻唑啉-6-磺酸)为底物, pH 值为3.0)。