Production and characterization of a novel alkaline protease from a newly isolated Neurospora crassa through solid-state fermentation
Production and characterization of a novel alkaline protease from a newly isolated Neurospora crassa through solid-state fermentation
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DOI：
10.1016/j.lwt.2019.108990
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发表时间：
2020-03
期刊：
影响因子：
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通讯作者：
Liufeng Zheng;Xinying Yu;Changhao Wei;Leyun Qiu;Chengwei Yu;Qian Xing;Ya-wei Fan;Z. Deng
中科院分区：
文献类型：
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作者：
Liufeng Zheng;Xinying Yu;Changhao Wei;Leyun Qiu;Chengwei Yu;Qian Xing;Ya-wei Fan;Z. Deng
微生物蛋白酶广泛用于制备具有促进健康的生物肽的蛋白质水解产物。本研究采用一株新分离的粗糙脉孢菌（命名为CGMCC3088），以豆渣为底物，通过固态发酵生产蛋白酶。最佳发酵条件为：豆渣10g；水，21 毫升；初始pH值，5.0；孵化温度，30°C；接种量，2 mL；发酵时间72 h，相应的蛋白酶活性为1959.82 U/g。通过硫酸铵沉淀进一步纯化蛋白酶，然后在 DEAE-Sepharose 和 Sephadex G-75 上进行离子交换层析。该蛋白酶的分子量为30 kDa，进一步的质谱分析清楚地表明它是一种新型蛋白酶。蛋白酶在55℃、pH 9时具有最佳活性。酶活性受到SDS和金属离子的部分抑制，而受有机溶剂的影响很小。该蛋白酶被苯甲基磺酰氟完全灭活，表明其丝氨酸蛋白酶活性占主导地位。该酶优先水解酪蛋白，动力学分析表明其Kman和Vmax分别为2.18 mg/mL和36.36 μg/mL/min。因此,粗糙脉孢菌CGMCC3088有潜力产生一种新型有机溶剂稳定的碱性蛋白酶,可应用于生物活性成分的制备。
Microbial proteases are widely used to prepare protein hydrolysates with health-promoting biopeptides. Here, a newly isolated strain ofNeurospora crassa(named as CGMCC3088) was used to produce proteases through solid-state fermentation of okara as the substrate. The optimal fermentation conditions are: okara, 10 g; water, 21 mL; initial pH, 5.0; incubation temperature, 30 °C; inoculation amount, 2 mL; fermentation time, 72 h, with a corresponding protease activity of 1959.82 U/g. The protease was further purified by ammonium sulphate precipitation, followed by ion-exchange chromatography on DEAE-Sepharose and Sephadex G-75. The molecular weight of the protease was 30 kDa, and further mass spectrometry analysis clearly indicated that it was a novel protease. The protease had the optimal activity at 55 °C and pH 9. The enzyme activity was partially inhibited by SDS and metal ions, whereas little affected by organic solvents. The protease was completely inactivated by phenylmethylsulfonyl fluoride, indicating its dominant serine protease activity. The enzyme preferably hydrolyzed casein, and kinetic analysis showed that its Kmand Vmaxwere 2.18 mg/mL and 36.36 μg/mL/min, respectively. Therefore,Neurospora crassaCGMCC3088 has the potential to produce a novel organic solvent-stable alkaline protease, which may be applied to the preparation of bioactive ingredients.