几丁质降解菌Acinetobacter sp. WML1的生物安全性和发酵优化

Biosafety and Fermentation Optimization of Acinetobacter sp. WML1 for Chitinase Production

摘要

Acinetobacter sp. WML1分离自淡水湖渔场沉积物, 能降解几丁质, 具有一定的应用价值。为了评价菌株WML1的生物安全性, 为菌株的安全使用提供参考和保障, 采用常规纸片扩散法检测耐药性, 用血琼脂平板划线法检测溶血性, 用溴甲酚紫显色法检测氨基酸脱羧酶活性, 用试剂盒测定硝酸还原酶活性。在菌株生物安全性分析的基础上, 通过单因素试验法和正交试验法进行菌株WML1产几丁质酶发酵条件的优化。结果表明, 菌株WML1对多种抗生素敏感, 无溶血性, 无硝酸还原酶活性, 有精氨酸脱羧酶活性及微弱的赖氨酸和鸟氨酸脱羧酶活性; 其最优产酶条件为0.4%黄豆粉、0.20%半乳糖、0.03% KH2PO4、0.07% K2HPO4、0.002% FeSO4、0.05% MgSO4·7H2O、0.001% ZnSO4、0.60%胶体几丁质、pH 9.5、5.0%的接种量、25 mL的装液量(对应250 mL容量瓶)、37 ℃的培养温度, 优化后的几丁质酶活为(2.72±0.09) U/mL。综上所述, 不动杆菌WML1在使用安全性上危害较小, 可防可控, 经发酵条件优化后其所产几丁质酶的活性提高了约4.61倍, 为产几丁质酶菌株的工业化应用提供了理论基础。

Abstract

Acinetobacter sp. WML1 was isolated from the fishery sediments of freshwater lakes and proved to have application value due to its ability to degrade chitin. Herein, the biosafety of the chitinase-producing strain WML1 was first evaluated for application. The drug resistance of the strain was detected with the disk diffusion method, haemolytic activity was analyzed by streaking the blood agar plate, amino acid decarboxy-lase and nitrate reductase activities were measured by the chromogenic assay with bromocresol purple and nitrate reductase activity assay kit, respectively. On the basis of biosafety analysis, the fermentation condi-tions of the strain were then optimized by single-factor and orthogonal tests for chitinase production. The re-sults showed that the strain WML1 was sensitive to most antibiotics, and had no haemolytic activity or ni-trate reductase activity, had arginine decarboxylase activity and weak lysine and ornithine decarboxylase ac-tivities. The optimal enzyme production conditions of strain WML1 were as follows: 25 mL medium containing 0.4% soybean powder, 0.20% galactose, 0.03% KH2PO4, 0.07% K2HPO4, 0.002% FeSO4, 0.05% MgSO4·7H2O, 0.001% ZnSO4, 0.60% colloidal chitin in a 250 mL flask, with the initial pH of 9.5, 5.0% inoculum size and culture temperature of 37 ℃. Under the optimized conditions, the chitinase activity reached (2.72±0.09) U/mL. In conclusion, Acinetobacter sp. WML1 is generally safe in application, with only low toxicity that is preven-table and controllable, and the enzyme activity could be about 4.61 times higher than that before optimiza-tion. The study provides a theoretical foundation for the industrial application of chitinase-producing bacteria.

关键词

几丁质降解菌 / 几丁质酶 / 不动杆菌WML1 / 生物安全性 / 发酵优化

Key words

chitinase-producing bacterium / chitinase / Acinetobacter sp. WML1 / biosafety / fermentation op-timization