Research Article: Mutagenesis and characterization of a Bacillus amyloliquefaciens strain for Cinnamomum camphora seed kernel oil extraction by aqueous enzymatic method

Date Published: July 17, 2017

Publisher: Springer Berlin Heidelberg

Author(s): Cheng Zeng, Rongbin Zhao, Maomao Ma, Zheling Zeng, Deming Gong.


The purpose of the present study was to increase the proteinase activity of the strain NCU116 by combining ultraviolet irradiation and N-methyl-N′-nitro-N-nitroso guanidine treatment, in order to enhance the efficiency of Cinnamomum camphora seed kernel oil (CCSKO) extraction by aqueous enzymatic method (AEM). The mutated strain, designated as NCU116-1, was screened out by the ratio of hydrolytic zone diameter to colony diameter on skim milk plate. The proteinase activity (9116.1 U/ml) of NCU116-1 was increased by 31.9% compared with the parental strain. The extracellular enzymes produced by NCU116-1 included proteinase, pectase, glucoamylase, cellulase and amylase. The proteinase had the maximum activity at 50 °C. Its optimum temperature and pH value were approximately 45 °C and 8.0 respectively. Mn2+ was an activator of neutral proteinase. The glucoamylase had the maximum activity at 35 °C, and was activated by Cu2+, Fe3+ and Mn2+. Its optimum temperatures and pH value were 35 °C and 8.0 respectively. The pectinase had the maximum activity at 40 °C, and was activated by Ca2+ and Mn2+. Its optimum temperatures and pH value were 35–40 °C and 6.0 respectively. The optimum conditions of CCSKO extraction by AEM were also investigated. The results suggested that the best amount of enzyme solution and enzymolysis time were 20% (v/v) and 4 h, respectively. The oil extraction rate was 95.2% under these conditions. Thus, a suitable mutated strain was selected for CCSKO extraction by AEM and the optimum extraction conditions were determined.

Partial Text

Cinnamomum camphora (Lauraceae) is a plant with high economic values (Babu et al. 2003). The content of medium chain fatty acid triglyceride (MCT) in Cinnamomum camphora seed kernel oil (CCSKO) was high (MCT > 94%), and MCT was reported to reduce the consumption and subsequent weight gain of animals (Mumme and Stonehouse 2015; Ferreira et al. 2013; Wang et al. 2016) and improve blood lipid and glucose levels (Page et al. 2009; Fu et al. 2015). There has been a growing interest in CCSKO and its extraction. Meanwhile, MCT was found to have a strong inhibitory effect on Gram-positive bacteria, Gram-negative bacteria, mould and enzymes (Hui et al. 2009; Luo et al. 2014).

Appearance and morphology of the colony in mutated strain under SEM had no significant difference from the original strain, but the extracellular enzyme activity changed greatly. The results may be due to the effects of physical and chemical mutagenesis on the original strain, the DNA which controlled extracellular traits has been mutated as well, but there was no significant change in the colony and cell morphology. Compared with the original strain, the activities of protease, pectinase, glucoamylase and amylase were increased by 107.2, 122.1, 163.5 and 23.8%, respectively. However, the activity of cellulase was reduced by approximately 49%. This may be due to the strong random mutation induced by physical and chemical mutagenesis, the negative mutation happened in cellulase. In some cases, mutagenesis can’t improve the enzyme activity of the strain, and the results have some randomness and contingency.




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