Construction Of A Recombinant Engineering Strain For High-efficiency Degradation Of Cyanogenic Glycosides And Detoxification Of Flaxseed By Fermentation

Flaxseed, the seed of an annual herb plant flax (Linum usitatissimum L.), is oneof the ten important oilseeds in the world. Flaxseed, being a rich source of α-linolenicacid, linoleic acid and mammalian lignan precursors, imparts many health benefits,including anti-tumor, reducing blood lipid, improving memory, anti-ageing andenhancing immunity. Flaxseed meal, a by-product of the flaxseed after oil extraction,is available to high-quality forages and food additives due to being rich in protein,amino acid, dietary fiber and other nutrients. However, cyanogenic glycosides (CGs)in flaxseed can release extremely poisonous hydrocyanic acid (HCN) because of thecatalytic decomposition effect of intestinal β-glucosidases. Obviously, the efficientremoval of CGs has become a key technical problem which should be resolvedbeforehand in applications of flaxseed.The detoxification of CGs has been tried by diverse conventional methods, e.g.,boiling, roasting, microwave and solvent extraction. In contrast with the conventionalmethods, the fermentation detoxification method owns many advantages of highefficiency, energy saving, safety, and being friendly to the environment, and has beensuccessfully used to remove the CGs in several dietary plants such as cassava.However, no report on an entire fermentative method to detoxify CGs in flaxseed haspresented so far.Based on the overview of the references on the detoxification of CGs, thetechnology of genetic engineering was employed to successfully construct arecombinant strain of Pichia pastoris GS115-Ch-Glu, which could simultaneously secrete the recombinant proteins of Bacillus sp. CN-22cyanide hydratase and humanliver β-glucosidase. Then, an enzymatic preparation including12.5%human liverβ-glucosidase and8.9%(w:w) Bacillus sp. CN-22cyanide hydratase, which wasproduced by this recombinant strain, was used to detoxify of CGs in flaxseed. Finally,response surface methodology (RSM) was used to optimize the fermentationconditions. The main results of this paper are described as below:(1) The concentrations of formate, formamide and ammonia were determined,and the influence of NADH on the degradability of cyanide was assayed in thedegradation of KCN. The results showed that the cyanide hydratase was a majordegrading enzyme of cyanide-biodegradation in strain Bacillus sp. CN-22.(2) Using the genomic DNA of Bacillus sp. CN-22as a template, PCR wasperformed to amplify cyanide hydratase (Ch) gene. Then, the gene Ch was insertedinto an expression vector pPIC9K to get a recombinant plasmid pPIC9K-Ch.(3) Using the human liver total RNA as a template, RT-PCR was performed toamplify β-glucosidase (Glu) gene. Then, the gene Glu was inserted into theexpression vector pPIC9K to get a recombinant plasmid pPIC9K-Glu.(4) The recombinant plasmids pPIC9K-Ch and pPIC9K-Glu were transformedinto Pichia pastoris GS115and GS115-Ch in succession. By the homologousrecombination between recombinant plasmids and genome DNA of Pichia pastoris,an an engineering strain GS115-Ch-Glu, which could simultaneously secrete therecombinant proteins of Bacillus sp. CN-22cyanide hydratase and human liverβ-glucosidase, was successfully obtained for the first time.(5) Under the optimal fermentation conditions of30°C and200rpm, therecombinant strain GS115-Ch-Glu was cultured in flasks for48h to examine thesupernatant for the target proteins after being induced by0.5%methanol. As assessedby SDS-PAGE analysis, the contents of cyanide hydratase and β-glucosidase could beup to12.5%and8.9%of the total proteins in the supernatant, respectively.(6) By setting CGs degradability and residual cyanide as two response values,RSM was used to optimize the fermentation detoxification conditions. The optimalconditions were the combination of25g flaxseed,1.27g enzymatic preparation,8.0gsterilized water,50mg MgCl₂,50mg MnCl₂,46.8°C, pH6.3, and detoxification time48h. Under these conditions, the maximum CGs degradability and the concentrationof reidual cyanide are99.26%and0.015mg g^-1, respectively. (7) The contents of lignans and fatty acids in the flaxseed samples before andafter fermentation detoxification were analyzed by high performance liquidchromatography (HPLC) and gas chromatography-mass spectrometer (GC-MS),respectively. The results show that the detoxified flaxseed can retain the beneficialnutrients of lignans and fatty acids at the same level as untreated flaxseed, indicatingthat the newly-established fermentation method is suitable to remove the CGs inflaxseed, and useful to prepare the safe flaxseed products with the same levels ofbeneficial bioactive ingredients.