【作者】 吴酬飞；

【导师】 王江海；

【作者基本信息】 中山大学 ， 生物技术， 2012， 博士

【摘要】 亚麻籽为一年生亚麻科草本植物亚麻（Linum usitatissimum L.）的种子，是世界十大油料之一。亚麻籽因富含α-亚麻酸和亚油酸等不饱和脂肪酸及木酚素而具有增强机体免疫力、延缓衰老、提高记忆力、降血脂血糖和抗肿瘤等保健功效。亚麻籽粕是亚麻籽榨油后副产物，它富含蛋白质和膳食纤维等营养成分，是优质的畜禽饲料和食品添加剂。然而，亚麻籽中含有生氰糖苷，它在β-葡萄糖苷酶的作用下可生成剧毒的氢氰酸。显然，亚麻籽中生氰糖苷的高效去除已成为亚麻籽开发利用时必须突破的关键技术问题。前人虽已建立多种去除亚麻籽中生氰糖苷的脱毒方法，如水煮法、烘烤法、微波法和溶剂提取法，但与传统的物理和化学脱毒方法相比，生物发酵脱毒法具有高效、节能、安全、环保等优点，并已被成功用于木薯生氰糖苷等的脱毒。然而，迄今为止尚未见到采用全生物发酵脱毒法去除亚麻籽中生氰糖苷的相关研究报道。作者在系统调研有关亚麻籽脱毒文献的基础上，选择对亚麻籽生氰糖苷具有高效降解能力的人肝脏β-葡萄糖苷酶和对氰根具有水解吸收作用的氰化物降解细菌Bacillus sp. CN-22的氰化物水合酶，采用基因工程技术成功构建了能同时分泌表达Bacillus sp. CN-22菌的氰化物水合酶和人β-葡萄糖苷酶的毕赤酵母工程菌株；在此基础上诱导该工程菌表达氰化物水合酶和β-葡萄糖苷酶，获得可用于亚麻籽脱氰的酶制剂及发酵脱毒的最优化条件，并取得了如下主要研究成果：（1）在降氰细菌Bacillus sp. CN-22降解氰化钾溶液的过程中，通过分析甲酸盐、甲酰胺、氨气和氰根浓度的变化以及NADPH对氰化钾降解速率的影响，获知氰化物水合酶为该细菌的主要氰化物降解酶。（2）以Bacillus sp. CN-22基因组DNA为模板，采用聚合酶链式反应（PCR）技术成功扩增得到氰化物水合酶基因（Ch）；并以质粒pPIC9K为表达载体，成功构建出毕赤酵母分泌表达载体pPIC9K-Ch。（3）以人肝脏组织总RNA为模板，采用反转录-聚合酶链式反应（RT-PCR）技术，扩增获得人β-葡萄糖苷酶基因（Glu）；并以质粒pPIC9K为表达载体，构建得到毕赤酵母分泌表达载体pPIC9K-Glu。（4）将毕赤酵母胞外分泌型表达载体pPIC9K-Ch和pPIC9K-Glu依次转化毕赤酵母GS115和重组菌株GS115-Ch的感受态细胞，通过载体与基因组DNA同源重组，首次成功构建可同时体外分泌表达氰化物水合酶和人β-葡萄糖苷酶的毕赤酵母工程菌株GS115-Ch-Glu。（5）以甲醇诱导表达目的蛋白氰化物水合酶和β-葡萄糖苷酶；SDS-PAGE分析结果表明，毕赤酵母工程菌株GS115-Ch-Glu的最佳诱导表达条件为0.5%甲醇、30°C、200rpm诱导表达48h；在该条件下，氰化物水合酶和β-葡萄糖苷酶分别占上清液总蛋白含量的8.9%和12.5%。（6）采用响应面分析方法，以亚麻籽生氰糖苷降解率和氰根残留浓度为响应值，对亚麻籽发酵脱毒体系和发酵脱毒条件进行优化研究，结果表明在25.0g亚麻籽，1.27g酶制剂，8.0g灭菌水，MgCl₂和MnCl₂各50mg于250mL广口锥形瓶内充分混合的脱毒体系下，将起始pH值调至6.3，当温度为46.8°C时，在密封静置发酵体系中脱毒48h后，亚麻籽中的生氰糖苷降解率高达99.26%，氰根残留量可降至0.015mg g^-1。（7）采用高效液相色谱（HPLC）和气相色谱-质谱（GC-MS）分析技术对发酵脱毒前后的亚麻籽样品中木酚素和脂肪酸含量进行比较研究，结果表明发酵脱毒前后亚麻籽样品中木酚素和脂肪酸含量无显著差异；这进一步证明本文所建立的全生物发酵脱毒方法不仅能高效去除亚麻籽中的生氰糖苷，而且还能有效保护亚麻籽中的生物活性物质在脱毒过程中免遭破坏。更多还原

【Abstract】 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.更多还原