【作者】 王丽娟；

【导师】 张充；

【作者基本信息】 南京农业大学 ， 食品科学与工程， 2023， 硕士

【摘要】 蛋白质谷氨酰胺酶（protein-glutaminase,酶学名EC 3.5.1.44,简称PG）是一种特异性作用于蛋白质谷氨酰胺基团,将其水解为谷氨酸的新型蛋白质改性工具。因其底物特异性、温和的反应条件与无毒无害的高安全性,已在多种植物蛋白中展现出良好的脱酰胺效果,应用前景十分广泛。目前的PG生产主要通过微生物发酵法获得,然而野生型PG生产菌株解朊金黄杆菌（Chryseobacterium proteolyticum）目前的产量仍然无法满足大规模工业化生产,因此亟需通过各种方法提升菌株的产酶水平。本研究以实验室保存的一株C.proteolyticum 1003作为出发菌株,建立物理化学复合诱变方法、抗性推理选育,获得一株高产突变菌株;外源筛选促进产酶的表面活性剂添加至发酵培养基并初步探究其作用机理;最后采用正交实验、响应面优化、神经网络耦合遗传算法等不同的组合优化方法,进一步提高产酶水平。本论文的主要研究内容及结果如下:1.复合诱变育种及丙二酸抗性推理选育将实验室保存的C.proteolyticum 1003作为出发菌株,探究不同处理时间的ARTP诱变与不同浓度的氯化锂添加量对菌株的影响,发现ARTP处理时间设定为30 s时菌株的正突变率最高,达到78.80%,氯化锂添加量为0.3%时菌株致死率在50%左右。由此确定复合诱变参数,开展菌株的诱变实验。从3420株突变菌株中筛选到一株PG产量为2.34 U/m L,较出发菌株提高20.1%的突变菌株WG12。通过对比C.proteolyticum 1003与WG12的生长产酶曲线,发现菌株产酶水平与其生长状况密切相关,因此选择对菌株生长有抑制效果的丙二酸加入固体平板,确定其临界浓度为5%,对突变菌株进行抗性压力筛选。在1620株单菌落中获得一株稳定遗传的高产菌株WG15,其PG产量较WG12增加0.362 U/m L,测定发现其PG基因编码序列未发生变化,但在大量产酶期胞内ATP含量提高。2.表面活性剂对WG15产酶影响表面活性剂具有增溶、乳化等良好的应用效果,近年来发现在一些微生物发酵中添加表面活性剂能够起到影响代谢产物分泌的作用。本研究在七种不同种类的表面活性剂中发现浓度为0.02%的茶皂素与0.05%的三乙醇胺分别能够使产酶量提升18.83%与9.96%,考察不同添加时间与二者协同处理后发现,在配制培养基时加入0.02%的茶皂素作用效果最佳,酶活产量提升至3.47 U/m L。随后通过实验初步探究茶皂素提升菌株产酶的作用机理,发现茶皂素的加入能够促进WG15的生长,从而使菌体在稳定期时能够产生更多的PG,菌株加入茶皂素后PG基因的转录水平也显著提高,使后续PG蛋白合成途径增强。另外细胞膜通透性的增加也促进了PG的胞外分泌。3.高产菌株发酵培养基组分的优化为进一步提升菌株WG15的产酶能力,首先通过单因素实验确定各组分添加量为:多聚蛋白胨1.5%、无水乳糖0.5%、Na H₂PO₄·2H₂O 20 mmol/L、KH₂PO₄20 mmol/L、Mg SO₄·7H₂O 10 mmol/L、茶皂素0.02%。后选取对菌株产酶影响较大的三种培养基组分Na H₂PO₄·2H₂O、KH₂PO₄与茶皂素,分别使用正交实验与响应面实验优化三者浓度,发现响应面优化效果更佳,将响应面模型作为构建BP神经网络的数据来源,不断进行训练,并结合遗传算法预测最佳配方,最终得到最优组合为Na H₂PO₄·2H₂O 17.311mmol/L,KH₂PO₄18.686 mmol/L,茶皂素0.015%。在此条件下的WG15产酶可达3.97U/m L。本研究利用复合诱变与抗性选育成功获得高产突变菌株,并且通过外源表面活性剂的添加与培养基优化,进一步提升发酵产酶水平,从出发菌株C.proteolyticum 1003的1.92 U/m L提升至3.97 U/m L,为推动PG的大规模生产提供一定技术借鉴。更多还原

【Abstract】 Protein-glutaminase（EC 3.5.1.44）is a novel protein modification tool that specifically acts on the protein glutamine group to hydrolyze it into glutamic acid.Because of its substrate specificity,mild reaction conditions and non-toxic and harmless high safety,it has shown good deamidation effect in a variety of plant proteins,and has a very wide application prospect.Currently,PG production is mainly obtained by microbial fermentation,but the current yield of wild-type PG-producing strains（Chryseobacterium proteolyticum）is still not sufficient for large-scale industrial production,so there is an urgent need to enhance the PG production level of the strains through various methods.In this study,a PG-producing C.proteolyticum 1003 preserved in our laboratory was used as the starting strain,and a high-yielding mutant strain was obtained by establishing a physicochemical combination mutagenesis method and resistance inference selection.Exogenous screening surfactants were added to the fermentation medium to further improve the level of enzyme production and preliminarily explore its mechanism of action.Finally,different combinations of optimization methods such as orthogonal experiments,response surface optimization and neural network coupled with genetic algorithm were used to improve the enzyme production level.The main findings and results of this paper are as follows:1.Compound mutation breeding and malonic acid resistance reasoning breeding.The effect of ARTP mutagenesis at different treatment time and different concentration of lithium chloride addition on the strain was investigated by using the laboratory preserved C.proteolyticum 1003 as the starting strain.It was found that the positive mutation rate of the strain was the highest when the ARTP treatment time was set at 30 s,reaching 78.80%,and the lethal rate of the strain was about 50%when the lithium chloride addition was 0.3%.The compound mutation parameters were determined and the mutation experiment was carried out.A mutant strain WG12 with a PG yield of 2.34 U/m L was screened from 3420 mutant strains,which was 20.1%higher than the original strain.By comparing the growth and enzyme production curves of C.proteolyticum 1003 and WG12,it was found that the enzyme production level of the strain was closely related to its growth status.Therefore,malonic acid,which has an inhibitory effect on the growth of the strain,was chosen to be added to the solid plates and a critical concentration of 5%was determined to screen the mutant strain for resistance pressure.A stable genetic high yielding strain WG15 was obtained from 1620 single colonies,with an increase in PG production of 0.362 U/m L compared to WG12.The PG gene coding sequence was determined to be unchanged,but the intracellular ATP content increased during the period of high enzyme production.2.Effects of surfactants on WG15 enzyme production.Surfactants have good application effects such as solubilization and emulsification.In recent years,it has been found that adding surfactants in some microbial fermentation can affect the secretion of metabolites.In this study,among seven different types of surfactants,it was found that 0.02%tea saponin and 0.05%triethanolamine could increase enzyme production by 18.83%and 9.96%,respectively.After investigating different adding time and cooperative treatment of the two,it was found that adding 0.02%tea saponin in the preparation of medium had the best effect,and the PG yield increased to 3.47 U/m L.Subsequently,the mechanism of tea saponin promoting enzyme production was preliminarily explored through experiments,and it was found that the addition of tea saponin could promote the growth of WG15,so that the bacteria could produce more PG at the stable stage.After addition of tea saponin,the PG gene transcription level was also significantly improved,which enhanced the subsequent PG protein synthesis pathway.In addition,the increase of membrane permeability also promoted the extracellular secretion of PG.3.Optimization of fermentation medium components for high-yield strains.In order to further improve the PG producing ability of strain WG15,the additive amount of each component was determined by single factor experiment as follows:Peptone1.5%,anhydrous lactose 0.5%,Na H₂PO₄·2H₂O 20 mmol/L,KH₂PO₄20 mmol/L,Mg SO₄·7H₂O 10 mmol/L,tea saponin 0.02%.After that,three trace elements Na H₂PO₄·2H₂O,KH₂PO₄and tea saponin,which had a great influence on the enzyme production of the strain,were selected to optimize the concentrations by orthogonal experiment and response surface experiment,respectively.It was found that the response surface optimization effect was better.The response surface model was used as the data source of BP neural network construction,continuous training was conducted,and the optimal formula was predicted by combining genetic algorithm.The optimal combination was Na H₂PO₄·2H₂O 17.311 mmol/L,KH₂PO₄18.686 mmol/L and tea saponin 0.015%.Under these conditions,the PG production of WG15 can reach 3.97 U/m L.In this study,a high-yield mutant strain was successfully obtained by compound mutagenesis and resistance breeding.Through the addition of exogenous surfactants and the optimization of culture medium,the fermentation enzyme production level was further improved,from 1.92 U/m L of the starting strain 1003 to 3.97 U/m L,providing certain support for the large-scale production of PG.更多还原