CN116463324B - 一种提高蛋白质谷氨酰胺酶生产酶活的方法 - Google Patents
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Abstract
本发明提供了提高蛋白质谷氨酰胺酶（PG）生产酶活的方法。该方法包括种子培养基配方：含鱼粉蛋白胨1.5%~2%，酵母浸膏1.5~2%，氯化钠0.5~1%，葡萄糖0.2~0.3%；发酵培养基配方：鱼粉蛋白胨1.5~2.5%，酵母浸膏0.5‑1.5%，碳酸钠0.05~0.1%，氯化镁0.002~0.004%，硫酸铁0.0003~0.0005%，乳糖0.2~0.4%；以及一种发酵工艺方法。本发明通过发酵后期添加甘氨酸和食品级吐温‑80，可将PG的产量提高244%，有利于PG的进一步工业化应用。
Description
技术领域
本发明涉及生物技术，具体涉及一种工艺优化方法，尤其涉及一种提高蛋白质谷氨酰胺酶生产酶活的方法。
背景技术
当今社会人们生活水平日益提高，比以往任何时候都要更加注重饮食安全和健康。作为关键的营养元素之一，蛋白质一直以来是食品领域的关注对象。其中，植物源蛋白来源丰富、相对廉价、环境友好且几乎不含饱和脂肪酸或胆固醇，对人们的健康比较有益，而成为食品工业的研究热点。当下，市场上已开发利用的植物蛋白主要包括大豆蛋白、小麦蛋白、玉米蛋白、大米蛋白和燕麦蛋白等，但这些蛋白水溶性都比较差，导致其乳化性、起泡性及凝胶性质都比较差，这大大影响了其在食品领域的应用。因此，提高蛋白质的溶解性至关重要。
蛋白质谷氨酰胺酶（EC 3.5.1.44，Protein-glutaminase，PG）是由日本学者Yamaguchi S等在解朊金黄杆菌（Chryseobacterium proteolyticum）中首次发现的（Yamaguchi S, Yokoe M. A novel protein-deamidating enzyme fromChryseobacterium proteolyticum sp. nov., a newly isolated bacterium from soil[J]. Applied and Environmental Microbiology, 2000, 66(8): 3337-3343.），可特异性水解短肽或蛋白质侧链的谷氨酰胺，并生成蛋白质L-谷氨酸盐和氨，是一种新型蛋白质脱酰胺酶。它专一性强，仅仅催化蛋白质侧链中的谷氨酰胺基团脱氨，不影响游离的谷氨酰胺，是一种比较安全有效的蛋白质改性工具酶。PG作用在植物蛋白上，可显著提高植物蛋白的溶解性、起泡性和乳化性等诸多性能，提高植物蛋白在食品领域的应用潜力。
根据报道，目前能自然生产PG的菌株主要为金黄杆菌属，包括解朊金黄杆菌、产吲哚金黄杆菌(Chryseobacterium indologenes)和黏金黄杆菌(Chryseobacterium gleum)等。我国已将解朊金黄杆菌来源的PG批准为“三新食品”，该产品的开发潜力巨大。然而，解朊金黄杆菌中PG的表达水平非常低，在原来的培养工艺及培养条件下，酶活仅为0.258 U/mL，这提高了后期产品处理的难度及生产成本，限制了其应用。
针对解朊金黄杆菌中PG酶的生产优化，目前主要通过两种途径实现：菌种优化及培养工艺优化。首先是菌种选育及优化，比如通过筛选新型解朊金黄杆菌，发酵酶活可以达到2.15 U/mL（专利公开号：CN114149934A）；再如从土壤中筛选出新的PG生产菌，其中最高产量达到0.65 U/mL，通过诱变及原生质体融合育种技术，将酶活提高到了1.14 U/mL（严文娟. 应用原生质体融合技术选育蛋白质谷氨酰胺酶高产菌株[D]. 华东师范大学.）。其次，选择适当的培养基或培养工艺，通常是生产优化的重要手段。据现有报道，有诸多培养优化方法可以提升PG的发酵酶活：通过在培养基中加入沸石作为pH稳定剂，可以将解朊金黄杆菌的PG发酵酶活提高到2.0 U/mL（专利公开号：CN107325977A）；通过在培养基中添加高分子聚合物PEG2000的方法，可将PG酶活提高60%~80%（专利公开号CN114317355A）；通过添加L-亮氨酸并优化培养基组分，可将PG的发酵酶活提高87%（Zhang K, Lyu Y, Zhang L, etal. Enhanced protein-glutaminase production from Chryseobacteriumproteolyticum by the addition of leucine[J]. Process Biochemistry, 2023, 130:401-408.）。以上技术方法均可大大提高PG发酵生产水平，但目前报道的解朊金黄杆菌生产酶活为5.43 U/mL（专利公开号：CN114317355A），仍有较大提升空间。
发明内容
针对以上问题，本发明提供了一种能够提高解朊金黄杆菌来源的PG产量的方法。
本发明的技术方案：
一种能够提高解朊金黄杆菌来源的PG产量的方法，该方法的步骤如下：接种解朊金黄杆菌于种子培养基中，置于33~36℃恒温摇床，180~230 r/min振荡培养14~16 h后，即得种子液。然后取新鲜的种子液，接种到装有发酵培养基的发酵罐中进行发酵培养。
优选地，所述的种子培养基，其特征为，含鱼粉蛋白胨1.5%~2%，酵母浸膏1.5~2%，氯化钠0.5~1%，葡萄糖0.2~0.3%，pH调整为6.8。
优选地，所述的发酵培养基，其特征为，含鱼粉蛋白胨1.5~2.5%，酵母浸膏0.5-1.5%，碳酸钠0.05~0.1%，氯化镁0.002~0.004%，硫酸铁0.0003~0.0005%，乳糖0.2~0.4%，pH调整为6.8。
优选地，所述的发酵培养，其特征在于，接种量控制在4.5%-5.5%，发酵温度控制在33~36℃，搅拌桨转速为350~750 r/min，发酵初始通气量控制在0.5 vvm，发酵中后期控制在2.5 vvm。
进一步地，所述的发酵培养，其特征还在于，在发酵起始后24~48h期间，向培养基中添加氨基酸。
优选地，所述的氨基酸为甘氨酸0.1%-2%，或L-苏氨酸0.1~2%，或L-亮氨酸0.1~2%等。
进一步地，所述的发酵培养，其特征还在于，在发酵起始后24~48h期间，向培养基中添加食品级吐温-80 0.006~0.02%。
本发明的有益效果如下：
解朊金黄杆菌中PG的表达产量较低，这限制了其工业化应用。根据文献报道，碳酸钠有助于提高溶液中PG的稳定性及活力（Sakai K, Sato Y, Okada M, et al. Enhancedactivity and stability of protein-glutaminase by Hofmeister effects[J].Molecular Catalysis, 2022, 517: 112054.）。本发明创造性地将较高浓度的碳酸钠应用于PG的发酵生产培养基中，这可稳定发酵液的PG，有益于发酵过程中PG的积累，进而提升PG的产量。
甘氨酸成本相较L-亮氨酸等更为低廉，可弱化细胞壁，从而改变细胞通透性，在细菌感受态制备过程中，添加一定浓度的甘氨酸可提升电击效率（McDonald I R, Riley PW, Sharp R J, et al. Factors affecting the electroporation of Bacillussubtilis[J]. Journal of applied bacteriology, 1995, 79(2): 213-218. Kim YH,Han KS, Oh S, et al. Optimization of technical conditions for thetransformation of Lactobacillus acidophilus strains by electroporation[J].Journal of Applied Microbiology, 2005, 99(1): 167-174. Löfblom J, KronqvistN, Uhlén M, et al. Optimization of electroporation-mediated transformation:Staphylococcus carnosus as model organism[J]. Journal of AppliedMicrobiology, 2010, 102(3): 736-747. ）。同时，吐温-80也可以增加细胞流动性，本发明首次创新性地将甘氨酸结合吐温-80应用在发酵后期过程中，从而影响PG的分泌效率，提高PG的生产水平。
附图简述
附图1为实施例1发酵过程中的酶活曲线。
具体实施方式
以下实施例所用的试剂和生物材料如未特别说明，均为商业化产品。
| 试剂 | CAS号 | 购自 |
| 鱼粉蛋白胨 | 91079-42-4 | 斯泰博上海生物科技有限公司 |
| 酵母浸膏 | 8013-01-2 | 斯泰博上海生物科技有限公司 |
| 氯化钠 | 7647-14-5 | 上海国药集团化学试剂有限公司 |
| 葡萄糖 | 14431-43-7 | 上海国药集团化学试剂有限公司 |
| 乳糖 | 63-42-3 | 上海国药集团化学试剂有限公司 |
| 碳酸钠 | 497-19-8 | 上海国药集团化学试剂有限公司 |
| 硫酸铁 | 15244-10-7 | 上海国药集团化学试剂有限公司 |
| 氯化镁 | 7791-18-6 | 上海国药集团化学试剂有限公司 |
| 食品级吐温-80 | 9005-65-6 | 斯泰博上海生物科技有限公司 |
| 甘氨酸 | 56-40-6 | 上海国药集团化学试剂有限公司 |
| CBZ-Gln-Gly | 6610-42-0 | Sigma-Aldrich |
下面结合实施例对本发明的技术方案进行详细说明。显然，所描述的实施例是用于说明本发明的一部分实施例，而不应视为限制本发明的范围。
实施例
一种能够提高解朊金黄杆菌来源的PG产量的方法，包括如下步骤：
步骤一、准备种子
挑取经活化稳定培养的解朊金黄杆菌单菌落，接种到种子培养基中，种子培养基组分如下：鱼粉蛋白胨1.5%，酵母浸膏1.5%，氯化钠1%，葡萄糖0.2%，pH调整为6.8。置于恒温摇床中，30℃，220 rpm过夜培养。
步骤二、接种
将上述过夜培养的种子液，接种到装有发酵培养基的3 L发酵罐中，接种量为2%。发酵培养基组分如下：鱼粉蛋白胨2%，酵母浸膏0.5%，碳酸钠0.1%，氯化镁0.004%，硫酸铁0.0005%，乳糖0.2%，pH调整为6.8。
步骤三、发酵控制
设置发酵温度为33℃，搅拌桨转速为700 rpm，发酵初始通气量控制在0.5 vvm，待培养6 h后，调整通气量为2.0 vvm。设置恒速补料，在前36 h内，添加葡萄糖总量为3%。在发酵第36 h，停止补充葡萄糖，添加甘氨酸终浓度为0.1%，同时添加食品级吐温-80至终浓度为0.01%。发酵进行60 h停止，过程中每隔12 h或6 h取一次样，检测PG的酶活。
酶活测定参照Yamaguchi S等（Yamaguchi S, Yokoe M. A novel protein-deamidating enzyme from Chryseobacterium proteolyticum sp. nov., a newlyisolated bacterium from soil[J]. Applied and Environmental Microbiology,2000, 66(8): 3337-3343.）的方法，并作部分调整：
（1）配制显色剂 1： 7.5 mg 亚硝基铁氰化钠和2.023 g 苯酚，溶于双蒸水并定容至50 mL；显色剂 2：2.5 g 氢氧化钾溶于双蒸水并定容至50 mL；显色剂3： 10.2 g 无水碳酸钾溶于双蒸水，滴加417 μL次氯酸钠溶液，加水定容至50 mL。
（2）取发酵结束的发酵液1 mL，12000 r/min，4℃离心5 min，取上清稀释相应倍数，以备检测其中PG的酶活力。
（3）将部分上清稀释液置于95℃水浴锅处理5 min 作为对照组。取1 mL 二肽Cbz-Gln-Gly 溶液于37℃水浴锅预热10 min，加入100 μL上清稀释液混匀，然后置于37℃水浴锅反应30 min，然后迅速加入1 mL 0.4 M 三氯乙酸溶液终止反应。
（4）采用苯酚法测定反应后溶液中氨的含量。取60 μL 反应液与240 μL 蒸馏水、300 μL 显色剂1、150 μL 显色剂2 和300 μL 显色剂3，充分混匀，置于37℃水浴锅中孵育20 min，冷却，测量体系在630 nm处的光吸收值（氨的浓度标准曲线根据同样的方法进行测定）。根据氨的量计算上清液中酶的活力，定义每分钟水解Cbz-Gln-Gly产生1μmol氨所需的酶的量为一个酶活单位。
如附图1所示，根据上述方法测定发酵液上清的酶活，最高出现在48h，为18. 69U/mL。比现有报道的解朊金黄杆菌的生产酶活（5.43 U/mL）提高了244%。
上述仅为本发明的部分优选实施例，本发明并不仅限于实施例的内容。对于本领域中的技术人员来说，在本发明技术方案的构思范围内可以有各种变化和更改，所作的任何变化和更改，均在本发明保护范围之内。
Claims (1)
1.一种提高蛋白质谷氨酰胺酶生产酶活的方法，其特征在于：提供了种子培养基配方，发酵培养基配方以及相应的发酵培养工艺；
所述种子培养基，其组分如下：鱼粉蛋白胨1.5%~2%，酵母浸膏1.5~2%，氯化钠0.5~1%，葡萄糖0.2~0.3%，pH调整为6.8；
所述发酵培养基，其组分如下：鱼粉蛋白胨1.5~2.5%，酵母浸膏0.5-1.5%，碳酸钠0.05~0.1%，氯化镁0.002~0.004%，硫酸铁0.0003~0.0005%，乳糖0.2~0.4%，pH调整为6.8；
所述的发酵培养工艺，其特征在于：在发酵起始后24~48h期间，向培养基中添加甘氨酸至终浓度为0.1%-2%，食品级吐温-80 至终浓度为0.006~0.02%。
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| 蛋白质谷氨酰胺酶的重组表达与发酵条件优化;李静竹等;食品与发酵工业;20210215;第47卷(第3期);294-301 * |
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