CN117286127A - 天冬氨酸脱羧酶及利用酿酒废弃物制备β-丙氨酸的方法 - Google Patents
天冬氨酸脱羧酶及利用酿酒废弃物制备β-丙氨酸的方法 Download PDFInfo
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- CN117286127A CN117286127A CN202311205969.8A CN202311205969A CN117286127A CN 117286127 A CN117286127 A CN 117286127A CN 202311205969 A CN202311205969 A CN 202311205969A CN 117286127 A CN117286127 A CN 117286127A
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及天冬氨酸脱羧酶及利用酿酒废弃物制备β‑丙氨酸的方法,属于生物工程技术领域。所述天冬氨酸脱羧酶突变体由野生型天冬氨酸脱羧酶氨基酸序列的第5位亮氨酸突变为蛋氨酸、第85位缬氨酸突变为谷氨酸、第91位丙氨酸突变为脯氨酸、第122位苏氨酸突变为脯氨酸所得。通过基因工程的手段将含有所述天冬氨酸脱羧酶突变体基因的载体导入宿主中获得重组菌,使其在pH=7的环境下依旧保持很高的活性,获得的重组菌在pH=7左右环境下对酿酒废弃物进行发酵,实现催化L‑天冬氨酸脱羧合成β‑丙氨酸。本发明以酿酒废弃物为发酵原料,提高了资源化利用率,降低了环境污染,并且产生了极高的经济价值。
Description
技术领域
本发明属于生物技术领域,具体涉及一种天冬氨酸脱羧酶及利用酿酒废弃物制备β-丙氨酸的方法。
背景技术
天然的β-丙氨酸是由某些含有天冬氨酸脱羧酶的微生物,将天然的非必要氨基酸天冬氨酸脱羧产生的。β-丙氨酸被广泛用于食品领域,其可以作为调味剂和pH调节剂添加到食品中,还可用作食品中的功能成分。例如,β-丙氨酸已被证明具有抗疲劳和增强耐力的作用,被广泛应用于运动营养品中。
除用于食品工业外,β-丙氨酸还可用作畜禽的饲料添加剂。β-丙氨酸可作为合成生物活性肽的前体物,具有抗氧化、抗炎、降压等多种生理功能。哺乳动物肌肉中的肌肽合成酶以β-丙氨酸和组氨酸(半必要氨基酸)为底物,在ATP存在的情况下,合成的肌肽。有研究表明:肌肽具有很强的抗氧化能力,可清除在氧化应激过程中使细胞膜的脂肪酸过度氧化而形成的活性氧自由基(ROS)以及α-β不饱和醛,对人体有益;肌肽还具有良好的抗糖化作用,通过与糖反应的形式,代替体内蛋白质与糖反应,从而帮助保护蛋白质不被糖基化;肌肽能延长纤维母细胞再生的周期,使持续制造胶原蛋白与弹力蛋白,预防阿滋海默症,防止神经和脑部退化。因此,β-丙氨酸是优质且天然的肌肽补充剂前体氨基酸。它可以提高动物的生长性能、肉质、免疫功能以及提高猪、鸡、和鱼瘦肉中的肌肽含量,提高养殖业的品质和附加值。
谷氨酸棒杆菌是工业上发酵生产L-谷氨酸以及L-天冬氨酸(谷氨酸衍生物)最常见的菌株,其具有食品安全性、发酵产量高,易培养,可基因编辑,耐底物浓度高,耐盐浓度高等诸多特点。然而,天冬氨酸脱羧酶在pH=7的环境下活性较低,而谷氨酸棒杆菌的最适生长pH值在7左右。因此,亟需解决在pH=7条件下天冬氨酸脱羧酶活性低的问题,同时找到合适且低廉的原料进行发酵,降低成本以扩大其应用。
发明内容
鉴于此,本发明提供了一种天冬氨酸脱羧酶及利用酿酒废弃物制备β-丙氨酸的方法。
本发明采用以下技术方案:
本发明目的之一在于提供一种天冬氨酸脱羧酶突变体,所述天冬氨酸脱羧酶突变体由野生型天冬氨酸脱羧酶氨基酸序列的第5位亮氨酸突变为蛋氨酸、第85位缬氨酸突变为谷氨酸、第91位丙氨酸突变为脯氨酸、第122位苏氨酸突变为脯氨酸所得。
本发明目的之二在于提供一种编码上述天冬氨酸脱羧酶突变体的基因。
本发明目的之三在于提供一种含有上述基因的重组表达载体、重组菌。
本发明目的之四在于提供一种谷氨酸棒杆菌,包含有上述基因。
本发明目的之五在于提供上述天冬氨酸脱羧酶突变体在生产β-丙氨酸中的应用。
本发明目的之六在于提供一种利用酿酒废弃物生产β-丙氨酸的方法,包括如下步骤:
将酿酒废弃物进行酸水解反应,得水解液;
将上述谷氨酸棒杆菌接种至所述水解液中进行发酵,即可得到β-丙氨酸。
进一步地,所述酸水解反应条件:盐酸终浓度为6.0~7.0M,温度120~130℃,时间10~12h。
进一步地,蒸发去除所述水解液中盐酸,调节pH至6.8~7.5,调节所述水解液中天冬氨酸浓度为10~13g/L,葡萄糖浓度为20~60g/L。
在一些实施例中,优选地,蒸发温度50~80℃、蒸发时间2~8h,利用铵盐调节pH。
进一步地,所述发酵条件:pH=6.8~7.0、温度28~35℃、转速200~400r/min、溶氧量30~100%、时间48~72h,当测得发酵液中葡萄糖质量浓度低于10g/L时,添加浓度为60%的葡萄糖溶液。
进一步地,在得到β-丙氨酸前还需对发酵液进行纯化,所述纯化步骤如下:
将发酵液过滤,调节滤液pH至2.0~6.0,利用树脂进行吸附;
用浓度为4-6%的氨水对吸附后的树脂进行洗涤,将洗涤液浓缩、脱色、干燥,即得β-丙氨酸。
在一些实施例中,优选地,利用陶瓷膜过滤发酵液,吸附树脂为强酸性阳离子交换树脂,脱色选自活性炭、树脂中任一种。
与现有技术相比,本发明的有益效果为:
(1)本发明构建的天冬氨酸脱羧酶突变体在pH=7环境下依旧保持很高的活性,并且转化至谷氨酸棒杆菌中获得的重组菌在pH=7左右环境下对酿酒废弃物进行发酵,实现催化L-天冬氨酸脱羧合成β-丙氨酸。
(2)本发明以酿酒废弃物为发酵原料,提高了资源化利用率,降低了环境污染,产生了经济价值。
(3)本发明方案制备的β-丙氨酸经纯化猴纯度高达98.7%,产品实际得率为84.3%。
附图说明
图1为本发明方案技术流程图;
图2为本发明实施例1中蛋白突变位点图;
图3为本发明实施例1中蛋白突变位点3D模拟图;
图4为本发明实施例3中pXMJ19-KEADC-L5M,V85E,A91P,T122P质粒图;
图5为本发明实施例4中发酵时间和β-丙氨酸浓度曲线图。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明,以使本领域的技术人员更加清楚地理解本发明。
关键试验材料来源及理化参数:
模板pET22b-KEADC、表达载体pXMJ19均购自Novagen公司;
大豆浸出液是采购于江苏立滨生物工程有限公司。
酿酒废弃物来自贵州省遵义市鸭溪镇酒厂其主要组分如下表:
表1酿酒废弃物主要组分
组分 | 纤维素 | 蛋白质(多肽) | 还原糖 | 有机酸 | 氨基酸 |
含量(%) | 34.54% | 22.77% | 0.55% | 3.96% | 0.33% |
本发明中未对具体原料进行说明均为已经存在物质,可以从市面上直接购买得到。
实施例1
天冬氨酸脱羧酶关键氨基酸位点的确定
使用计算酶学软件进行模拟:来源于Kroppenstedtia eburnea的ADC(天冬氨酸脱羧酶)晶体结构为模板,运用Modeller 9.11程序(http://salilab.org/modeller/)进行三维结构的同源建模及优化,并模拟在不同pH环境下的结构、稳定性和活性变化。
构建天冬氨酸的三维结构,并经Chem3D Ultra 12.0模块的MMFF94分子力学对其进行能量最小化。以模拟后的底物及突变体的三维结构为受体,运用AutoDock 4.2(http://autodock.scripps.edu)程序,将天冬氨酸对接至酶活性中心的合适位置。采用PyMOL(http://pymol.org/)分析ADC及突变体的三维结构。
通过计算,确定L5,V85,A91,T122氨基酸是潜在的氨基酸位点。
实施例2
获得在pH=7环境下具有较高催化活性的天冬氨酸脱羧酶突变体酶活性检测:
采用McIlvaine缓冲体系0.2mol/L,pH4.7,含0.1mmol/L PLP,10mmol/L底物L-天冬氨酸钠,200μL底物溶液和100μL酶液在30℃反应后,稀释10倍,经0.22μm混合纤维树酯微孔滤膜过滤用Agilent 1100液相色谱仪定量测定其他氨基酸和生成的β-丙氨酸,一个酶活力单位(U)定义为在测定条件下每分钟产生1μmolβ-丙氨酸所需的酶量。
设计饱和突变引物:
根据筛选出的关键氨基酸位点,设计相应的饱和突变引物,具体如下:
L5M-F:GTACCGCACCATGATGAAGTCCAAGATC
L5M-R:CGGTGTCACCTGGCTGCACCAGG
V85E-F:AGTCCAAGATCCACCGCGCCACC
V85E-R:GTAGGAGATGATGATTTCGGTGTCACCT
A91P-F:CCACCGTGACCGAAGCAAACCTG
A91P-R:CTTCCTTGGTCATCATCGGGTAGGAGATG
T122P-F:GGTGCAGGTGGTGAACAACAACAACG
T122P-R:GTCACGCCGTGCGGTTCTTCGCC
构建饱和突变库:
以pET22b-KEADC为模板、利用上述引物,,采用全质粒PCR(Whole-plasmid PCR)技术分别对L5,V85,A91,T122密码子实施饱和突变。将pET22b-KEADC突变体用后转化E.coliBL21(DE3),构建突变转化子文库。
筛选高活性突变体:
从上述突变转化子文库中挑选97个转化子(E.coli/KEADC-L5X-V85X-A91X-T122X,X代表任意氨基酸)接种于1mL LB抗性(Chl氯霉素)培养基中,37℃、220r/min培养12h,以2%(体积比)接种量转接至1mL相同培养基,培养至OD600约为0.6-0.8时,加入0.4mmol/L IPTG、20℃诱导表达8h,4℃、8000r/min离心5min收集菌体,用Tris-HCl缓冲液(50mmol/L、pH=7.0)悬浮清洗菌体2次,加入1mg/mL溶菌酶溶液10μL后,反复冻融破碎细胞,4℃、12000r/min离心5min收集上清液,即为粗酶液。
根据上述酶活性检测方法,以催化β-丙氨酸产量为指标,筛选出活性高于KEADC的转化子,并对其进行DNA测序,确定优良突变转化子。
实验结果表明,最佳突变为L5M,V85E,A91P,T122P,蛋白位点如图2和图3所示。
通过以上实验方案,我们可以得到在pH=7环境下具有较高催化活性的天冬氨酸脱羧酶突变体KEADC-L5X,V85X,A91X,T122X。
实施例3
重组菌的构建
将实施例2中获得的天冬氨酸脱羧酶突变体KEADC-L5X,V85X,A91X,T122X利用无缝克隆到表达载体pXMJ19中,构建成重组质粒pXMJ19-KEADC-L5X,V85X,A91X,T122X,酶基因上下游的酶切位点分别为:BamH I和EcoR I,质粒图谱如图4所示。
将重组质粒pXMJ19-KEADC-L5X,V85X,A91X,T122X通过电转化至谷氨酸棒杆菌的感受态细胞中,将该重组细胞命名为CG-58591122。
验证谷氨酸棒杆菌CG-58591122的活性:
将上述谷氨酸棒杆菌CG-58591122在培养基中于30℃,200r/min连续培养24h,然后离心收集菌体,按照实施例2中酶活性检测方法验证谷氨酸棒杆菌CG-58591122活性。
其中,谷氨酸棒杆菌CG-58591122发酵培养基制备如下:120g/L葡萄糖、1g/LKH2PO4、0.4g/L MgSO4、10mg/L FeSO4、10mg/L MnSO4、40g/L(NH4)2SO4、10mL/L大豆浸出液、0.7g/L DL-甲硫氨酸、50μg/L生物素、300μg/L维生素B1、0.25g/L L-异亮氨酸,NaOH调pH为7.2,摇瓶发酵需预先加入4g/100mL CaCO3以中和发酵过程产酸,115℃灭菌15min。
实施例4
利用谷氨酸棒杆菌CG-58591122和将酿酒废弃物生产β-丙氨酸
酿酒废弃物强酸水解:
将酿酒废弃物加入适量的浓盐酸进行酸水解,以水解液中天冬氨酸和葡萄糖浓度不再增加为终点。经实验优化水解条件为:盐酸终浓度为6.0~7.0M,水解温度120~130℃,水解时间10~12h。
通过蒸发除掉盐酸,蒸发温度50~80℃,蒸发时间为2~8h,蒸发以后水解液中天冬氨酸浓度为10~13g/L,葡萄糖浓度20~60g/L;
用铵盐将水解液pH调至6.8~7.5,得到适宜微生物生长的酿酒废弃物水解液。
β-丙氨酸的合成:
将谷氨酸棒杆菌CG-58591122种子液接种至上述酿酒废弃物水解液中进行发酵,将发酵液纯化处理,即得β-丙氨酸。
菌种活化:将冻存于-70℃的甘油管保藏菌株于LB固体培养基上划线,28~35℃培养20~36h。
一级种子培养:用接种环挑取活化好的新鲜菌落,接种于装有50mL种子培养基的250mL三角瓶中,28-35℃、220r/min培养12~20h。经优化的种子液为:葡萄糖2~4%,硫酸铵0.5~1%,K2HPO4 0.1~0.2%,MgSO4·7H2O 0.01~0.05%,MnSO4 0.5~2mg/L,pH6.8~7.0。
二级种子液培养:培养好的一级种子液初始OD600为0.8~1.6,按3%~8%接种于装有500mL发酵培养基的2L三角瓶,28~35℃、220r/min培养8~16h。经优化的种子培养基为:葡萄糖2~4%,糖蜜0.3~1%,硫酸铵0.5~1%,K2HPO4 0.1~0.2%,MgSO4·7H2O 0.01~0.05%,MnSO40.5~2mg/L,pH6.8~7.0。
发酵罐发酵酿酒废弃物水解液:将20L水解处理后的酿酒废弃物水解液置于50L液体发酵罐中,灭菌后将温度降至28~35℃,pH控制在6.8~7.0,按3~8%的接种量将二级种子液接入发酵液中,种子液OD600值控制在0.8~1.6;发酵过程中,通过流加50%的氨水或者醋酸的方式自动控制发酵培养基pH为6.8-7.0,控制温度为28~35℃,通过发酵罐搅拌转速(200~400r/min)和通气量(1.5vvm)的偶联控制溶氧水平保持30~100%;当测得发酵液中葡萄糖质量浓度低于10g/L时,流加质量浓度为60%的葡萄糖溶液,以保证菌株生长和产β-丙氨酸所必需的葡萄糖供应,发酵培养48~72h,最终产物β-丙氨酸浓度为12~15g/L。
产物纯化:
待发酵结束后,将发酵液用陶瓷膜过滤除去菌体及大分子蛋白;将除去菌体的发酵液pH调至2~6,以一定的流速通过强酸性阳离子交换树脂,β-丙氨酸被交换至树脂上,发酵液中的色素、可溶蛋白及阴离子随废液被洗出;用4~6%的氨液将β-丙氨酸从树脂上洗出,将洗出液浓缩后用活性炭或者树脂脱色,并干燥得到产品。经过HPLC分析鉴定,产品的纯度为98.7%。产品纯化实际得率为84.3%。
对比例1
本对比例提供一种利用野生的Kroppenstedtia eburnea的ADC(天冬氨酸脱羧酶)构建的谷氨酸棒杆菌催化酿酒废弃物生产β-丙氨酸的方法,其构建谷氨酸棒杆菌的步骤与实施例3基本相同,利用酿酒废弃物生产β-丙氨酸和实施例4基本相同,区别在于:将谷氨酸棒杆菌CG-58591122种子液用Kroppenstedtia eburnea的ADC(天冬氨酸脱羧酶)基因构建的谷氨酸棒杆菌种子液替代。
实验证明野生型的ADC(天冬氨酸脱羧酶)构建到谷氨酸棒杆菌中后,在pH=7左右环境下,重组菌可以正常生长,但该酶活性完全丧失,不具备在中性pH环境下催化天冬氨酸合成β-丙氨酸的能力。但该野生ADC酶在pH=5左右,可以实现催化该反应,只是其宿主无法在弱酸环境下生长。因此,野生ADC酶的催化环境与宿主的生长环境不匹配,无法实现发酵酿酒废弃物水解液生产β-丙氨酸的工艺。
以上仅为本发明的较佳实施方案,并非用于限定本发明的保护范围。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种天冬氨酸脱羧酶突变体,其特征在于,所述天冬氨酸脱羧酶突变体由野生型天冬氨酸脱羧酶氨基酸序列的第5位亮氨酸突变为蛋氨酸、第85位缬氨酸突变为谷氨酸、第91位丙氨酸突变为脯氨酸、第122位苏氨酸突变为脯氨酸所得。
2.编码权利要求1所述天冬氨酸脱羧酶突变体的基因。
3.一种含有权利要求2所述基因的重组表达载体、重组菌。
4.一种谷氨酸棒杆菌,其特征在于,包含有权利要求2所述基因。
5.权利要求1所述天冬氨酸脱羧酶突变体在生产β-丙氨酸中的应用。
6.一种利用酿酒废弃物生产β-丙氨酸的方法,其特征在于,包括如下步骤:
将酿酒废弃物进行酸水解反应,得水解液;
将权利要求4所述谷氨酸棒杆菌接种至所述水解液中进行发酵,即可得到β-丙氨酸。
7.根据权利要求6所述的方法,其特征在于,所述酸水解反应条件:盐酸终浓度为6.0~7.0M,温度120~130℃,时间10~12h。
8.根据权利要求6所述的方法,其特征在于,蒸发去除所述水解液中盐酸,调节pH至6.8~7.5,调节所述水解液中天冬氨酸浓度为10~13g/L,葡萄糖浓度为20~60g/L。
9.根据权利要求6所述的方法,其特征在于,所述发酵条件:pH=6.8~7.0、温度28~35℃、转速200~400r/min、溶氧量30~100%、时间48~72h,当测得发酵液中葡萄糖质量浓度低于10g/L时,添加浓度为60%的葡萄糖溶液。
10.根据权利要求6所述的方法,其特征在于,在得到β-丙氨酸前还需对发酵液进行纯化,所述纯化步骤如下:
将发酵液过滤,调节滤液pH至2.0~6.0,利用树脂进行吸附;
用浓度为4-6%的氨水对吸附后的树脂进行洗涤,将洗涤液浓缩、脱色、干燥,即得β-丙氨酸。
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