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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (3): 56-64    DOI: 10.13523/j.cb.20190308
技术与方法     
L-氨基酸脱氨酶的分子改造及其用于全细胞催化法生产α-酮戊二酸条件的优化 *
王越,李江华,堵国成,刘龙()
江南大学生物工程学院 无锡 214122
Molecular Modification of L-amino Acid Deaminase and Optimization of α-ketoglutaric Acid Production by Whole-cell Biocatalysis
Yue WANG,Jiang-hua LI,Guo-cheng DU,Long LIU()
Key Laboratory of Carbohydrate Chemistry and Biotechnology Ministry of Education Jiangnan University, Wuxi 214122, China
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摘要：

α-酮戊二酸(α-ketoglutaric acid,α-KG)是谷氨酸脱氨基的酮酸产物,作为一种重要的有机酸广泛用于食品、医药、精细化工等领域。为提高L-氨基酸脱氨酶全细胞催化法合成α-KG的效率及产量,首先通过优化全细胞催化剂制备条件及全细胞转化反应条件,包括发酵过程中的温度、诱导剂浓度、诱导剂添加时刻、诱导时间等;全细胞转化过程中的温度、pH、细胞量、转化时间。各个条件优化后以200g/L谷氨酸钠为底物时,产量最终提高了54.9%,摩尔转化率为39.6%。其次,通过定点饱和突变对L-氨基酸脱氨酶进行定向进化以提高其催化能力。经过多次突变、筛选,最优突变体E.coli BL21-pET-20b(+)-pm1152催化200g/L谷氨酸钠生成α-KG最高产量为100.9g/L,摩尔转化率为64.7%,较最初对照菌株提高了66.3%。结果表明,条件优化和饱和突变可有效提高重组大肠杆菌全细胞转化合成α-KG的能力。
关键词： L-氨基酸脱氨酶;  条件优化;  定点饱和突变;  全细胞催化    
Abstract:

Alpha-ketoglutaric acid (α-KG), which is a keto acid product deaminated by glutamic acid, is widely used in food, medicine, fine chemicals and other fields as an important organic acid. To improve the yeild and the efficiency of biotransformation for the synthesis of α-ketoglutaric acid. First, by optimizing the conditions of whole-cell biocatalyst preparation and whole-cell biocatalysis conditions. Optimization conditions include the temperature, pH, inducer concentration, induction time in the whole-cell biocatalyst preparation process and the temperature, pH, biocatalyst concentration, biocatalytic time in the whole-cell biocatalysis process. Determine the optimal conditions of each item by detecting the amount of product α-KG. After the conditions were optimized, the maximum yield was increased by 54.9 % and the molar conversion was 39.6 %. Secondly, the directed evolution of L-amino acid deaminase by site-directed mutagenesis increased its catalytic ability. Through multiple mutations, screening, the yield of α-ketoglutaric acid biocatalytic synthesized by monosodium glutamate with optimal mutant E.coli BL21-pET-20b (+)-pm1152 was 100.9 g/L, and the molar conversion rate was 64.7 %, an increase of 66.3% compared to the control strain. The maximal yield and molar conversion of L-glutamic acid to α-KG was reached under the following optimal conditions: 20 g/L whole-cell biocatalyst, 30 ℃, pH 6.0, and 60-h biocatalysis, strain: E.coli BL21-pET-20b (+)-pm1152. The results showed that the conditional optimization and saturation mutation could effectively increase the whole-cell biocatalyst of recombinant E. coli to synthesize α-ketoglutaric acid.
Key words: L-amino    acid    deaminase    Condition    optimization    Site-saturation    mutagenesis    Whole-cell    biocatalyst
收稿日期: 2018-09-06 出版日期: 2019-04-12
ZTFLH:  Q819  
基金资助: * 国家自然科学基金优秀青年基金(31622001);江苏省重点研发计划-社会发展资助项目(BE2016638)
通讯作者: 刘龙     E-mail: longliu@jiangnan.edu.cn
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引用本文:

王越,李江华,堵国成,刘龙. L-氨基酸脱氨酶的分子改造及其用于全细胞催化法生产α-酮戊二酸条件的优化 *[J]. 中国生物工程杂志, 2019, 39(3): 56-64.

Yue WANG,Jiang-hua LI,Guo-cheng DU,Long LIU. Molecular Modification of L-amino Acid Deaminase and Optimization of α-ketoglutaric Acid Production by Whole-cell Biocatalysis. China Biotechnology, 2019, 39(3): 56-64.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190308    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I3/56

引物名称 碱基序列(5'-3')
I100-F NNNATTAGTTACCAAACATCACCAGAAATCTTCCC
I100-R TTGGCTGTATGCACGGCCTGATTG
G206-F NNNACACCTGCACTCGCTCGTTATG
G206-R TGTTTCAGGATCAACAGAGCCTGAAT
G235-F NNNAAAATCTCTGATGTGGTGAGTGAG
G235-R ACCCGCAGTTTCAATACCTCTTAC
P272-F NNNACGCTCAATGTATATCTATCACAACAACGT
P272-R GATATCAATACCCATATTGCCCATAAATAAACGTGAC
V276-F NNNTATCTATCACAACAACGTGTCTCAGGG
V276-R ATTGAGCGTTGGGATATCAATACCCATATTGC
L278-F NNNTCACAACAACGTGTCTCAGGG
L278-R ATATACATTGAGCGTTGGGATATCAATACCC
V283-F NNNTCAGGGGTTCCTGGTGCACCAC
V283-R ACGTTGTTGTGATAGATATACATTGAGCGTTG
E340-F NNNTTACCGTTGGAATTCTCTATTGGTG
E340-R TCCGCCACCTAATAAGTGCATAAATTTAGG
L363-F NNNGATGAAAAAACACCATTCGAACAATTCCG
L363-R ATTCCAAGAGGTCGGCATTTTAAATG
N416-F NNNGAATTACCTATCATTTCTGAGGTCAAAGAATAC
N416-R AAATGTTGGACTCACAACGGCACCCCAAC
表1  定点饱和突变引物
图1  诱导温度优化
图2  诱导剂添加时刻优化及培养结束时菌体OD600值
图3  诱导时间优化
图4  IPTG浓度优化及培养结束时菌体OD600值
图5  种龄对催化合成α-酮戊二酸的影响
图6  温度对催化合成α-酮戊二酸的影响
图7  pH对催化合成α-酮戊二酸的影响
图8  转化时间对催化合成α-酮戊二酸的影响
图9  细胞量对催化合成α-酮戊二酸的影响
图10  L-氨基酸脱氨酶与L-谷氨酸对接结果
图11  定点突变正向结果突变体
图12  复合突变正向结果
图13  突变后催化条件对α-酮戊二酸的影响
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