地衣芽孢杆菌KD-1β-甘露聚糖酶定点突变提高酶活性及稳定性

doi:10.13560/j.cnki.biotech.bull.1985.2021-0106

摘要/Abstract

摘要：

对地衣芽孢杆菌β-甘露聚糖酶进行定向突变获得酶活性和稳定性提高的酶。应用PCR方法对Bacillus licheniformis KD-1 β-甘露聚糖酶基因manBl进行基因克隆和定点突变,在枯草芽孢杆菌B. subtilis DB104中进行分泌表达,采用二硝基水杨酸（DNS）法进行酶活性测定。β-甘露聚糖酶ManBl及6个突变体最适pH 6.0,最适温度60℃,6个突变体比活力和pH 6.0-10.0 之间的pH稳定性均高于野生型,突变体ManBl（T112R/K291E）比活力是野生型的2.6倍,为（9 742±370.0）U/mg,K_m值为2.67 mg/mL,70℃的半衰期为80 min;C-末端带有His标签后,ManBl和ManBl（T112R/K291E）的酶活性和热稳定性降低。β-甘露聚糖突变体ManBl（T112R/K291E）酶活性和稳定性高,适合在养殖业、食品加工和洗涤业等多种工业领域中应用。

关键词: β-甘露聚糖酶, 地衣芽孢杆菌, 定点突变, 酶活性, 稳定性

Abstract:

The β-mannanase gene from Bacillus licheniformis strain KD-1 was directed mutated for improving its activity and stability. The β-mannanase gene manBl from B. licheniformis strain KD-1 was cloned and directed mutated by PCR. The gene and its mutants were expressed in B. subtilis DB104. The activities of the enzymes were measured by dinitrosalicylic acid,and DNS method. The β-mannanase ManBl and its 6 variants had the same optimal pH 6.0 and optimal temperature 60℃. All the variants had higher specific activity and pH stability between pH 6.0-10.0 than wild-type ManBl. The specific activity of the mutant ManBl（T112R/K291E）was（9 742±370.0）U/mg,which was 2.6 times of wild-type ManBl. The K_m value of ManBl（T112R/K291E）was 2.67 mg/mL. The half-life of ManBl（T112R/K291E）was 80 min at 70℃. The two enzymes with 6×His-tagged at the C terminal had lower specific activity and thermostability than the corresponding enzyme without His-tag. The β-mannanase mutant ManBl（T112R/K291E）shows potential application in feed,food and laundry industrial fields with its high specific activity,pH stability and thermostability.

Key words: β-mannanase, Bacillus licheniformis, directed mutation, enzyme activity, stability

田庚, 高伟强, 陈晓波, 张春晓. 地衣芽孢杆菌KD-1β-甘露聚糖酶定点突变提高酶活性及稳定性[J]. 生物技术通报, 2021, 37(10): 100-109.

TIAN Geng, GAO Wei-qiang, CHEN Xiao-bo, ZHANG Chun-xiao. Directed Mutagenesis of β-mannanase Gene from Bacillus licheniformis KD-1 for Improving Enzyme Activity and Stability[J]. Biotechnology Bulletin, 2021, 37(10): 100-109.

图/表 10

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质粒Plasmids	备注Notes	来源Source
pWB980		[20]
pWB-manBl	pWB980 with manBl gene	本研究This study
pWB-manBl（K291E）	pWB980 with manBl gene mutant K291E	本研究This study
pWB-manBl（T112R）	pWB980 with manBl gene mutant T112R	本研究This study
pWB-manBl（L211I）	pWB980 with manBl gene mutant L211I	本研究This study
pWB-manBl- T112R/K291E	pWB980 with manBl gene mutant T112R/K291E	本研究This study
pWB-manBl- L211I/ K291E	pWB980 with manBl gene mutant L211I/K291E	本研究This study
pWB-manBl- T112R/L211I	pWB980 with manBl gene mutant T112R/L211I	本研究This study
pWB-manBl-His	pWB980 with manBl gene fused with 6*His tagged at C terminal	本研究This study
pWB-manBl（T112R/K291E）-His	pWB980 with manBl（T112R/K291E）gene fused with 6*His tagged at C terminal	本研究This study

质粒Plasmids	备注Notes	来源Source
pWB980		[20]
pWB-manBl	pWB980 with manBl gene	本研究This study
pWB-manBl（K291E）	pWB980 with manBl gene mutant K291E	本研究This study
pWB-manBl（T112R）	pWB980 with manBl gene mutant T112R	本研究This study
pWB-manBl（L211I）	pWB980 with manBl gene mutant L211I	本研究This study
pWB-manBl- T112R/K291E	pWB980 with manBl gene mutant T112R/K291E	本研究This study
pWB-manBl- L211I/ K291E	pWB980 with manBl gene mutant L211I/K291E	本研究This study
pWB-manBl- T112R/L211I	pWB980 with manBl gene mutant T112R/L211I	本研究This study
pWB-manBl-His	pWB980 with manBl gene fused with 6*His tagged at C terminal	本研究This study
pWB-manBl（T112R/K291E）-His	pWB980 with manBl（T112R/K291E）gene fused with 6*His tagged at C terminal	本研究This study

菌株 Strain	备注Notes	来源Source
B. subtilis DB104	his,nprR2,nprE18,ΔaprA3	[21]
B. licheniformis KD-1		本研究This study
WB980	B. subtilis DB104（pWB980）,Kan^r	本研究This study
MTV13	B. subtilis DB104（pWB-manBl）,Kan^r	本研究This study
ABV3	B. subtilis DB104（pWB-manBl-K291E）,Kan^r	本研究This study
T2	B. subtilis DB104（pWB-manBl-T112R）,Kan^r	本研究This study
L4	B. subtilis DB104（pWB-manBl-L211I）,Kan^r	本研究This study
LK2	B. subtilis DB104（pWB-manBl-L211I /K291E）,Kan^r	本研究This study
TL1	B. subtilis DB104（pWB-manBl-T112R/L211I）,Kan^r	本研究This study
TK7	B. subtilis DB104（pWB-manBl-T112R/K291E）,Kan^r	本研究This study
A3	B. subtilis DB104（pWB-manBl-His6）,Kan^r	本研究This study
C5	B. subtilis DB104（pWB-manBl-T112R/L211I-His6）,Kan^r	本研究This study

菌株 Strain	备注Notes	来源Source
B. subtilis DB104	his,nprR2,nprE18,ΔaprA3	[21]
B. licheniformis KD-1		本研究This study
WB980	B. subtilis DB104（pWB980）,Kan^r	本研究This study
MTV13	B. subtilis DB104（pWB-manBl）,Kan^r	本研究This study
ABV3	B. subtilis DB104（pWB-manBl-K291E）,Kan^r	本研究This study
T2	B. subtilis DB104（pWB-manBl-T112R）,Kan^r	本研究This study
L4	B. subtilis DB104（pWB-manBl-L211I）,Kan^r	本研究This study
LK2	B. subtilis DB104（pWB-manBl-L211I /K291E）,Kan^r	本研究This study
TL1	B. subtilis DB104（pWB-manBl-T112R/L211I）,Kan^r	本研究This study
TK7	B. subtilis DB104（pWB-manBl-T112R/K291E）,Kan^r	本研究This study
A3	B. subtilis DB104（pWB-manBl-His6）,Kan^r	本研究This study
C5	B. subtilis DB104（pWB-manBl-T112R/L211I-His6）,Kan^r	本研究This study

引物名称 Primer name	引物序列Prime sequence (5'-3')
Bl man2-F1	gtgaaaaaaaRcatcgtttg
BL man2-R1	ttattccacRacaggcgtcaaag
BL man2-F2	CACACCGTTTCTCCGGTGAAC
manBT-2	GTAAGTCCCGTCTAGCCTTGCCCTTATTCCACAACAGGCGTCAAAG
amyT-F	TGTTGTGGAATAAGGGCAAGGCTAGACGGGACTTAC
amyT-R	CTAAATCGTGTCTTTCTTGGAACTTCCAGGGTATGTTTCTCTTTGATGTC
spmanB-F	AGGCGCAACTCAAGCTTTTGCCCACACCGTTTCTCCGGTGAAC
Pvf-1	ACATCAAAGAGAAACATACCCTGGAAGTTCCAAGAAAGACACGATTTAG
Pvf-2	GTTCACCGGAGAAACGGTGTGGGCAAAAGCTTGAGTTGCGCCT
R-manBl-F	GTTTCTCCGGTGAACCCGAATGCCCA
R-manBl-R	CGCCGTCCCATATCTCTCCTTTATTCA
V-manBl-F	TGAATAAAGGAGAGATATGGGACGGCG
V-manBl-R	TGGGCATTCGGGTTCACCGGAGAAAC
DR-manBl-R	GAAGTACGTCGTTTCCGGGTATTTTTCTTTG
DR-manBl-F	CAAAGAAAAATACCCGGAAACGACGTACTTC
BL-L211I-R	CGCATACACCCACAAGATATGATCCAGGCCTCT
BL-L211I-F	AGAGGCCTGGATCATATCTTGTGGGTGTATGCG
BL-T112R-R	ATAATGACCCGATCTAAACGCGGGGTTTGCGAG
BL-T112R-F	CTCGCAAACCCCGCGTTTAGATCGGGTCATTAT
V-His-F	CACCATCACCATCACCATTAAGGGCAAGGCTAGACGGGACTTAC
V-manBl-R	TGGGCATTCGGGTTCACCGGAGAAAC
R-manBl-F	GTTTCTCCGGTGAACCCGAATGCCCA
manBl-His-R	TTGCCCTTAATGGTGATGGTGATGGTGTTCCACAACAGGCGTCAAAG