定向进化提高来源于Arthrobacter ramosus 的MTHase的热稳定性

doi:10.13560/j.cnki.biotech.bull.1985.2020-0910

摘要/Abstract

摘要：

麦芽寡糖基海藻糖水解酶（maltosyltrehalose hydrolase,MTHase）是以淀粉或麦芽糊精为底物制备海藻糖的关键酶之一。来源于Arthrobacter ramosus的MTHase,表达量好,比活高,但热稳定性差,限制了其工业化应用。采用定向进化技术,筛选得到L137M和A216T两个突变体,在60℃下,野生型和两个突变体的半衰期（t_1/2）分别为15.5、20.5和23.3 min,L137M和A216T的t_1/2分别比野生型提高1.3和1.5倍。进一步考察了L137M、A216T和野生型酶在60℃分别和同样来源于Arthrobacter ramosus的麦芽寡糖基海藻糖合成酶（maltosyltrehalose synthase,MTSase）复配制备海藻糖,在相同的加酶量下,野生型、L137M和A216T的海藻糖转化率分别为51.3%,52.6%和55.7%,两个突变体的转化率都比野生型提高,说明突变体提高的热稳定性有利于海藻糖的转化率。

关键词: 热稳定性, 麦芽寡糖基海藻糖水解酶, 定向进化, Arthrobacter ramosus, 海藻糖

Abstract:

Maltosyltrehalose hydrolase（MTHase）is one of the key enzymes for trehalose production using starch or maltodextrin as the substrate. MTHase from Arthrobacter ramosus has a good-level expression and high specific activity,but poor thermostability,which limits its industrial application. In this study,mutants of L137M and A216T were screened using the directed evolution. The half-life（t_1/2,at 60℃）of wild-type,L137M and A216T were 15.5,20.5 and 23.3 min,respectively. The half-period t_1/2 of L137M and A216T were 1.3- and 1.5-fold than that of the wild type,respectively. The performances of L137M and A216T and wild-type enzymes were further investigated by their trehalose yields from MTSase at 60℃. Under the same amount of enzyme,the trehalose conversion rates by wild-type,L137M and A216T were 51.3%,52.6% and 55.7%,respectively. The trehalose conversion rates by the two mutants were higher than that by the wild-type,indicating that the improved thermostability of the mutants is beneficial for industrial trehalose production.

Key words: thermostability, maltosyltrehalose hydrolase, directed evolution, Arthrobacter ramosus, trehalose

陈春, 宿玲恰, 夏伟, 吴敬. 定向进化提高来源于Arthrobacter ramosus 的MTHase的热稳定性[J]. 生物技术通报, 2021, 37(3): 84-91.

CHEN Chun, SU Ling-qia, XIA Wei, WU Jing. Improved the Thermostability of MTHase from Arthrobacter ramosus by Directed Evolution[J]. Biotechnology Bulletin, 2021, 37(3): 84-91.

图/表 10

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MgCl₂/（mmol·L^-1）	MnCl₂/（mmol·L^-1）	核苷酸易错率/kb
2.5	0.1	0-4
	0.2	4-8
	0.3	7-10
5	0.1	8-11
	0.2	10-14
	0.3	11-15

MgCl₂/（mmol·L^-1）	MnCl₂/（mmol·L^-1）	核苷酸易错率/kb
2.5	0.1	0-4
	0.2	4-8
	0.3	7-10
5	0.1	8-11
	0.2	10-14
	0.3	11-15

纯化步骤		总蛋白含量/mg	总酶活/U	比活力/ （U· mg^-1）	纯化倍数	回收率/%
粗酶液	野生型	262	29 370	112.3	1.0	100
	L137M	248	27 914	112.6	1.0	100
	A216T	251	29 048	115.6	1.0	100
硫酸铵沉淀	野生型	56	19 972	359.4	3.2	68
	L137M	49	18 256	371.7	3.3	65
	A216T	52	19 171	369.9	3.2	66
MonoQ阴离子交换柱	野生型	15	8 635	572.8	5.1	29
	L137M	12	7 537	608.3	5.4	27
	A216T	14	9 005	647.3	5.6	31

纯化步骤		总蛋白含量/mg	总酶活/U	比活力/ （U· mg^-1）	纯化倍数	回收率/%
粗酶液	野生型	262	29 370	112.3	1.0	100
	L137M	248	27 914	112.6	1.0	100
	A216T	251	29 048	115.6	1.0	100
硫酸铵沉淀	野生型	56	19 972	359.4	3.2	68
	L137M	49	18 256	371.7	3.3	65
	A216T	52	19 171	369.9	3.2	66
MonoQ阴离子交换柱	野生型	15	8 635	572.8	5.1	29
	L137M	12	7 537	608.3	5.4	27
	A216T	14	9 005	647.3	5.6	31

	K_m /（mmol·L^-1）	k_cat（S^-1）	k_cat/K_m /（S^-1mmol·L^-1）
野生型	5.03±0.37	1 278±40	254±12
L137M	5.01±0.35	1 244±53	248±13
A216T	5.15±0.41	1 325±47	257±15