Improved the Thermostability of MTHase from Arthrobacter ramosus by Directed Evolution

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

Abstract

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

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.

Figures/Tables 10

References 22

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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