重组耐热β-葡萄糖苷酶的固定化及其对黄酮糖苷的定向转化

doi:10.3969/j.issn.1673-5854.2021.03.002

生物质化学工程 ›› 2021, Vol. 55 ›› Issue (3): 10-16.doi: 10.3969/j.issn.1673-5854.2021.03.002

重组耐热β-葡萄糖苷酶的固定化及其对黄酮糖苷的定向转化

解静聪, 徐浩, 张宁(), 杨静, 赵剑

中国林业科学研究院林产化学工业研究所; 生物质化学利用国家工程实验室; 国家林业和草原局林产化学工程重点实验室; 江苏省生物质能源与材料重点实验室, 江苏南京 210042

收稿日期:2020-06-16 出版日期:2021-05-30 发布日期:2021-05-20
通讯作者: 张宁 E-mail:zhangning@icifp.cn
作者简介:张宁, 副研究员, 博士, 硕士生导师, 从事生物质能源研究; E-mail: zhangning@icifp.cn
解静聪(1988-), 男, 浙江嘉兴人, 助理研究员, 博士, 主要从事生物质资源利用及酶工程研究
基金资助:
国家重点研发计划资助项目(2017YFD0601001)

Immobilization of Thermostable Recombinant β-Glucosidase and Its Application in Transformation of Flavonol Glycosides

Jingcong XIE, Hao XU, Ning ZHANG(), Jing YANG, Jian ZHAO

Institute of Chemical Industry of Forest Products, CAF; National Engineering Lab. for Biomass Chemical Utilization; Key Lab. of Chemical Engineering of Forest Products, National Forestry and Grassland Administration; Key Lab. of Biomass Energy and Material, Jiangsu Province, Nanjing 210042, China

Received:2020-06-16 Online:2021-05-30 Published:2021-05-20
Contact: Ning ZHANG E-mail:zhangning@icifp.cn

摘要/Abstract

摘要：

为提高黄酮糖苷类化合物酶法转化的效率，降低酶使用成本，对重组耐热β-葡萄糖苷酶的固定化条件进行了研究，比较了固定化酶和游离酶的酶学性质差异，同时研究了固定化酶转化3种黄酮糖苷的时效曲线及转化效率。结果表明：重组耐热β-葡萄糖苷酶的固定化最优条件为在pH值6.0，室温条件下，酶活力2 U/mL，海藻酸钠质量分数1.5%，氯化钙质量分数2.5%，戊二醛质量分数1.25%，颗粒硬化时间2.5 h，此条件下固定化酶颗粒的酶活力为0.64 U/mg，酶固定化率可达62.5%。固定化酶较游离酶具有更优的酸碱稳定性和热稳定性，且重复使用20次后仍具有55%以上的残余酶活力。固定化酶能够高效定向转化异槲皮素、大豆苷以及淫羊藿次苷I等3种黄酮糖苷，生成活性更强的黄酮苷元槲皮素、大豆苷元和淫羊藿素，其摩尔转化率分别为99.37%、97.21%和97.93%，产率分别为0.776、1.091和0.714 g/（L·h）。

关键词: 黄酮糖苷, 耐热β-葡萄糖苷酶, 固定化

Abstract:

In order to increase the efficiency of the enzymatic conversion of flavonol glycosides and reduce the cost of the enzyme, the immobilization conditions of recombinant thermostable β-glucosidase were investigated. The differences of enzymatic properties between immobilized enzyme and free enzyme were compared, and the conversion curves and conversion efficiency of the immobilized enzyme on transforming three kinds of flavonol glycosides were obtained. The results showed that the optimal immobilization conditions were sodium alginate 1.5%, calcium chloride 2%, glutaraldehyde 1.25%, and pellets solidifying time 2.5 h under room temperature and pH 6.0. Under these conditions, enzyme activity was 0.64 U/mg, and the immobilization rate was 62.5%. The pH stability and temperature stability of the immobilized β-glucosidase were better than those of the free β-glucosidase and it retained more than 55% residual activity after reusing for 20 times. The isoquercetin, daidzin, and icariside I were transformed into their aglycones with high efficiency though immobilized β-glucosidase and the conversion rates were 99.37%, 97.21% and 97.93%, respectively, and the yields of quercetin, daidzein and icariin were 0.776, 1.091 and 0.714 g/(L·h), respectively.

Key words: flavonol glycosides, thermostable β-glucosidase, immobilization of enzyme

中图分类号:

TQ35

解静聪, 徐浩, 张宁, 杨静, 赵剑. 重组耐热β-葡萄糖苷酶的固定化及其对黄酮糖苷的定向转化[J]. 生物质化学工程, 2021, 55(3): 10-16.

Jingcong XIE, Hao XU, Ning ZHANG, Jing YANG, Jian ZHAO. Immobilization of Thermostable Recombinant β-Glucosidase and Its Application in Transformation of Flavonol Glycosides[J]. Biomass Chemical Engineering, 2021, 55(3): 10-16.

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固定化条件 conditions		固定化酶活力/(U·mg^-1) activity of immobilized enzyme	酶固定化率/% rate of enzyme recovery
海藻酸钠质量分数 mass fraction of sodium alginate	1.0%	0.54	35.0±2.6
	1.5%	0.55	54.0±2.5
	2.0%	0.35	44.4±2.1
	2.5%	0.21	32.3±1.8
	3.0%	0.16	29.8±1.2
	3.5%	0.13	27.1±2.9
氯化钙质量分数 mass fraction of calcium chloride	1.0%	0.33	31.3±2.6
	1.5%	0.46	45.0±3.2
	2.0%	0.57	55.0±2.8
	2.5%	0.60	56.6±2.7
	3.0%	0.44	43.0±2.3
	3.5%	0.42	39.3±2.0
颗粒硬化时间 pelletssolidifying time	1.0 h	0.26	23.0±1.6
	1.5 h	0.35	35.4±2.1
	2.0 h	0.47	43.4±1.3
	2.5 h	0.56	55.7±2.7
	3.0 h	0.59	55.4±2.3
	3.5 h	0.53	54.4±1.3
	4.0 h	0.56	53.4±2.3
戊二醛质量分数 mass fraction of glutaraldehyde	0%	0.32	30.1±2.3
	0.5%	0.44	43.7±3.6
	1.0%	0.57	53.5±2.3
	1.25%	0.64	62.5±2.4
	1.5%	0.60	57.7±1.6
	2.0%	0.59	55.3±3.1

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重组耐热β-葡萄糖苷酶的固定化及其对黄酮糖苷的定向转化

Immobilization of Thermostable Recombinant β-Glucosidase and Its Application in Transformation of Flavonol Glycosides

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