Identification of a cold-adapted lipase-producing yeast and its enzyme characterization

doi:10.16085/j.issn.1000-6613.2021-2475

Abstract

Abstract:

The cold-adapted lipase, which has become a good candidate for industrial low-temperature processes, plays an important role in the fields of biomass energy, food, leather products and wastewater treatment. In this study, a high-yielding strain, NYNU 19160, was screened from 55 cold-adapted lipase-producing yeast strains preserved in our laboratory. The strain NYNU 19160 was identified as Papiliotrema fonsecae by morphology, physiological characteristics and ITS and 26S rDNA sequence analysis. The properties of the cold-adapted lipase were studied after purification by ammonium sulphate fractionation, dialysis and concentration. Results showed that it belonged to low temperature alkaline lipase. The optimal reaction temperature of the lipase was 20℃, and the optimal reaction pH was 7.5. Cu²⁺ significantly promoted the hydrolysis activity of the enzyme, while Li⁺ showed significant indigenous inhibition. Organic solvents, including acetonitrile, methanol and acetic acid, had a great promotion effect on the enzyme activity, while benzene and n-hexane inhibited the enzyme activity. The enzyme showed strong specificity for p-nitrophenyl butyrate (pNPC₄) substrate.

Key words: yeast, cold-adapted lipase, identification, purification, enzymatic property

摘要：

低温脂肪酶已成为工业低温工艺良好候选物，在生物质能源、食品、皮制品、废水处理等领域发挥重要作用。本文从实验室保藏的55株产低温脂肪酶酵母菌株中筛选出一株高产菌株NYNU 19160，通过形态学、生理生化以及ITS和26S rDNA序列分析，将该菌株鉴定为Papiliotrema fonsecae。经过硫酸铵分级沉淀、透析、浓缩将该脂肪酶纯化后，对酶学性质进行了研究。结果表明，该脂肪酶最适反应温度为20℃，最适作用pH为7.5，属于低温碱性脂肪酶；Cu²⁺显著促进该酶的水解活性，而Li⁺表现为显著抑制作用；有机溶剂乙腈、甲醇、乙酸对酶活性有较强的促进作用，而苯和正己烷则抑制了该酶的活性；该酶对对硝基苯酚丁酸脂（pNPC₄）底物表现出较强特异性。

关键词: 酵母菌, 低温脂肪酶, 鉴定, 纯化, 酶学性质

CLC Number:

SHI Chengfeng, JIA Ranran, YAN Zhenli, HUI Fengli. Identification of a cold-adapted lipase-producing yeast and its enzyme characterization[J]. Chemical Industry and Engineering Progress, 2022, 41(10): 5541-5548.

史程风, 贾冉冉, 阎振丽, 惠丰立. 一株产低温脂肪酶酵母菌的鉴定及酶学性质[J]. 化工进展, 2022, 41(10): 5541-5548.

Figures/Tables 12

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菌株编号	初筛酶活性	复筛酶活性/U·mL^-1	菌株编号	初筛酶活性	复筛酶活性/U·mL^-1
NYNU 18124	++++	17.72±4.95	NYNU 18335	+++	9.13±3.78
NYNU 19166	++++	19.51±3.08	NYNU 18316	+++	9.05±4.17
NYNU 18314	+++	12.88±1.75	NYNU 18457	++	8.88±3.04
NYNU 18320	++	9.26±3.82	NYNU 1833	++	8.72±5.88
NYNU 1812141	+++	20.43±2.32	NYNU 18315	++	8.55±1.55
NYNU 19160	++++	34.47±3.03	NYNU 18455	++	7.88±2.98

菌株编号	初筛酶活性	复筛酶活性/U·mL^-1	菌株编号	初筛酶活性	复筛酶活性/U·mL^-1
NYNU 18124	++++	17.72±4.95	NYNU 18335	+++	9.13±3.78
NYNU 19166	++++	19.51±3.08	NYNU 18316	+++	9.05±4.17
NYNU 18314	+++	12.88±1.75	NYNU 18457	++	8.88±3.04
NYNU 18320	++	9.26±3.82	NYNU 1833	++	8.72±5.88
NYNU 1812141	+++	20.43±2.32	NYNU 18315	++	8.55±1.55
NYNU 19160	++++	34.47±3.03	NYNU 18455	++	7.88±2.98

糖发酵	结果	氮源同化	结果	碳源同化	结果	生长测试	结果
D-葡萄糖	-	乙胺	+	D-葡萄糖	+	维生素基础培养基	+
D-半乳糖	-	尸胺	-	D-半乳糖	+	生长在4℃	+
L-山梨糖	-	肌酐	-	L-山梨糖	-	生长在10℃	+
麦芽糖	-	硝酸盐	+	蔗糖	+	生长在15℃	+
α-D-吡喃葡糖苷	-	D-色氨酸	+	木糖醇	+	生长在20℃	+
蔗糖	-	亚硝酸盐	+	D-麦芽糖	+	生长在25℃	+
D-海藻糖	-	L-赖氨酸	-	5-酮-D-葡糖糖盐	+	生长在30℃	+
蜜二糖	-	氨基葡萄糖	+	甲醇	-	生长在35℃	+
乳糖	-	咪唑	w	D，L-乳酸	-	生长在40℃	-
纤维二塘	-	肌酸	+	水杨苷	+	生长在45℃	-
松三糖	-			核糖醇	d	0.01%放线菌酮	w
D-木糖	-			L-阿拉伯糖醇	+	0.1%放线菌酮	-
D-绵子糖	-			柠檬酸	d	1%乙酸	-
菊粉	-			乙醇	d
可溶性淀粉	-			D-阿拉伯糖	w

糖发酵	结果	氮源同化	结果	碳源同化	结果	生长测试	结果
D-葡萄糖	-	乙胺	+	D-葡萄糖	+	维生素基础培养基	+
D-半乳糖	-	尸胺	-	D-半乳糖	+	生长在4℃	+
L-山梨糖	-	肌酐	-	L-山梨糖	-	生长在10℃	+
麦芽糖	-	硝酸盐	+	蔗糖	+	生长在15℃	+
α-D-吡喃葡糖苷	-	D-色氨酸	+	木糖醇	+	生长在20℃	+
蔗糖	-	亚硝酸盐	+	D-麦芽糖	+	生长在25℃	+
D-海藻糖	-	L-赖氨酸	-	5-酮-D-葡糖糖盐	+	生长在30℃	+
蜜二糖	-	氨基葡萄糖	+	甲醇	-	生长在35℃	+
乳糖	-	咪唑	w	D，L-乳酸	-	生长在40℃	-
纤维二塘	-	肌酸	+	水杨苷	+	生长在45℃	-
松三糖	-			核糖醇	d	0.01%放线菌酮	w
D-木糖	-			L-阿拉伯糖醇	+	0.1%放线菌酮	-
D-绵子糖	-			柠檬酸	d	1%乙酸	-
菊粉	-			乙醇	d
可溶性淀粉	-			D-阿拉伯糖	w

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