一株产低温脂肪酶酵母菌的鉴定及酶学性质

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

摘要/Abstract

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

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

中图分类号:

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.

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.

图/表 12

参考文献 29

1	DAVIS B G, BOYER V. Biocatalysis and enzymes in organic synthesis[J]. Natural Product Reports, 2001, 18(6): 618-640.
2	JOSEPH B, RAMTEKE P W, THOMAS G. Cold active microbial lipases: some hot issues and recent developments[J]. Biotechnology Advances, 2008, 26(5): 457-470.
3	陆小波, 魏云林. 低温酶及其冷适应性机制研究进展[J]. 生物技术通报, 2006, 58(4): 51-53, 58.
	LU Xiaobo, WEI Yunlin. Progress in the study of cold-adapted enzyme and their cold-adaptation mechanisms[J]. Biotechnology Bulletin, 2006, 58(4): 51-53, 58.
4	RASHID N, SHIMADA Y, EZAKI S, et al. Low-temperature lipase from psychrotrophic Pseudomonas sp. strain KB700A[J]. Applied and Environmental Microbiology, 2001, 67(9): 4064-4069.
5	达文燕, 杨霞霞, 张爱梅. 低温酶及产低温脂肪酶微生物研究概述[J]. 甘肃农业, 2013(13): 14-16.
	Wenyan DA, YANG Xiaxia, ZHANG Aimei. A review of cryogenic enzymes and microorganisms producing cryogenic lipase[J]. Gansu Agriculture, 2013 (13): 14-16.
6	张学林, 唐湘华, 李俊俊, 等. 低温脂肪酶酶促酯交换制备生物柴油[J]. 中国油脂, 2013, 38(2): 66-68.
	ZHANG Xuelin, TANG Xianghua, LI Junjun, et al. Preparation of biodiesel by enzymatic transesterification with low temperature lipase[J]. China Oils and Fats, 2013, 38(2): 66-68.
7	薛永强, 张辉华, 王达, 等. 短链脂肪酸对肠道健康的调控机制及在动物生产中的应用[J]. 饲料工业, 2020, 41(19): 18-22.
	XUE Yongqiang, ZHANG Huihua, WANG Da, et al. Regulation mechanism of short-chain fatty acids on intestinal health and their application in animal production[J]. Feed Industry, 2020, 41(19): 18-22.
8	蔡懿鑫, 蒋慧姣, 孔祥峰, 等. 短链脂肪酸的生理功能及其在仔猪生产中的应用[J]. 中国畜牧杂志, 2019, 55(11): 30-34.
	CAI Yixin, JIANG Huijiao, KONG Xiangfeng, et al. Physiological function of short-chain fatty acids and their application in piglet production[J]. Chinese Journal of Animal Science, 2019, 55(11): 30-34.
9	ARAVINDAN R, ANBUMATHI P, VIRUTHAGIRI T. Lipase applications in food industry[J]. Indian Journal of Biotechnology, 2007, 6(2): 141-158.
10	陈贵元, 魏云林. 低温脂肪酶的研究现状与应用前景[J]. 生物技术通报, 2006 (2): 29-32.
	CHEN Guiyuan, WEI Yunlin. The research status and application prospect of cdd-adapted lipase[J]. Biotechnology Bulletin, 2006(2): 29-32.
11	HASSAN S W M, LATIF H H ABD EL, ALI S M. Production of cold-active lipase by free and immobilized marine Bacillus cereus HSS: application in wastewater treatment[J]. Frontiers in Microbiology, 2018, 9: 2377.
12	BOETIUS A, ANESIO A M, DEMING J W, et al. Microbial ecology of the cryosphere: sea ice and glacial habitats[J]. Nature Reviews Microbiology, 2015, 13(11): 677-690.
13	唐兵, 唐晓峰, 彭珍荣. 嗜冷菌研究进展[J]. 微生物学杂志, 2002, 22(1): 51-53.
	TANG Bing, TANG Xiaofeng, PENG Zhenrong. Andvance in psychrophiles study[J]. Journal of Microbiology, 2002, 22(1): 51-53.
14	谢玉婷, 查代明, 石红璆, 等. Burkholderia sp. JXJ-16低温耐有机溶剂脂肪酶产酶条件优化及粗酶酶学性质[J]. 食品工业科技, 2017, 38(4): 207-213.
	XIE Yuting, Daiming CHA, SHI Hongqiu, et al. Optimization of enzyme production conditions and enzymatic properties of cold-adapted and organic solvent-tolerant lipase from Burkholderia sp. JXJ-16[J]. Science and Technology of Food Industry, 2017, 38(4): 207-213.
15	王春雨, 迟乃玉, 张庆芳, 等. 低温脂肪酶的分离纯化及酶学性质[J].食品与生物技术学报, 2013, 32(8): 809-813.
	WANG Chunyu, CHI Naiyu, ZHANG Qingfang, et al. Purification and enzymatic properties of cryogenic lipase[J]. Journal of Food Science and Biotechnology, 2013, 32(8): 809-813.
16	PARK S Y, KIM J Y, BAE J H, et al. Optimization of culture conditions for production of a novel cold-active lipase from Pichia lynferdii NRRL Y-7723[J]. Agricultural and Food Chemistry, 2013, 61(4): 882-886.
17	ABHAS K, MAHARANA, SHIV M. A cold and organic solvent tolerant lipase produced by antarctic strain Rhodotorula sp. Y-23[J]. Journal of Basic Microbiology, 2018, 58(4): 331-342.
18	王培培, 刘晓宁, 林国秀, 等. 低温脂肪酶产生菌的筛选、产酶发酵及粗酶性质研究[J]. 工业微生物, 2011, 41(6): 78-84.
	WANG Peipei, LIU Xiaoning, LIN Guoxiu, et al. Screening, fermentation and crude enzyme properties of low temperature lipase producing bacteria[J]. Industrial Microbiology, 2011, 41(6): 78-84.
19	陈贵元, 季秀玲, 林连兵, 等. 低温脂肪酶产生菌筛选与鉴定、产酶条件及酶学性质研究[J]. 云南大学学报（自然科学版）, 2010, 32(1): 108-113.
	CHEN Guiyuan, JI Xiuling, LIN Lianbing, et al. Screening and identification of low temperature lipase-producing bacteria, enzyme-producing conditions and enzymatic properties[J]. Journal of Yunnan University, 2010, 32(1): 108-113.
20	王兴吉, 贾仁洁, 王克芬, 等. 低温脂肪酶菌株的诱变筛选及酶学特性研究[J]. 安徽农业科学, 2019, 47(1): 1-3, 9.
	WANG Xingji, JIA Renjie, WANG Kefen, et al. Mutagenesis screening of low temperature lipase strain and its enzymatic characteristics[J]. Anhui Agricultural Sciences, 2019, 47(1): 1-3, 9.
21	TANAKA D, YONEDA S, YAMASHIRO Y, et al. Characterization of a new cold-adapted lipase from Pseudomonas sp. TK-3[J]. Applied Biochemistry and Biotechnology, 2012, 168(2): 327-338.
22	刘元利, 陈吉祥, 李彦林, 等. 一株产低温脂肪酶沙雷氏菌的鉴定、基因表达及酶学性质[J]. 中国食品学报, 2018, 18(6): 121-129.
	LIU Yuanli, CHEN Jixiang, LI Yanlin, et al. Identification, gene expression and enzyme characterization of Serratia sp. producing cold adapted lipase[J]. Journal of Chinese Institute of Food Science and Technology, 2018, 18(6): 121-129.
23	萧能, 余瑞元, 袁明秀, 等. 生物化学实验原理和方法[M]. 北京: 北京大学出版社, 2005.
	XIAO neng, YU ruiyuan, YUAN mingxiu, et al. Principles and methods of biochemical experiments[M]. Beijing: Peking University Press, 2005.
24	MARGESIN R, FELLER G, HÄMMERLE M, et al. A colorimetric method for the determination of lipase activity in soil[J]. Biotechnology Letters, 2002, 24(1): 27-33.
25	KURTZMAN C P, FELL J W, BOEKHOUT T, et al. Methods for isolation, phenotypic characterization and maintenance of yeasts[M]// The Yeasts. Amsterdam: Elsevier, 2011: 87-110.
26	FONSECA Á, BOEKHOUT T, FELL J W. Cryptococcus Vuillemin (1901)[M]. 5th ed. Amsterdam: Elsevier, 2011: 1659-1737.
27	THOMPSON J D, GIBSON T J, PLEWNIAK F, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools nucleic[J]. Nucleic Acids Research, 1997, 25(24): 4876-4882.
28	李司宇, 刘雪, 王文婧, 等. 微生物进化树构建方法[J]. 现代农业科技, 2019(19): 249-250, 253.
	LI Siyu, LIU Xue, WANG Wenjing, et al. Construction of microbial evolutionary trees[J]. Modern Agricultural Science and Technology, 2019(19): 249-250, 253.
29	SAITOU N, NEI M. The neighbor-joining method: a new method for reconstructing phylogenetic trees[J]. Molecular Biology and Evolution, 1987, 4(4): 406-425.

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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