Screening of Acidic Protease Producing Strains and Its Application in Seed Germination

doi:10.13304/j.nykjdb.2022.0420

Abstract

Abstract:

Acid protease is of great demand in industrial production. At present， the varieties of acidic protease producing strains in China are relatively rare， so it is urgently needed to develop new microbial protease and select high-yield protease strains. A strain producing protease in acidic environment was screened from mangrove soil in Haikou. The protease activity was determined by folinol method. It was identified as Serratia rubidaea by morphological observation， physiological and biochemical experiment and 16S rRNA sequence blast. The strain was used to ferment fish waste to produce biological liquid fertilizer. The effect of the fermentation liquid as biological liquid fertilizer was evaluated through seed germination promotion experiment. The results showed that the protease producing strain R3 isolated in this study could be used for the fermentation of fish waste. When the fermentation broth was diluted 5 to 25 times， the treatment for 60 h could promote the germination of cabbage seeds， and the germination rate reached the effect of liquid fertilizer. In this study， the isolated strain producing acid protease was used to ferment fish waste to biological liquid fertilizer， which utilized the waste fish protein as the resources and increased the value of fish products.

Key words: acidic protease, strain identification, biological liquid fertilizer, seed germination

摘要：

酸性蛋白酶在工业生产中需求量巨大，但我国产酸性蛋白酶菌株的品种较为单一，因此，亟需开发新型的微生物源蛋白酶和选育高产蛋白酶菌种。从海口红树林土壤中筛选出一株可以在酸性环境下产蛋白酶的菌株，采用福林酚法测定蛋白酶活，经过形态学观察、生理生化实验及16S rRNA序列比对鉴定为深红沙雷氏菌。利用该菌株发酵鱼下脚料制备生物液肥，通过种子萌发实验，研究发酵液的促生效果。结果显示，分离出的产酸性蛋白酶菌株R3可以用于鱼下脚料的发酵，在发酵液稀释5~25倍时，处理60 h均可以促进菜心种子的萌发，与市场购买的液肥效果相同。利用分离出的产酸性蛋白酶菌株发酵鱼下脚料制备生物液肥，实现了对废弃鱼蛋白资源的重新利用，增加了鱼类产品的附加值。

关键词: 酸性蛋白酶, 菌株鉴定, 生物液肥, 种子萌发

CLC Number:

S182

Xin PENG, Can FENG, Xiang MA, Hong LI, Yanqiong TANG, Juanjuan LI, Zhu LIU. Screening of Acidic Protease Producing Strains and Its Application in Seed Germination[J]. Journal of Agricultural Science and Technology, 2022, 24(9): 88-95.

彭欣, 冯璨, 马香, 李宏, 唐燕琼, 李娟娟, 刘柱. 产酸性蛋白酶深红沙雷氏菌的筛选及其在种子萌发中的应用[J]. 中国农业科技导报, 2022, 24(9): 88-95.

Figures/Tables 11

Table 1 Components of buffer solution with different pH

pH	Na₂HPO₄/mL	柠檬酸 Citric acid/mL	水Water/mL
3	4.11	15.89	—
4	7.71	12.29	—
5	10.3	9.70	—
6	12.63	7.37	—
7	16.47	3.53	—
8	19.45	0.55	—
9	0.88	5.00^*	14.12
10	3.2	5.00^*	11.8

Fig.1 Enzyme activity and D/d of acid protease producing strainNote： Different lowercase letters in the figure indicate significant difference in enzyme activity at P<0.05 level.

Fig.2 Morphological characteristics of R3 strain

Table 2 Physiological and biochemical characteristics of R3 strain

指标Index	特征Characteristics
葡萄糖 Glucose	+
V-P试验 V-P test	+
尿素 urea	-
甲基红试验 Methyl red test	-
吲哚试验 Indole test	-
硫化氢 Hydrogen sulfide	-
蔗糖 Sucrose	+
麦芽糖 Malt dust	+
柠檬酸盐试验 Citrate test	+

Fig. 3 Phylogenetic tree among R3 strain and other similar strains based on 16S rRNA gene sequence

Fig. 4 Enzyme activity at different fermentation daysNote：Different lowercase letters in the figure indicate significant difference at P<0.05 level.

Fig. 5 Enzymatic activity at different pHNote：Different lowercase letters in the figure indicate significant difference at P<0.05 level.

Fig. 6 Enzyme activity at different temperatureNote：Different lowercase letters in the figure indicate significant difference at P<0.05 level.

Fig. 7 Enzyme activity under different proportion of fish to waterNote：Different lowercase letters in the figure indicate significant difference at P<0.05 level.

Table 3 Seed germination rate

处理Treatment		24 h	36 h	48 h	60 h
清水Water		0.00 f	0.00 e	17.14±1.31 d	37.14±1.56 e
发酵稀释液 Fermentation dilution	1×	8.57±0.27 e	8.57±0.34 d	17.14±1.23 d	31.43±1.62 e
	5×	17.14±1.13 d	20±1.09 c	42.86±1.35 c	45.71±1.54 de
	10×	37.14±1.31 b	51.43±2.31 b	71.43±1.32 bc	71.43±2.12 c
	15×	28.57±1.12 bc	48.57±1.73 bc	77.14±2.43 b	80±2.57 b
	20×	25.71±1.24 c	51.43±1.81 b	80±1.94 b	88.57±2.67 a
	25×	0.00 f	0.00 e	20±1.41 d	51.43±2.02 d
氨基酸液肥 Amino acid liquid fertilizer		0.00 f	62.86±2.07 a	88.57±2.41 a	91.43±2.47 a

Fig. 8 Root length and bud length of different treatment groupsNote：Different lowercase letters in the figure indicate significant difference at P<0.05 level.

References 16

1	王兰梦,郑立昊,郎多勇,等.微生物肥料及其在中药生态农业中的前景分析[J].北方园艺,2022(2):141-147.
	WANG L M, ZHENG L H, LANG D Y, et al.. Microbial fertilizer and its prospect in traditional Chinese medicine ecological agriculture [J] Northern Hortic., 2022 (2): 141-147.
2	KARIM A A, BHAT R. Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins [J]. Food Hydrocoll., 2009, 23(3):563-576.
3	BAI J B, TIAN M, LI J Q. Control system of liquid fertilizer variable-rate fertilization based on beetle antennae search algorithm [J/OL]. Processes, 2022,10(2):357 [2022-05-29]. .
4	FEDERICO A G, ROBERTO C G, REINER R R, et al.. Formulation of a liquid fertilizer for sorghum (Sorghum bicolor (L.) Moench) using vermicompost leachate [J]. Bioresour. Technol., 2008, 99(14):6174-6180.
5	申惠波.生物液肥对辣椒生长发育的影响[J].北方园艺,2007(9):51-53.
	SHEN H B. Effects of biological liquid fertilizer on the growth and development of pepper [J] Northern Hortic., 2007 (9): 51-53.
6	苑艳辉.综合利用鱼下脚料的研究近况[J].科学养鱼,2004(2):64.
	YUAN Y H. Recent research on comprehensive utilization of fish offal [J]. Sci. Fish Culture, 2004 (2): 64.
7	蔡路昀,张滋慧,李秀霞,等.鱼类下脚料在工业中应用的研究进展[J].食品工业科技,2017,38(8):356-363.
	CAI L Y, ZHANG Z H, LI X X, et al.. Research Progress on the application of fish residues in industry [J] Food Ind. Sci. Technol., 2017,38 (8): 356-363.
8	高英,俞玉忠.福林酚法测定脑蛋白水解物溶液中的多肽含量[J].海峡药学,2004(6):57-58.
	GAO Y, YU Y Z. Determination of polypeptide content in brain protein hydrolysate solution by Folin phenol method [J] Strait Pharmacy, 2004 (6): 57-58.
9	陈瑜,孔海深.伯杰鉴定细菌学手册第9版简介[J].国外医学,1995,18(6):32,48.
	CHEN Y, KONG H S. Berger’s Handbook of identification bacteriology, 9th Edition [J] Foreign Med., 1995,18 (6): 32,48.
10	WEI S, LEVY B, HOFFMAN N, et al. A rapid and simple bead-bashing-based method for genomic DNA extraction from mammalian tissue. [J]. BioTechniques,2020,68(5):240-244.
11	张婧,刘广娜,左蔚琳.土壤微生物基因组DNA不同提取方法的比较及PCR扩增体系的建立[J].吉林农业,2018(16):55-56.
	ZHANG J, LIU G N, ZUO W L. Comparison of different extraction methods of soil microbial genomic DNA and establishment of PCR amplification system [J] Jilin Agric., 2018 (16): 55-56.
12	倪红,万娟,刘诚,等. 一株具有富磷、降解有机磷及抑制植物病原真菌的沙雷氏菌:CN106119168B[P]. 2019-06-18.
13	杜秉海,丁延芹,汪城墙,等. 一种具有促生效果的深红沙雷氏菌及其应用:CN110564644B[P]. 2019-12-13.
14	GUTTERMAN Y. Seed germination in desert plants [J]. Endeavour, 2014,19(1):47-47.
15	赵艳萍,魏佳乐.生长素的生理作用研究[J].乡村科技,2019(9):102-103.
	ZHAO Y P, WEI J L. Study on the physiological function of auxin [J] Rural Sci. Technol., 2019 (9): 102-103.
16	杨洪兵.高温胁迫下外源氨基酸对荞麦种子萌发及幼苗生长的影响[J].河南农业科学,2014,43(11):20-23.
	YANG H B. Effects of exogenous amino acids on buckwheat seed germination and seedling growth under high temperature stress [J]. Henan Agric. Sci., 2014,43(11):20-23.

[1]	ZHANG Shengzhen, MA Yanzhi. Effects of CaCl2 on Seed Germination and Seedling Physiological Characteristics of Schizonepeta tenuifolia Briq. under Salt Stress [J]. Journal of Agricultural Science and Technology, 2021, 23(7): 65-71.
[2]	WANG Zhiheng, YANG Xiuliu, ZOU Fang, HUANG Siqi, ZHOU Wuyan, XU Zhongwei, WEI Yuqing*. Effects of Salt and Drought Cross Stress on Germination and Physiological Characteristics of Sweet Sorghum Seeds [J]. Journal of Agricultural Science and Technology, 2021, 23(2): 37-49.
[3]	WANG Zhiheng, HUANG Siqi, ZOU Fang, YANG Xiuliu, WEI Yuqing*. Effects of Temperature and NaCl on Seed Germination and Seedling Antioxidant Enzyme Activities of Sweet Sorghum [J]. Journal of Agricultural Science and Technology, 2020, 22(9): 42-51.
[4]	YANG Ying1, WANG Yuelin2, YAN Jingqiuzi1, LI Gangtie1*. Effects of Salt Stress on Seed Germination and Seedling Physiological Characteristics of Hemiptelea davidii [J]. Journal of Agricultural Science and Technology, 2020, 22(4): 53-60.
[5]	YAN Jingqiuzi1, LI Gangtie1*, WANG Yuelin2, MA Yunxia1, YANG Ying1. Effects of Salt Stress on Seed Germination and Seedling Physiological Characteristics of Morus mongolica [J]. Journal of Agricultural Science and Technology, 2020, 22(1): 28-37.
[6]	WANG Rui, ZHENG Weidong, LIU Duanmei, LIANG Li. Study on the Seed Germination and Seedling Growth Characteristics of Different Genotype Rapeseed Varieties [J]. Journal of Agricultural Science and Technology, 2019, 21(4): 25-34.
[7]	SHAN Hao, ZHANG Hu, CUI Aimin, WANG Yunfeng, ZHANG Jiugang*. Effect of Exogenous Growth Regulator on Seed Germination of Maize under Salt Stress [J]. Journal of Agricultural Science and Technology, 2018, 20(8): 82-90.
[8]	YAN Shilong1§, CAI Bo2,3§, TANG Hualing4, YUAN Yang5, AO Su2,3*. Allelopathy of Different Parts of Momordica charantia [J]. Journal of Agricultural Science and Technology, 2018, 20(8): 100-107.
[9]	JING Yupeng1,2, LIAN Haifei1, LI Huanchun1, SHI Pei1, DU Chao3, LIU Mei4, CHANG Xinjuan5. Study on Saline-alkaline Tolerance Ability and Evaluation Index Selection for Corn [J]. Journal of Agricultural Science and Technology, 2018, 20(11): 94-104.
[10]	MA Yanzhi. The Effects of MeJA and CS Mixture on Seed Germination of Bupleurum Chinense DC. [J]. Journal of Agricultural Science and Technology, 2017, 19(4): 38-44.
[11]	ZHANG Meng-huan1, QI Jian-jun1, LI Xian-en1, KAISA Sulaiman2*. Study on Germination Characteristics of Three Ferula Seed [J]. Journal of Agricultural Science and Technology, 2016, 18(5): 49-53.
[12]	Zhang Shigong Gao Jiyin Song Jingzhi. New Discovery of Crop Salt-resistance:effect of Salicylic Acid on Salt-resistance in Wheat(Triticum aestivum L. ) [J]. , 1999, 1(1): 32-35.