海研站菌S52菌株Cr(Ⅵ)还原酶活性和稳定性及其还原Cr(Ⅵ)的条件优化

doi:10.13481/j.1671-587x.20210106

摘要/Abstract

摘要： 目的

研究温度和pH值对海研站菌S52菌株六价铬［Cr（Ⅵ）］还原酶活性和稳定性的影响，优化最佳还原条件，并探讨金属离子和小分子物质对其还原能力的影响。

方法

将S52菌株的种子液接种于LB培养基中过夜培养，将菌液离心过滤后得到的胞外活性物质、超声波冰浴破菌得到胞内活性物质和对照组S52全菌液分别加入到含50 mg·L^－1 Cr（Ⅵ）的LB培养基中，在37 ℃、pH值为8.0条件下培养，于0、3、6、12、24和48 h分别用二苯碳酰二肼分光光度法测定各组溶液中的Cr（Ⅵ）浓度，计算不同时刻Cr（Ⅵ）还原率、细胞内外还原酶的相对活性及相对稳定性。设定温度为25 ℃、30 ℃、35 ℃、40 ℃、45 ℃和50 ℃，pH值为5.0、6.0、7.0、8.0、9.0和10.0，采用混合实验设计，计算细胞内外还原酶在0、3、6和12 h对Cr（Ⅵ）的还原率，分析不同温度和pH值处理组中细胞内外Cr（Ⅵ）还原酶对Cr（Ⅵ）还原率的变化，探讨最佳还原条件。分别将浓度为0.2 mmol·L^－1的Cu²⁺、Mn²⁺和Cd²⁺溶液， 1 mmol·L^－1的十二烷基磺酸钠（SDS）和乙二胺四乙酸（EDTA），1%的Triton×100和吐温80加入到培养基中作为处理组，以未作处理的培养基为对照组，在最优还原条件下与50 mg·L^－1 Cr（Ⅵ）反应3 h，测定并计算Cr（Ⅵ）还原率，比较不同金属离子和小分子物质处理组中细胞内外还原酶对Cr（Ⅵ）的还原率。

结果

3 h时，胞内酶和胞外酶对Cr（Ⅵ）的还原率达到最高，且胞内酶的还原能力高于胞外酶（P<0.05）。当温度一定时，pH值从5.0到6.0，胞内酶和胞外酶对Cr（Ⅵ）的还原率均明显升高；随着pH值逐渐升高，其还原率大幅降低。当pH值一定时，随温度升高，胞内酶对Cr（Ⅵ）的还原率逐渐提高，胞外酶对Cr（Ⅵ）的还原率逐渐降低。不同温度、pH值组间Cr（Ⅵ）还原率比较差异有统计学意义（P<0.05），温度与pH值之间具有明显的交互效应（P<0.05）。与对照组比较，Cu²⁺处理组胞内酶和胞外酶对Cr（Ⅵ）的还原率明显升高（P<0.05），而Cd²⁺和Mn²⁺处理组胞内酶和胞外酶对Cr（Ⅵ）的还原率明显降低（P<0.05）；与对照组比较，小分子物质TritonX-100处理组胞内酶和胞外酶对Cr（Ⅵ）的还原率差异无统计学意义（P>0.05），而吐温80、EDTA和SDS处理组胞内酶和胞外酶对Cr（Ⅵ）的还原率明显降低（P<0.05）。

结论

S52菌株的Cr（Ⅵ）还原酶在细胞内和细胞外均可发挥还原作用，且胞内酶的还原作用更强。胞内酶最适温度为35 ℃~45 ℃，最适pH值为7.0；胞外酶最适温度为25 ℃~30 ℃，最适pH值为7.0。不同金属离子和小分子物质对Cr（Ⅵ）还原酶活性有不同的影响。

关键词: 海研站菌S52菌株, 六价铬, 还原酶, 温度, pH值, 金属离子, 小分子物质

Abstract: Objective

To explore the effects of temperature and pH value on the enzyme activity and stability of Mesonia sp. S52 Cr（Ⅵ） and optimize the reduction conditions， and to study the effects of metal ions and small molecules on the reducing ability of the reductase.

Methods

The seed solution of the S52 strain was inoculated and cultured overnight in LB medium. The extracellular active substance obtained by centrifugal filtration of the bacterial solution， the intracellular active substance obtained by ultrasonic ice-breaking bacteria and the S52 whole bacterial solution were added to the LB medium containing 50 mg·L^－1 Cr（Ⅵ） and cultured at 37 ℃， pH 8.0. Benzyl dihydrazide spectrophotometry was used to determine the Cr （Ⅵ） concentrations in the solution at 0， 3， 6，12， 24 and 48 h， and then the reduction rates of Cr（Ⅵ）， the relative activities and relative stabilities of intracellular and extracellular enzymes were calculated. Using a multi-factor mixed experiment design， the temperatures were set as 25 ℃， 30 ℃， 35 ℃， 40 ℃， 45 ℃， 50 ℃， and the pH values were 5.0， 6.0， 7.0， 8.0， 9.0， 10.0； the reduction rates of Cr（Ⅵ） by reductases at 0， 3， 6 and 12 h were calculated， then the effects of different temperatures and pH values on the reduction rate of reductase were analyzed， and the optimal reduction conditions were optimized. 0.2 mmol·L^－1 Cu²⁺， Mn²⁺ and Cd²⁺ solution， 1mmol·L^－1SDS and EDTA， 1% Triton X-100， Tween 80 were added to the medium separately as treatment groups， and the untreated culture medium was used as control group. They were reacted with 50 mg·L^－1 Cr（Ⅵ） for 3 h under the optimal reduction conditions. The reduction rates of Cr（Ⅵ） were measured and calculated， and the changes of reduction rates in different metal ions and small molecules treatment groups were compared.

Results

At 3 h， the reduction rates of Cr（Ⅵ） by intracellular and extracellular enzymes reached the highest， and the reduction ability of intracellular enzymes was higher than that of extracellular enzymes （P<0.05）. When the temperature was constant and the pH value was from 5.0 to 6.0， the reduction rates of Cr（Ⅵ） by intracellular and extracellular enzymes were increased significantly； when the pH values were gradually increased， the reduction rates of Cr（Ⅵ） were decreased significantly. Under certain pH condition， with the increase of temperature， the intracellular enzyme reduction rates were gradually increased， and the extracellular enzyme reduction rates were decreased. The differences in reduction rates between different temperature and pH groups were statistically significant （P<0.05）， and the interaction between temperature and pH was significant （P<0.05）. Compared with control group， the enzymatic reduction rate in Cu²⁺ treatment group reached to 46.4% （P<0.05），the reduction rates in Cd²⁺ and Mn²⁺ treatment groups were decreased（P<0.05）； there was no statistically significant difference in the reduction rate of Cr（Ⅵ） between small molecular substance Triton X-100 and control group （P>0.05）， while the reduction rates of Cr（Ⅵ） in Tween 80， EDTA and SDS treatment groups were lower than that in control group（P<0.05）.

Conclusion

The Cr（Ⅵ） reductase of strain S52 can play a reducing role both inside and outside the cells， and the reducing effect of intracellular enzyme is stronger than that of extracellular enzyme. The optimal temperature of intracellular enzymes is 35 ℃－45 ℃， and the optimal pH value is 7.0； the optimal temperature of extracellular enzymes is 25 ℃－30 ℃， and the optimal value is 7.0. Different metal ions and small molecules have different effects on the activities of reductases.

Key words: Mesonia sp. S52, Cr（Ⅵ）, reductase, temperature, pH value, metal ions, small molecule substances

中图分类号:

R123

李佳瑶,李晓夏,安秋颖,范春,郭东北,唐晨,张敏,桑都哈西·歇里亚孜旦null,赵苒. 海研站菌S52菌株Cr(Ⅵ)还原酶活性和稳定性及其还原Cr(Ⅵ)的条件优化[J]. 吉林大学学报(医学版), 2021, 47(1): 44-52.

Jiayao LI,Xiaoxia LI,Qiuying AN,Chun FAN,Dongbei GUO,Chen TANG,Min ZHANG,Xieryazdan SANGDUHAX,Ran ZHAO. Activity and stability of reductase of Mesonia sp. S52 Cr(Ⅵ) and its optimization of Cr(Ⅵ) reduction conditions[J]. Journal of Jilin University(Medicine Edition), 2021, 47(1): 44-52.

图/表 7

表1

图1

图2

表2

表3

表4

表5

参考文献 31

1	STURM G， BRUNNER S， SUVOROVA E， et al. Chromate resistance mechanisms in Leucobacter chromiiresistens ［J］. Appl Environ Microbiol， 2018， 84（23）： e02208-e02218.
2	KABIR M M， FAKHRUDDIN A N M， CHOWDHURY M A Z， et al. Isolation and characterization of chromium（Ⅵ）-reducing bacteria from tannery effluents and solid wastes ［J］. World J Microbiol Biotechnol， 2018， 34（9）： 126.
3	FERNÁNDEZ P M， VIÑARTA S C， BERNAL A R， et al. Bioremediation strategies for chromium removal： Current research， scale-up approach and future perspectives ［J］. Chemosphere， 2018， 208： 139‐148.
4	张云江，何杨，张瑶瑶，等.辽宁省一般人群全血和尿液中铬水平分析［J］.中国公共卫生，2016，32（5）： 639-642.
5	FERREIRA L M R， CUNHA-OLIVEIRA T， SOBRAL M C， et al. Impact of carcinogenic chromium on the cellular response to proteotoxic stress ［J］. Int J Mol Sci， 2019， 20（19）：E4901.
6	ZHAO R， YAN S S， LIU M， et al. Seafood consumption among Chinese coastal residents and health risk assessment of heavy metals in seafood ［J］. Environ Sci Pollut Res， 2016， 23（16）： 16834-16844.
7	RAINBOW P S， LUOMA S N. Metal toxicity， uptake and bioaccumulation in aquatic invertebrates--modelling zinc in crustaceans ［J］. Aquat Toxicol， 2011， 105（3/4）： 455‐465.
8	陆秋艳，吕华东，邱秀玉，等.福建省水产品中铅镉铬蓄积量检测［J］.中国公共卫生，2009，25（1）： 67-68.
9	寇琰，于素芳，易超，等.6 价铬对人胚肺细胞P53和Bcl-2蛋白表达影响［J］.中国公共卫生，2006，22（9）： 1119.
10	JOBBY R， JHA P， GUPTA A， et al. Biotransformation of chromium by root nodule bacteria Sinorhizobium sp. SAR1 ［J］. PLoS One， 2019， 14（7）： e0219387.
11	JOBBY R， JHA P， YADAV A K， et al. Biosorption and biotransformation of hexavalent chromium［Cr（Ⅵ）］： A comprehensive review ［J］. Chemosphere， 2018， 207： 255‐266.
12	WANG Y T， XIAO C S. Factors affecting hexavalent chromium reduction in pure cultures of bacteria ［J］. Water Res， 1995， 29（11）： 2467-2474.
13	TAHRI JOUTEY N， BAHAFID W， SAYEL H， et al. Hexavalent chromium removal by a novel Serratia proteamaculans isolated from the bank of Sebou River （Morocco）［J］. Environ Sci Pollut Res， 2014，21 （4）， 3060-3072.
14	郭东北，唐晨，张敏，等.金属离子和小分子物质对耐铬（Ⅵ）菌株M52还原能力的影响［J］.吉林大学学报（医学版），2019，45（5）：1003-1008.
15	肖伟，王磊，李倬锴，等.六价铬还原细菌Bacillus cereus S5.4还原机理及酶学性质研究［J］.环境科学，2008，29（3）：751-755.
16	夏险，李明顺，武士娟，等.微生物铬转化和抗性机制与生物修复研究进展［J］.微生物学通报，2017，44（7）：1668-1675.
17	焦仕林，朱培蕾，姜朴，等.蜡样芽孢杆菌还原六价铬效果分析［J］.中国公共卫生，2016，32（10）： 1326-1329.
18	THATOI H， DAS S， MISHRA J， et al. Bacterial chromate reductase， a potential enzyme for bioremediation of hexavalent chromium： a review［J］. J Environ Manage， 2014， 146： 383-399.
19	ZHAO X L， DRLICA K. Reactive oxygen species and the bacterial response to lethal stress ［J］. Curr Opin Microbiol， 2014， 21： 1‐6.
20	BALDIRIS R， ACOSTA-TAPIA N， MONTES A， et al. Reduction of hexavalent chromium and detection of chromate reductase （ChrR） in Stenotrophomonas maltophilia ［J］. Molecules， 2018.DOI：10.3390/molecules 23020406. doi: 10.3390/molecules 23020406
21	MYERS C R. The effects of chromium（Ⅵ） on the thioredoxin system： implications for redox regulation［J］. Free Radic Biol Med， 2012， 52（10）： 2091‐2107.
22	ELANGOVAN R， ABHIPSA S， ROHIT B， et al. Reduction of Cr（Ⅵ） by a Bacillus sp. ［J］.Biotechnol Lett， 2006， 28： 247-252
23	BAE W C，LEE H K，CHOE Y C， et al. Purification and characterization of NADPH-dependent Cr（Ⅵ） reductase from Escherichia coli ATCC 33456 ［J］．J Microbiol， 2005， 43 （1）： 21-27．
24	魏斐，杨丽荣，薛保国，等.还原六价铬细菌及其还原酶的研究［J］.中国生物工程杂志，2012，32（4）：53-59.
25	杨胜男，刘娜，宋东辉. 菌藻混合体系去除Cr（Ⅵ）的条件优化及Cr（Ⅵ）还原酶活性的测定［J］.生物技术通报，2019，35（9）：83-92.
26	TANRIKULU A C，ABAKAY A，EVLIYAOGLU O，et al. Coenzyme Q10，copper， zinc， and lipid peroxidation levels in serum of patients with chronic obstructive pulmonary disease ［J］. Biol Trace Elem Res， 2011， 143（2）： 659-667.
27	XU W H， JIAN H， LIU Y G， et al. Bioreduction of chromate by an isolated bacillus anthracis Cr-4 with soluble Cr（Ⅲ） product ［J］. Water Air Soil Pollut， 2015， 226 （3）： 82.
28	TAN H， WANG C，ZENG G Q， et al. Bioreduction and biosorption of Cr（Ⅵ） by a novel Bacillus sp. CRB-B1 strain ［J］. J Hazard Mater， 2020， 386：121628
29	刘玉沛. 赤红球菌与小球藻互生关系对苯酚降解影响的研究［D］.秦皇岛：燕山大学， 2015.
30	SAFONOVA E， KVITKO K V， IANKEVITCH M I， et al. Biotreatment of industrial wastewater by selected algal-bacterial consortia ［J］. Engineer Life Sci， 2010， 4（4）： 347-353.
31	PRADHAN D， SUKLA L B， MISHRA B B， et al. Biosorption for removal of hexavalent chromium using microalgae Scenedesmus sp. ［J］. Cleaner Production， 2019， 209： 617-629.

Group	Reduction rate of Cr(Ⅵ)
Group	(t/h) 3	6	12	24	48
Control	1.8±0.5	6.1±1.2	11.8±1.9	26.9±3.1	73.1±4.3
Intracellular enzyme	18.1±1.3^*△	3.2±0.9^*	0.2±0.1^*	0.0±0.0^*	0.6±0.1^*
Extracellular enzyme	10.8±1.5^*	2.7±0.5^*	0.1±0.2^*	0.8±0.1^*	0.0±0.0^*

pH	Reduction rate of Cr(Ⅵ)
pH	(θ/℃) 25	30	35	40	45	50
5.0	6.2±0.9^*?#	7.9±1.1^*?#	8.3±1.0^*?#	12.9±1.8^*	14.4±4.0^*	10.0±1.1^*?#
6.0	12.3±1.4^?#	24.5±2.1^?#	30.6±3.2^?#	38.2±3.3	39.7±4.2	33.4±3.3^?#
7.0	12.3±1.9^*?#	23.0±3.0^*?#	29.2±2.3^*?#	33.6±4.1^*	36.0±5.1^*	28.6±4.3^*?#
8.0	7.4±0.4^*?#	11.6±2.4^*?#	17.4±3.6^*?#	19.2±3.5^*	19.9±1.9^*	16.1±5.5^*?#
9.0	5.8±1.1^*?#	6.0±1.3^*?#	8.0±1.5^*?#	9.1±3.0^*	9.3±1.1^*	6.1±1.2^*?#
10.0	3.4±0.1^*?#	4.3±0.2^*?#	5.8±0.8^*?#	6.8±0.9^*	7.1±0.8^*	5.2±0.9^*?#

pH	Reduction rates of Cr (Ⅵ)
pH	(θ/℃) 25	30	35	40	45	50
5.0	8.0±0.5^*?#	9.6±1.8^*?	5.8±1.5^*?#	4.4±1.5^*?#	4.5±1.1^*?#	2.7±0.9^*?#
6.0	17.6±3.1^#	19.8±2.2	16.1±1.2^#	15.7±4.9^#	12.0±2.6^#	6.6±2.2^#
7.0	17.0±2.2^#	19.5±3.6	14.6±2.5^#	11.6±2.1^#	9.9±2.1^#	5.7±1.4^#
8.0	11.6±3.1^*?#	12.0±1.4^*?	11.2±3.3^*?#	10.4±3.2^*?#	8.4±1.2^*?#	2.5±0.8^*?#
9.0	8.1±1.4^*?#	9.0±1.0^*?	3.8±0.7^*?#	3.8±0.4^*?#	2.4±1.0^*?#	1.0±0.2^*?#
10.0	5.8±0.9^*?#	5.7±1.1^*?	3.7±0.6^*?#	2.3±0.1^*?#	1.1±0.9^*?#	0.7±0.5^*?#

Group	Resuction rate of Cr(Ⅵ)
Group	Intracellular enzyme	Extracellular enzyme
Control	38.6±3.2	18.9±2.8
Cd²⁺	28.5±4.3^*	16.1±1.9^*
Cu²⁺	46.4±4.1^*	21.4±3.5^*
Mn²⁺	35.2±3.8^*	13.7±2.7^*

Group	Reduction rate of Cr(Ⅵ)
Group	Intracellular enzyme	Extracellular enzyme
Control	37.9±3.8	18.2±1.8
EDTA	19.8±3.2^*	11.7±3.4^*
Triton X-100	36.3±2.5	17.7±2.0
Tween 80	24.2±3.9^*	13.5±0.8^*
SDS	25.6±1.0^*	10.2±2.1^*