微生物学报  2019, Vol. 59 Issue (1): 169-180   DOI: 10.13343/j.cnki.wsxb.20180099.
http://dx.doi.org/10.13343/j.cnki.wsxb.20180099
中国科学院微生物研究所,中国微生物学会,中国菌物学会
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文章信息

赵婉琳, 叶静, 张娜, 肖美添, 赵鹏, 黄雅燕. 2019
Wanlin Zhao, Jing Ye, Na Zhang, Meitian Xiao, Peng Zhao, Yayan Huang. 2019
褐藻胶降解菌的筛选、鉴定及产酶条件优化
Screening, identification and fermentation optimization of an alginate-degrading strain
微生物学报, 59(1): 169-180
Acta Microbiologica Sinica, 59(1): 169-180

文章历史

收稿日期:2018-03-10
修回日期:2018-07-05
网络出版日期:2018-08-20
褐藻胶降解菌的筛选、鉴定及产酶条件优化
赵婉琳1 , 叶静1,2 , 张娜1,2 , 肖美添1,2 , 赵鹏1,2 , 黄雅燕1,2     
1. 华侨大学化工学院, 福建 厦门 361021;
2. 厦门市海洋资源综合利用工程技术研究中心, 福建 厦门 361021
摘要[目的] 筛选一株能降解褐藻胶的菌株,并优化产酶条件以提高褐藻胶裂解酶活力。[方法] 从漳州海域采集到海水和海泥,以海藻酸钠为唯一碳源,通过富集培养、初筛、复筛筛选到一株能够降解褐藻胶的菌株。依据16S rRNA序列分析、生理生化特征、菌体形态及菌落特征对该菌进行鉴定。通过单因素和正交试验对该菌的产酶条件进行优化。[结果] 该菌属于海科贝特氏菌,命名为Cobetia marina HQZ08。该菌株最佳的产酶培养基组成为:海藻酸钠7.00 g/L、蛋白胨3.00 g/L、NaCl 30.00 g/L,K2HPO4·3H2O 1.25 g/L。最佳发酵条件为:接种量2%,接种龄12 h,培养基起始pH为7.0,培养温度25℃,培养时间24 h。优化后褐藻胶裂解酶活力达到68.5 U/mL,TLC法分析酶解产物为褐藻胶寡糖。[结论] HQZ08菌株可以用于降解褐藻胶,产生聚合度为2-6的褐藻胶寡糖。
关键词褐藻胶裂解酶    海科贝特氏菌    褐藻胶寡糖    
Screening, identification and fermentation optimization of an alginate-degrading strain
Wanlin Zhao1 , Jing Ye1,2 , Na Zhang1,2 , Meitian Xiao1,2 , Peng Zhao1,2 , Yayan Huang1,2     
1. College of Chemical Engineering, Huaqiao University, Xiamen 361021, Fujian Province, China;
2. Xiamen Engineering and Technological Research Center for Comprehensive Utilization of Marine Biological Resources, Xiamen 361021, Fujian Province, China
Received 10 March 2018; Revised 5 July 2018; Published online 20 August 2018
*Corresponding author: Peng Zhao, Tel: +86-592-6161589, E-mail: zhaopeng@hqu.edu.cn
Yayan Huang, E-mail: yyhuang@hqu.edu.cn
Supported by the Special Fund for Marine Research in the Public Interest (201505026-5), by the Major Project of Industry-University Collaboration in Fujian Province (2018N5008) and by the Major Project of Science and Technology Plan of Quanzhou (2015Z140)
Abstract: [Objective] To screen an alginate-degrading strain and to improve the activity of its alginate lyase by optimizing the fermentation conditions. [Methods] A strain capable of degrading alginate was screened by the steps of enrichment culture, screening, and re-screening by using seawater and sea mud collected from the sea area of Zhangzhou (Fujian, China). Then, the strain was identified according to its 16S rRNA sequence analysis, physiological and biochemical characteristics, mycelium morphology and colony characteristics. The conditions of enzyme production of the strain were optimized by single factor and orthogonal test. [Results] The strain, belonging to the genus Cobetia marina, was named Cobetia marina HQZ08. The optimal medium of the strain was composed of 7.00 g/L sodium alginate, 3.00 g/L peptone, 30.00 g/L NaCl, 1.25 g/L K2HPO4·3H2O. The optimal fermentation conditions were as follows:the optimum inoculum was 2%, the inoculation age was 12 h, the initial pH of the culture medium was 7.0, the culture temperature was 25℃, and the incubation time was 24 h. The enzymatic activity of alginate lyase reached 68.5 U/mL after optimization. Enzymatic hydrolysates were determined to be alginate oligosaccharides. [Conclusion] HQZ08 strain can be used to degrade alginate to produce alginate oligosaccharides with a degree of polymerization of 2-6.
Keywords: alginate lyase    Cobetia marina    alginate oligosaccharides    

褐藻胶包括水溶性的海藻酸钠和水不溶性的海藻酸及其与2价以上金属离子结合的褐藻酸盐类,主要以海藻酸盐的形式存在于褐藻细胞壁中。它由α-1, 4-L古罗糖醛酸(G)和它的C5差向异构体β-1, 4-D甘露糖醛酸(M)两种糖醛酸单体聚合而成,组成方式主要有3种:MM片段,GG片段和杂聚的MG片段[1-3]。由于褐藻胶水溶性较差,不易被人体吸收,限制了褐藻胶的应用。而近年来低分子量的褐藻胶寡糖逐渐受到人们的关注,其多种生物活性被逐渐挖掘。

褐藻胶经降解可得到褐藻胶寡糖,和褐藻胶相比具有分子量低、溶解性强、机体吸收利用率高、稳定性好及安全无毒等理化特性[4]。研究表明褐藻胶寡糖能够抑制原代培养的皮肤成纤维细胞的增殖及胶原蛋白的表达[5],刺激人类角质细胞的增殖[6],另外在免疫调节、抗肿瘤、抑菌、抗炎及促生长方面的研究也取得一定的进展[7-11]。褐藻胶寡糖不但可以保湿,而且具有清除人体细胞多余的自由基、有效抵抗人体衰老的作用[12-13]。褐藻胶寡糖的制备方法主要有酶降解法、物理和化学降解法。酶解法是一种条件温和可控性强和特异性高的生物降解方法,降解产物活性高。褐藻胶裂解酶通过β-消除反应催化褐藻胶1-4糖苷键的断裂,在六元碳环的C4、C5之间产生双键[14]。酶降解法中的褐藻胶裂解酶主要来源于海洋细菌、真菌、海洋软体动物、海洋藻类和土壤微生物[15]。目前已发现的产褐藻胶裂解酶的海洋细菌有50多种,如假单胞菌(Pseudomonas sp. QD03)[16]、固氮菌(Azotobacter vinelandii)[17]、黄杆菌(Flavobacterium sp. LXA)[18]、交替假单胞菌(Pseudoalteromonas sp. CY24)[19]、鞘氨醇单胞菌(Sphingomonas sp. strain A1)[20]、弧菌(Vibrio sp. QY102)[21]等。

本研究从海水和海泥中筛选出1株具有褐藻胶裂解酶活力的菌株,利用16S rRNA鉴定其为海科贝特氏菌,并对其产酶条件进行优化,为褐藻胶寡糖的酶法制备提供支持。

1 材料和方法 1.1 材料

1.1.1 样品来源: 海水及海泥从漳州海域采集。

1.1.2 培养基[22-23]: (1) 富集培养基(g/L):海藻酸钠5.00,硫酸铵5.00,氯化钠25.00,MgSO4·7H2O 1.00,K2HPO4·3H2O 2.00,FeSO4·7H2O 0.01,pH 7.2。(2)初筛培养基(g/L):海藻酸钠5.00,硫酸铵5.00,氯化钠25.00,MgSO4·7H2O 1.00,K2HPO4·3H2O 2.00,FeSO4·7H2O 0.01 g,琼脂15 g,pH 7.2。(3)种子和初始发酵培养基(g/L):海藻酸钠5,蛋白胨5,酵母提取粉1,氯化钠30,pH 7.5。

1.1.3 主要试剂和仪器: Sillca gel 60F254硅胶板(Merck公司),其他试剂均为国产分析纯试剂。离心机(湖南湘仪实验室仪器开发有限公司,H1650-W),光学显微镜(日本Olympus Corporation,BX51),紫外可见分光光度计(上海美普达仪器有限公司UV-1800),扫描电子显微镜(日本日立,S4800),恒温培养箱(广东省医疗器械厂,LRH-250-Z),振荡培养箱(上海欣蕊自动化设备有限公司)。

1.2 菌种的筛选

富集培养:在250 mL锥形瓶中装入50 mL富集培养基,取少量海泥加入无菌水振荡30 min,然后将振荡液和采集的海水各5 mL分别接种到50 mL富集培养基中,30 ℃培养48 h。

初筛:初筛培养基倒平板,将富集培养的菌液进行10–1–10–7梯度稀释,对10–3–10–7稀释度的菌液进行涂布,30 ℃培养48 h。观察菌落周围透明圈大小,选取生长状况良好且透明圈较大的菌株为出发菌株,进行平板划线分离纯化。

复筛:在250 mL的锥形瓶中装入50 mL液体种子培养基,将初筛得到的菌种分别接种到种子培养基中,30 ℃恒温培养24 h后,以2%的接种量接种到发酵产酶培养基中,30 ℃恒温培养24 h后,离心收集上清液,使用DNS法测定酶活力。将酶活力最高的菌株摇瓶培养,其菌液和40%甘油1:1混合,于–80 ℃冰箱存放。

1.3 褐藻胶裂解酶活力的测定方法

参照DNS (3, 5-二硝基水杨酸)法[24]并做了适当修改:100 μL粗酶液,加入900 μL 0.3%的褐藻胶溶液(25 mmol/L的磷酸盐缓冲液,pH 7.5)中,在40 ℃恒温水浴锅中反应5 min,加入2 mL的DNS试剂停止反应,沸水煮沸5 min。流水冷却,定容到10 mL,测定540 nm处的吸光度。酶活力(U/mL)定义为:1 mL粗酶液每分钟催化产生1 μg还原糖所需的酶用量定义为1个酶活力单位。

1.4 菌株的鉴定

1.4.1 细菌的形态结构观察及生理生化特征试验: 固体平板培养基上观察菌落形态,在光学显微镜和扫描电子显微镜下观察菌体形态。部分生理生化特征鉴定参照《常见细菌系统鉴定手册》[25]

1.4.2 16S rRNA序列鉴定[26]: 菌株Cobetia marina HQZ08基因组采用细菌基因组试剂盒进行提取,之后采用通用27F/1492R引物(正向引物5′-AGAGTTTGATCCTGGCTCAG-3′;反向引物5′-GGTTACCTTGTTACGACTT-3′)进行16S rRNA的PCR扩增,PCR扩增条件为:95 ℃ 5 min;95 ℃ 30 s,55 ℃ 30 s,72 ℃ 60 s,35个循环;72 ℃ 10 min。测序得到的16S rRNA序列提交到NCBI核酸序列库进行BLAST比对,从GenBank搜索与该菌株亲缘关系较近的菌株确定其种属,并使用MEGA 5.1软件中的Neighbor-Joining法构建进化树。

1.5 菌株产酶条件的优化

对培养基组分进行优化,包括碳源种类、最适碳源浓度、氮源种类、最适氮源浓度、NaCl浓度和金属离子;并进一步对褐藻胶、蛋白胨、NaCl、K2HPO4·3H2O四个因素进行正交实验,在优化的产酶培养基基础上改变接种量、接种龄、培养基初始pH、培养温度、培养时间,探究其对菌株产酶能力的影响。除考察培养时间因素外,其余条件的发酵时间均为24 h,所有试验均设3个平行,前一步优化的条件用于下一步试验中。

1.6 酶解产物分析

取1%褐藻胶溶液35 ℃酶解44 h,使用TLC法对酶解产物进行分析。展开剂为正丁醇:甲酸:水=4:6:1,显色剂为苯胺/二苯胺/磷酸溶液(1 mL HCl、2 mL苯胺、2 g二苯胺、10 mL 85%磷酸溶液溶于100 mL丙酮中)。将酶解液在硅胶板上点样,室温吹干,放入层析缸中用展开剂将其展开,待展开剂接近硅胶板上侧1 cm左右时取出吹干,放入显色剂中,取出,热风吹干显色。

2 结果和分析 2.1 褐藻胶降解菌株的筛选

通过初筛,从海水和海泥中共筛选出9株能在以海藻酸钠为唯一碳源的固体平板培养基上生长且形成透明圈的菌株,对9种菌株进行摇瓶复筛,取上清测其酶活力。结果表明菌株HQZ08发酵液酶活力最高,经过5代传代培养后证明该菌株产酶能力较稳定,可以作为后续实验的研究对象。

2.2 菌株HQZ08的鉴定

2.2.1 生理生化及形态学鉴定: 菌株HQZ08在平板上培养24 h后菌落形态如图 1-A所示,菌落呈圆形,表面光滑,边缘整齐,颜色为乳白色,革兰氏染色鉴定结果为阴性。扫描电子显微镜观察菌体长度约1 μm,呈直杆状,图 1-B为该菌扫描电子显微镜镜检图片。HQZ08与Arahal等[27]描述的海科贝特氏菌模式菌株Cobetia marina DSM 4741T的生理生化特征比较如表 1所示。二者的生理生化特征绝大多数相似,而利用“lactose”发酵时结果却不同,可能是因为二者的同源相似性虽然达99%,但仍有些微差异,该差异导致利用“Lactose”的结果不同。刘旭梅等[28]筛选的菌株Cobetia sp.利用Lactose的结果与本实验结果相同,另外也有文献[29]表明筛出的菌株和伯杰手册中描述的菌株生化特征不同的情况。

图 1 菌株HQZ08的形态学特征 Figure 1 Morphological characteristics of strain HQZ08. A: Colony morphology; B: SEM of HQZ08.

表 1. 菌株HQZ08的生理生化特征 Table 1. Physiological and biochemical characteristics of strain HQZ08
Test item HQZ08 Cobetia marina
Growth with Na+ + +
Straight rod shaped + +
Nitrate reduction - -
O/F + ND
Catalase + ND
Gelatinase - -
Ethanol oxidation - ND
M.R - -
V-P - ND
Lactose + -
Saccharose - -
Arabinose - -
D-fructose + +
Maltose + +
D-glucose + +
D-Mannitol + +
+: positive or growth; -: negative or not growth; ND: not determined.

2.2.2 16S rRNA序列及系统发育树: 测序得到菌株HQZ08的16S rRNA基因序列含有1436 bp,通过BLAST比对发现菌株HQZ08 (MG917730)与菌株Cobetia marina strain JCM21022同源性最高,其相似度达到99%,系统发育树如图 2所示。结合菌株的形态学特征和生理生化特征,确定其为海科贝特氏菌(Cobetia marina),并命名为Cobetia marina HQZ08。该菌株保藏于中国典型培养物保藏中心,保藏编号为CCTCC No. M 2017409。

图 2 菌株HQZ08的16S rRNA序列系统发育树 Figure 2 Phylogenetic tree of the 16S rRNA sequence of strain HQZ08. Numbers in parentheses represent accession numbers in GenBank. Numbers at each branch point represent the bootstrap values on Neighbor-joining analysis of 1000 replication deta sets. Bar 0.5 is the sequence divergence.

2.3 培养基组成的优化

2.3.1 碳源对菌株产酶的影响: 分别将5 g/L的海藻酸钠、岩藻聚糖、葡萄糖和淀粉作为HQZ08菌株生长的唯一碳源,研究不同碳源对该菌株产酶的影响。图 3-A表明以葡萄糖、淀粉和岩藻聚糖为唯一碳源时,菌株的产酶能力较弱,以海藻酸钠为唯一碳源时酶活最高,故选择海藻酸钠作为后期优化碳源。在此基础上进一步研究海藻酸钠浓度(1、3、5、7、9 g/L)对菌株HQZ08的产酶影响。由图 3-B可知,菌株的产酶能力随着海藻酸钠浓度的增加而增加,当海藻酸钠浓度为7 g/L时菌株产酶能力最强。浓度低时可能因为细菌所需要的营养成分不足导致产酶能力较低,浓度过高时可能因为培养基粘度太高影响细菌的产酶能力[30]

图 3 碳源对菌株HQZ08产酶的影响 Figure 3 Effect of carbon source on enzyme production of strain HQZ08. A: Type of carbon source; B: Sodium alginate concentration.

2.3.2 氮源对菌株产酶的影响: 以7.0 g/L的海藻酸钠作为碳源,分别选取5.0 g/L的蛋白胨、酵母提取粉、牛肉提取粉、NH4NO3、(NH4)2SO4、5.0 g/L蛋白胨+2.5 g/L酵母提取粉、5.0 g/L蛋白胨+2.5 g/L牛肉提取粉为氮源(编号分别为A–G),考察不同氮源对菌株产酶的影响。由图 4-A可知,几种氮源都可以被菌株HQZ08利用,以蛋白胨作为氮源时细菌产酶能力最强。随后以蛋白胨作为菌株发酵产酶的最佳氮源,探讨不同蛋白胨浓度(1、3、5、7、9 g/L)对菌株产酶的影响。由图 4-B可知,当蛋白胨浓度为5 g/L时菌株发酵液酶活最高,而浓度过高时可能影响菌体按比例吸收营养物质,从而影响褐藻胶裂解酶的积累[31]

图 4 氮源对菌株HQZ08产酶的影响 Figure 4 Effect of nitrogen source on enzyme production of strain HQZ08. A: Type of nitrogen source; B: Peptone concentration.

2.3.3 NaCl浓度及金属离子对菌株产酶的影响: 进一步考察NaCl浓度(10、15、20、25、30、35 g/L)对菌株产酶的影响(图 5)。当NaCl浓度达到30 g/L,菌株的产酶能力最强。可能是因为该菌株的分离源来自于海洋,最适盐浓度也和原来的生存环境较接近。在此基础上考察K+、Mg2+、Ca2+、Fe2+四种金属离子对菌株产酶的影响(图 6-A),结果发现K+对菌株的产酶有促进作用,Mg2+、Ca2+、Fe2+均有轻微的抑制作用。随后探讨不同K+浓度(0.50、0.75、1.00、1.25、1.50、1.75 g/L)对菌株产酶的影响,由图 6-B可知,当K2HPO4·3H2O浓度为1.25 g/L时菌株发酵液酶活最高。

图 5 NaCl浓度对菌株HQZ08产酶的影响 Figure 5 Effect of NaCl concentration on enzyme production of strain HQZ08.

图 6 金属离子对菌株HQZ08产酶的影响 Figure 6 Effects of metal ion on enzyme production of strain HQZ08. A: Type of metal ion; B: K2HPO4·3H2O concentration.

2.3.4 正交试验: 根据单因素实验结果,选择海藻酸钠、蛋白胨、NaCl、K2HPO4·3H2O四个因素进行正交实验,结果见表 2。由表中可以看出,各因素对菌株产酶的影响大小为:海藻酸钠 > 蛋白胨 > NaCl > K2HPO4·3H2O,并确定培养基组成为海藻酸钠7 g/L,蛋白胨3 g/L,NaCl 30 g/L,K2HPO4·3H2O 1.25 g/L。

表 2. 正交试验结果 Table 2. Results of orthogonal experiment
Run No. Alginate/(g/L) Peptone/(g/L) NaCl/(g/L) K2HPO4·3H2O/(g/L) Alginate lyase activity/(U/mL)
1 5 3 25 1.00 48.23
2 5 5 30 1.25 41.50
3 5 7 35 1.50 36.40
4 7 3 30 1.50 55.52
5 7 5 35 1.00 52.13
6 7 7 25 1.25 52.73
7 9 3 35 1.25 30.60
8 9 5 25 1.50 27.82
9 9 7 30 1.00 29.45
K1 120.73 128.95 123.38 124.41
K2 160.38 121.45 126.47 124.83
K3 87.87 118.58 119.13 119.74
R 72.51 10.37 7.34 5.09

2.4 菌株HQZ08产酶的条件优化

本实验考察了不同接种量(0.5%–3.0%)对菌株产酶的影响,接种后培养基总体积为50 mL。由图 7-A可知,2%的接种量是菌株HQZ08发酵产酶的最佳接种量。将种子液分别培养至6、9、12、15、21 h之后接种到发酵培养基中,比较不同接种龄酶活力的变化,结果表明接种龄为12 h时酶活力最高(图 7-B所示)。培养基的初始pH不仅对菌体细胞膜的通透性有直接影响,还能影响其稳定性以及代谢产物酶的活力[32]。本实验比较了培养基不同初始pH (6.0–8.5)对菌株产酶的影响。如图 7-C所示,当发酵培养基初始pH为7.0时酶活力达到最大值。温度对细菌的生长和产酶能力影响较显著,将菌株在20、25、28、30、32、35 ℃下培养进行产酶能力的考察,如图 7-D所示,当温度为25 ℃时酶活力达到最大值。随着温度升高,酶活力明显下降,说明该菌不适宜在较高温度下培养。产酶培养的过程中,随着培养时间的增加,菌群的生长达到稳定期,产酶量开始增加,而培养时间过长时可能会导致产生的酶逐渐失活。分别培养18–42 h,考察培养时间对菌株产酶的影响,由图 7-E可以看出,当培养时间达到24 h时酶活力最高为68.5 U/mL,超过24 h酶活力明显下降。

图 7 发酵条件对菌株HQZ08产酶的影响 Figure 7 Effect of fermentation conditions on enzyme production of strain HQZ08. A: Inoculation amount; B: Inoculation age; C: pH; D: Temperature; E: Time.

目前已有很多有关细菌产褐藻胶裂解酶的报道,例如芽孢杆菌、弧菌等。李悦明等[33]从土壤中分离出的芽孢杆菌酶活力为23.4 U/mL,郭恩文等[34]筛选到的弧菌Vibrio sp. QY107及傅晓妍等[21]筛选到的弧菌Vibrio sp. QY102经发酵120 h酶活力分别为12.32 U/mL和10.2 U/mL,吴海歌等[35]从腐烂海带中分离到的假交替单胞菌属优化后的酶活力达到51.498 U/mL。而关于Cobetia marina产褐藻胶裂解酶的报道很少,2016年,Yagi等[36]从褐藻中分离出Cobetia sp. NAP1菌株,其酶活力仅有4 U/mL,本实验筛选的菌株和文献报道的相比具有一定的优势。

2.5 酶解产物分析

褐藻胶被发酵液上清降解44 h后,产物进行TLC分析,与褐藻胶寡糖标准品二糖、三糖、四糖、六糖对比可知,酶解产物主要是聚合度为2–6的褐藻胶寡糖(图 8)。

图 8 褐藻胶降解产物的TLC分析 Figure 8 TLC analysis of the degradation products of alginate. 0: alginate oligosaccharide standard (dimmer、trimer、tetramer、hexamer); 1: enzymatic product of alginate.

3 结论

本文选用以海藻酸钠作为唯一碳源的选择培养基,从海水和海泥中筛选出多株具有褐藻胶裂解酶活力的菌株,通过液体发酵培养基复筛测其酶活,筛选一株酶活最高的菌株HQZ08,经生理生化特征实验及16S rRNA序列鉴定其为海科贝特氏菌。通过单因素和正交实验对培养基组分及发酵产酶条件进行优化,优化后的发酵液粗酶活提升到68.5 U/mL,本研究的固体粗酶活力为15800 U/g,略高于目前市面上仅有Sigma公司出售的褐藻胶裂解酶产品(酶活力≥10000 U/g),若要进一步提高HQZ08菌株的酶活力还可以进一步对装液量、摇床转速等进行考察,并对粗酶液进行分离纯化。TLC法测定该菌的酶解产物为聚合度2–6的褐藻胶寡糖,降解后寡糖的得率为32.15%,可为后续开发低聚合度的褐藻胶寡糖奠定基础。

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