摘要
目的
从两栖动物肠道中分离并筛选能够有效分解几丁质的菌株,研究其发酵条件、酶学特性,并进行细菌全基因组测序及功能分析。
方法
对高原林蛙肠道内容物进行筛选,分离出1株具有产几丁质酶能力的菌株,并进行形态学和分子生物学鉴定;通过单因素试验和响应面试验对产酶条件进行优化,并研究其酶学性质;开展全基因组测序,鉴定几丁质酶基因家族。
结果
筛选到1株可有效产几丁质酶的菌株JD-3,经鉴定为麦芽香肉食杆菌(Carnobacterium maltaromaticum)。最佳产酶条件为:发酵时间2.47 d,发酵温度31.4 ℃,初始pH 4.9,接种量4%,酶活性达到12 mU/mL。酶学性质分析表明,该酶的最适反应温度为20 ℃,pH 3.0,在室温、酸性条件下表现出良好的稳定性。全基因组分析显示,JD-3基因组全长为4 195 636 bp,包含6个环状重叠群(contig)、63个tRNA基因、19个rRNA基因和3 864个蛋白质编码序列(coding sequence, CDS)。在基因组中鉴定出2个几丁质酶基因,均属于GH18家族,系统发育分析将其归为2个不同的类别。
结论
从高原两栖动物肠道中分离出1株具有常温耐酸特性的几丁质分解菌,经形态学和系统发育鉴定为麦芽香肉食杆菌,这为动物消化系统微生物资源的开发利用提供了新思路。
几丁质(chitin)是由N-乙酰氨基葡萄糖(N-acetylgluco-samine, GlcNAc)单元通过β-1,4-糖苷键连接而成的多聚物,在自然界中的含量仅次于纤维
动物消化道内寄居着复杂且稳定的微生物群
高原林蛙(Rana kukunoris)隶属于蛙科(Ranidae)林蛙属(Rana),是青藏高原广泛分布的两栖动
1 材料与方法
1.1 材料
供试菌株分离自青海省西宁市大通县向化乡北川河源湿地(37.105 9°N,101.463 5°E,3 100 m)。采集高原林蛙6只,双毁髓处死后取高原林蛙肠道内容物,迅速放入液氮中,运送到青海民族大学实验室,置于-80 ℃冰箱中保存备用。动物实验通过青海民族大学科技伦理审查(批准号为:2024-015)。
1.2 溶液及培养基
5%胶体几丁质溶液:称取20 g几丁质粉末,加入250 mL的85%的磷酸,在一定时间内完全溶解后,将酸解后的液体倒入预冷的2.5 L蒸馏水中,搅拌均匀,4 ℃静置过夜;8 000 r/min离心10 min,将上清液倒出,加入预冷的蒸馏水重悬,再静置,多次反复,直到悬浊液pH为6.5;定容至400 mL,121 ℃灭菌后置于4 ℃保存备用。
初筛培养基(g/L):胶体几丁质10.00,K2HPO4 0.70,KH2PO4 0.30,MgSO4·7H2O 0.50,ZnSO4 0.01,琼脂20.00,pH自然。121 ℃灭菌20 min。
复筛培养基(发酵培养基) (g/L):胶体几丁质10.00,K2HPO4 0.70,KH2PO4 0.30,MgSO4·7H2O 0.50,ZnSO4 0.01,pH自然。121 ℃灭菌20 min。
LB培养基(g/L):胰蛋白胨10.00,NaCl 5.00,酵母膏5.00,pH 7.0-8.0。121 ℃灭菌20 min。
1.3 菌株筛选
1.3.1 初筛
取0.1 g高原林蛙肠道内容物加入25 mL富集培养基中,30 ℃、160 r/min培养3 d,富集后的样品梯度稀释至1
1.3.2 复筛
将初筛得到的菌株接种于发酵培养基中,30 ℃、180 r/min培养3 d。随后采用3,5-二硝基水杨酸(3,5-dinitrosalicylicacid, DNS)比色法测定粗酶液的酶活性,选取酶活性高的菌株进行下一步鉴定。
1.4 酶活性测定
测定方法参照沈愿
1.5 菌种鉴定
将分离纯化出来的单菌落接种到已灭菌的LB培养基中,30 ℃倒置培养48 h,观察菌落特征,进行革兰氏染色,利用光学显微镜观察菌株染色状况。采用扫描电子显微镜观察细胞形态。
采用DNA试剂盒提取菌株的全基因组DNA作为模板,使用细菌16S rRNA基因通用引物27F (5′-AGAGTTTGATCCTGGCTCAG-3′)和1492R (5′-GGTTACCTTGTTACGACTT-3′)对待测菌株的16S rRNA基因序列进行PCR扩增。PCR反应体系(25 μL):2×phanta Max Mix (p515) 12.5 µL,上、下游引物(10 µmol/L)各1 µL,DNA模板0.5 µL,ddH2O 10 µL。PCR反应条件:95 ℃预变性5 min;95 ℃变性30 s,60 ℃退火30 s,72 ℃延伸2 min,共35次循环;72 ℃终延伸5 min。PCR扩增产物经1%琼脂糖凝胶电泳检测后,送至生工生物工程(上海)股份有限公司进行测序。将获得的序列通过BLAST与GenBank中的已知序列进行比对,并利用MEGA v.11.0基于最大似然法构建菌株系统发育树,以确定菌株的属种。
1.6 产酶条件优化
初始发酵培养条件:初始pH 7.0,温度30 ℃,转速160 r/min,装液量100 mL (250 mL三角瓶),接种量5%。单因素试验:在初始培养条件下,分别考察发酵时间(1、2、3、4和5 d)、发酵温度(26、28、30、32和34 ℃)、接种量(2%、4%、6%、8%和10%)以及培养基的初始pH (4.0、5.0、6.0、7.0和8.0)对产几丁质酶活性的影响,培养72 h后测定几丁质酶活性。
在单因素试验的基础上,选取发酵时间、发酵温度、初始培养基pH作为主要的3个影响因子,以这3个因子为自变量,几丁质酶酶活性(Y)为响应值,采用Design-Expert v.11.0设计3因素3水平的Box-Behnken (BB)响应面试验,BB试验因素与水平见
Levels | Factors | ||
---|---|---|---|
T/℃ | t/d | pH | |
-1 | 30 | 1 | 4.0 |
0 | 32 | 2 | 5.0 |
1 | 34 | 3 | 6.0 |
1.7 几丁质粗酶液酶学性质探究
选用优化后的培养条件:培养温度32 ℃,培养基初始pH 5.0,培养时间2 d,产酶发酵培养基初始接种量4%。
1.7.1 最适温度及稳定性
通过在不同温度(20-60 ℃)下测定酶活性,确定几丁质粗酶的最适温度。同时,将粗酶液在不同温度下预先孵育2 h后测定酶活性,以评估其稳定性。
1.7.2 最适pH及稳定性
通过在不同pH体系(pH 3.0-8.0)下测定酶活性,确定几丁质粗酶的最适pH。同时,将粗酶液在不同pH体系(pH 3.0-8.0)中于4 ℃放置2 h后测定酶活性以评估其pH稳定性。
1.8 反应进程曲线及动力学参数测定
在最适温度和最适pH条件下,将1 mL几丁质粗酶液与1 mL 1%几丁质胶体混合,分别在10、20、30、40、50、60、90、120、150、180 min的时间点反应1 h后,按照DNS比色法测定反应体系中的还原糖含量,并绘制反应进程曲线。
为测定几丁质粗酶的反应动力学参数,记录酶在不同浓度几丁质胶体中的初始反应速度。根据上述实验所获得的最佳反应温度和最佳反应pH,将几丁质粗酶与不同浓度(0.25%、0.5%、1%、2%、4%)的几丁质胶体分别反应1 h,采用DNS比色法测定几丁质粗酶酶活性和各体系中的还原糖含量。通过Lineweaver-Burk双倒数作图法进行动力学参数模型测定,计算米氏常数(Km)和最大反应速度(Vmax)值。
1.9 菌株全基因组测序及分析
将菌株接种在LB液体培养基中,30 ℃、160 r/min培养24 h后,置于冷冻离心机中4 ℃、10 000 r/min离心10 min,去除上清液,迅速将菌体置于液氮中保藏,并通过干冰运输至上海凌恩生物科技有限公司进行测序。采用三代测序搭建框架和二代测序进行补洞纠错的方法完成菌株JD-3全基因组的测序,使用Flye软件拼接,获得完整的细菌基因组完成图。利用QUAST和TABLET软
1.10 几丁质酶基因家族鉴定
使用HMMER v.3.4.0软件筛选出符合条件的几丁质酶蛋白。对筛选得到的几丁质酶家族蛋白序列构建最大似然进化树,bootstrap值设定为1 000,其他参数保持默认,以探讨其系统发育关系。利用基因组Gff注释文件,通过比对获得几丁质酶家族蛋白的定位信息,使用TBtools软件进行染色体定位图的绘制和共线性分析,并通过MEME软件预测保守结构基序。
2 结果与分析
2.1 高原林蛙肠道产几丁质酶菌株的筛选
利用几丁质酶筛选培养基,从高原林蛙肠道中分离得到6株几丁质分解菌(

图1 产几丁质酶菌株的筛选
Figure 1 Screening of chitinase-producing strains. A: Preliminary screening results; B: Growth results of strain cultured in culture medium for 4 d.
2.2 JD-3菌株的鉴定
将菌株JD-3接种至LB平板上,于30 ℃恒温培养3 d后,观察到菌落形状为圆形,无色透明,黏稠且易挑起,革兰氏染色为阳性,扫描电镜观察显示菌体呈短棒状(

图2 菌株JD-3的扫描电镜图
Figure 2 Scanning electron microscopy (SEM) of strain JD-3.
将菌株JD-3的16S rRNA测序结果上传至NCBI进行BLAST比对,发现其与麦芽香肉食杆菌(Carnobacterium maltaromaticum) TMW 2.1581的比对率为100%。[核酸序列数据存储在国家微生物科学数据中心,链接为https://nmdc.cn/resource/genomics/sequence/detail/NMDCN0007N9M,编号为NMDCN0007N9M]。使用MEGA 11.0软件构建系统发育树(

图3 菌株JD-3基于16S rRNA基因序列构建的系统发育树
Figure 3 Phylogenetic tree of strain JD-3 based on the 16S rRNA gene sequence. The evolutionary history was inferred using the maximum likelihood method based on the Kimura 2-parameter model. The bootstrap consensus tree inferred from 1 000 replicates is taken to represent the evolutionary history of the taxa analyzed.
2.3 菌株JD-3产酶条件的优化
随着发酵时间的延长,菌株JD-3产生的几丁质粗酶酶活性在第2天时达到最大值0.020 U/mL,随后逐渐下降(

图4 单因素试验对几丁质粗酶酶活性的影响
Figure 4 Effect of single factor experiment on the activity of chitin crude enzyme. A: Fermentation time; B: Initial pH; C: Initial inoculum size; D: Fermentation temperature. The different lowercase letters in the picture indicate significant differences among treatments at P<0.05.
在单因素试验的基础上,确定发酵温度(A)、发酵时间(B)、培养基初始pH (C)为主要影响因子,以这3个影响因子为考察因素,几丁质酶酶活性(Y)为响应值,采用Design-Expert v.11.0设计3因素3水平的Box-Behnken响应面试验,对数据进行拟合分析,得到的二次多项回归方程为:Y=0.011 4+0.000 4A+0.001 0B+0.000 4C-0.000 3AC-0.000 5BC-0.000 6

图5 双因素交互关系的响应曲面图
Figure 5 Response surface diagrams of the interactions between two factors. A: Temperature and pH; B: Temperature and time; C: Time and pH.
2.4 菌株JD-3粗酶液的酶学性质测定
菌株JD-3在最优培养条件下产生的几丁质粗酶活性随温度变化展现出不同的趋势。随着温度的升高,酶活性逐渐下降,在20 ℃时为最大值0.013 U/mL,随温度继续升高而逐渐降低,当温度超过50 ℃时,几丁质酶的活性显著下降,表明其在较低温度下具有较高的活性。结合菌株的培养条件推测几丁质酶在20 ℃内可能达到最大活性(

图6 酶学性质研究
Figure 6 Study on enzymatic properties. A: Temperature; B: Temperature stability; C: pH; D: Acid-base stability. The different lowercase letters in the picture indicate significant differences among treatments at P<0.05.
2.5 反应进程曲线及动力学参数的测定
在最适反应条件下,几丁质酶的反应进程曲线如

图7 反应进程曲线(A)及Lineweaver-Burk曲线(B)
Figure 7 Reaction proceeding curve (A) and Lineweaver-Burk curve (B).
2.6 全基因组分析
为获取高质量的细菌全基因组序列,本研究采用三代PacBio测序结果构建框架,并用二代Illumina测序数据进行补洞和纠错。共获得370 Mb的Illumina二代测序数据和460 Mb的PacBio三代测序数据(原始数据存储在国家微生物科学数据中心,链接为https://nmdc.cn/resource/genomics/sra/detail/NMDC40066841,编号为NMDC40066841)。C. maltaromaticum JD-3菌株全基因组全长为4 195 636 bp,包含6个contig (

图8 菌株JD-3全基因结构注释
Figure 8 JD-3 gene structure annotation. A: Characterization and characteristics of the JD-3 genome (Orbital 1 represents the contig length, orbital 2 represents the gene density, orbital 3 represents the gene G+C content, orbital 4 represents the gene G+C offset, and orbital 5 represents the N base content in the gene); B: Phylogenetic tree based on predicted ribosomal RNA.
Sequence index | Sequence description | Number of repetitive sequences |
---|---|---|
1 | Contig 1 pilonontig 1 pilon | 130 |
2 | Contig 2 pilonontig 1 pilon | 7 |
3 | Contig 3 pilonontig 1 pilon | 34 |
4 | Contig 4 pilonontig 1 pilon | 2 |
5 | Contig 5 pilonontig 1 pilon | 6 |
6 | Contig 6 pilonontig 1 pilon | 2 |
2.7 几丁质酶基因家族鉴定
在细菌全基因组中,鉴定出2个属于GH18家族的几丁质酶家族基因,命名为gh18和gh19。这些基因分布在contig 1的特定区域(

图9 几丁质酶基因家族鉴定
Figure 9 Chitinase gene family identification. A: Chromosome distribution of chitinase gene family; B: Ten motif sequences predicted by chitinase gene family; C: Phylogenetic map of conserved motifs of 3-20 chitinase gene family.
3 讨论
3.1 几丁质分解菌的分离鉴定及功能分析
几丁质分解菌的研究始于20世纪初,最初主要集中在从土壤、废弃物和水体中筛选产酶微生
3.2 C.maltaromaticum JD-3产酶条件及酶学性质分析
为评估C. maltaromaticum 菌株JD-3对几丁质的分解能力,本研究对其产酶条件进行了系统的优化。单因素试验结果显示,发酵温度、时间和培养基初始pH对C. maltaromaticum JD-3几丁质酶活性的影响尤为显著。在最优条件下,C. maltaromaticum JD-3所产的几丁质酶活性为12 mU/mL,不及盐单胞菌(Halomonas)等高产几丁质酶微生
3.3 C.maltaromaticum JD-3全基因组测序及分析
Dang
几丁质酶基因家族根据氨基酸序列的相似性被划分为不同的家族,其中GH18和GH19是最重要的几丁质酶家族。GH18家族的成员通常表现出内切酶活性,能够随机切割几丁质链,而GH19家族成员通常具有外切酶活性,从几丁质链的非还原端开始切
综上所述,菌株JD-3拥有更复杂的基因组结构,可能具有更广泛的代谢能力和环境适应性。基因组的测定为C. maltaromaticum JD-3的深入研究提供了数据支持,也为两栖动物肠道微生物在几丁质分解和资源回收等领域的应用提供了理论依据。
4 结论
从高原林蛙肠道内容物中分离出一株可分解几丁质的菌株JD-3,鉴定为麦芽香肉食杆菌(C. maltaromaticum)。该菌在常温酸性环境中酶活性最高,证实其在极端环境中的适应性。全基因组测序发现C. maltaromaticum JD-3基因组结构复杂,含有2个GH18几丁质酶基因。研究结果丰富了对肉食杆菌属分解几丁质功能的认识,为阐释其分解几丁质提供了数据支撑,也为蛙源微生物资源在生物分解和资源回收领域的应用提供了理论基础。
作者贡献声明
王蕊:论文撰写和修改,数据处理和分析;林星荣:实验执行,数据收集和处理分析;王婉婷:协助实验操作,参与论文讨论;沈迎芳:实验构思和设计,参与论文讨论和修改;张湑泽:实验构思和设计,论文撰写和修改,数据处理和分析。
利益冲突
作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。
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