Cloning, expression and application of a chitinase gene from Micromonospora aurantiaca
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摘要:
从海洋微生物橙黄小单孢菌 (Micromonosprra aurantiaca) 的基因组DNA中克隆到一条新型的碳水化合物18家族几丁质酶基因Machi3,并成功在大肠杆菌 (Escherichia coli) 中表达。该酶的最适反应温度和pH分别为55 ℃和7.0,在低于55 ℃和pH 6.0~9.0范围内稳定性较好。镁离子 (Mg2+)、钙离子 (Ca2+)、吐温40 (Tween 40) 和吐温80 (Tween 80) 对MaChi3的酶活力有轻微的促进作用。该重组酶对α-几丁质、胶体几丁质、虾壳粉、不同脱乙酰度 (50%~95%) 的壳聚糖、淀粉及纤维素均具有水解活性,其中以胶体几丁质为底物时酶活力最高 (2.24 U·mg−1)。由扫描电镜结果可知,几丁质经盐酸 (HCl) 预处理后得到的胶体几丁质纤维结构变得松散,更有利于MaChi3的水解作用。以胶体几丁质为底物时动力学参数Km和Vmax值分别为5.93 mg·mL−1和8.58 μmol·(min·mg)−1。此外,胶体几丁质经MaChi3酶解后生成的主产物为N, N-二乙酰基壳二糖,产率 (几丁质) 为285.54 mg·g−1。该酶展现出良好的酶学特性,为其在几丁质资源中的开发和应用奠定了基础。
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关键词:
- 橙黄小单孢菌 /
- 几丁质酶 /
- 克隆表达 /
- 酶学性质 /
- N, N-二乙酰基壳二糖
Abstract:In this study, a novel carbohydrate 18 family chitinase gene Machi3 was cloned from the genomic DNA of marine microorganism Micromonospra aurantiaca and successfully expressed in Escherichia coli. The optimal reaction temperature and pH for MaChi3 were 55 ℃ and 7.0, respectively. MaChi3 showed good stability below 55 ℃ and at pH of 6−9. The activity of MaChi3 was slightly promoted by Mg2+, Ca2+, Tween 40 and Tween 80. The recombinant chitinase showed hydrolytic activity toward α-chitin, colloidal chitin, shrimp shell powder, chitosan (50%−95% of deacetylation), starch and cellulose, among which the highest activity of 2.24 U mg−1 was observed in colloidal chitin. The results of scanning electron microscopy suggests that the fiber structure of chitin became loose after pretreatment with HCl, so it was more favorable to the hydrolysis of MaChi3. The Km and Vmax values of MaChi3 toward colloidal chitin were 5.93 mg·mL−1 and 8.58 μmol·(min·mg)−1, respectively. In addition, the main product of colloidal chitin hydrolyzed by MaChi3 was N, N-diacetyl chitobiose with a yield of 285.54 mg·g−1. MaChi3 shows good catalytic activity, which is beneficial for its development and application in biotransformation of chitin.
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透皮促进剂 (Penetration enhancers, PE) 是指通过改变角质层的屏障功能[1],以达到增加药物经皮肤到达体内的通透率[2]和减少给药次数和剂量[3]的物质,使用方式通常是将PE与药物混匀,均匀涂抹到表皮组织或喷洒到黏膜组织[4−5],或通过药浴给药[6−7]。常见的PE有石菖蒲 (Acorus tatarinowii) 挥发油和水溶性氮酮。石菖蒲的主要功能成分为挥发油,主要活性成分为细辛醚类[8],具有抗氧化、抗血栓、抗抑郁和药物促透等作用,由于其促透效果卓著而备受关注[9−11]。水溶性氮酮为一种合成的PE,因其促透率高、副作用小,已被广泛应用于医药、农业和日化行业。水产养殖种苗捕捞、运输过程中,通常使用安全可靠的麻醉剂。丁香酚和间氨基苯甲酸乙酯甲磺酸盐 (三卡因、MS-222) 是2种常用的麻醉剂[12−13],但因其存在安全隐患而被多次报道[14−19]。目前,PE在畜禽中使用较多,而在水产中研究甚少。本文以中国花鲈 (Lateolabrax maculatus) 幼鱼为实验对象,结合鳃组织的抗氧化指标分析,评价了石菖蒲挥发油和水溶性氮酮对丁香酚或MS-222的促透效果,明确最佳促透剂含量,以期为鱼类麻醉应激的防护和PE在水产上的应用提供参考。
1. 材料与方法
1.1 实验材料
中国花鲈幼鱼购自广东省珠海市某育苗厂。运输至实验室后先经1 g·m−3的碘制剂消毒,再置于室内水泥池 (3 m×4 m×1 m) 暂养2周以上,暂养密度为10尾·m−3。暂养期间,投喂海水鱼配合饲料 (广州海龙饲料有限公司),每日早晚各1次,投喂质量为鱼体质量的 (3±1)%。日换水1次,换水率30%。水温 (28±0.5)℃,溶解氧始终高于6 mg·L−1,pH 7.6±0.2,自然光周期。实验所用石菖蒲挥发油 (挥发油≥99%) 和水溶性氮酮 (纯度≥99%) 均购自湖北省康纯香料有限公司;丁香酚 (纯度≥98%) 购于北京迈瑞达科技有限公司;MS-222 (纯度≥99%) 购于青岛文鼎商贸有限公司。
1.2 实验方法
1.2.1 促透实验
MS-222与碳酸氢钠以质量比1∶1溶于纯水,配制成母液,为防止光分解,保存于棕色玻璃瓶中备用[20];以体积比1∶10将丁香酚溶于乙醇,制成母液备用 (MS-222和丁香酚各制备9份母液)。参照白一岑等[11]方法,将石菖蒲挥发油和水溶性氮酮以1∶10体积比溶于乙醇,向配制好的MS-222和丁香酚母液中分别加入质量分数为1%、4%、7%和10%的石菖蒲挥发油和1%、3%、5%和10%的水溶性氮酮 (PE与麻醉剂质量比) 配成促透麻醉液,同时设置乙醇为对照组。参照Wang等[14]的分类标准和麻醉剂量,将麻醉诱导过程简化为深度镇静阶段(对强刺激有反应;鳃盖张合频率减少;平衡正常)和麻醉阶段(完全丧失肌肉张力和平衡;鳃盖张合频率慢),复苏评判标准为完全复苏(对外界刺激有反应;鳃盖张合和肌肉收缩正常)。选用10 mg·L−1丁香酚或50 mg·L−1MS-222对幼鱼进行麻醉 (此浓度的MS-222或丁香酚可诱导至麻醉阶段,但不能诱导至深度麻醉阶段)。平均体质量为 (98.1±7.3) g的幼鱼停食24 h后用于麻醉实验。先向白色塑料桶中加入10 L曝气2 d以上的自来水;然后加入麻醉液或促透麻醉液,使丁香酚或MS-222质量浓度分别达到10 mg·L−1或50 mg·L−1;最后将幼鱼置于麻醉桶中,记录每条幼鱼进入镇静和麻醉状态所用时间 (每个处理组10次重复,每尾鱼单独观察并只使用1次)。麻醉后幼鱼被转移至无麻醉液的复苏桶中进行复苏,记录复苏所用时间。此外,将幼鱼分别置于5 mg·L−1石菖蒲挥发油和5 mg·L−1水溶性氮酮溶液 (本实验用到最大剂量的PE),观察幼鱼能否进入镇静状态或麻醉状态,以排除2种促皮渗透剂是否对幼鱼具有麻醉效果。
1.2.2 麻醉剂减量实验
根据促透实验得出质量分数为7%石菖蒲挥发油和3%水溶性氮酮促进麻醉效果最佳,将8 mg·L−1 丁香酚或40 mg·L−1MS-222与质量分数为7%石菖蒲挥发油或3%水溶性氮酮混合,同时设置无促透剂的对照组。进行麻醉实验,记录镇静、麻醉和复苏所用时间。
1.2.3 生化指标测定
设置对照组 (AC)、10 mg·L−1丁香酚 (EC)、8 mg·L−1丁香酚+7%石菖蒲挥发油 (ES)、8 mg·L−1 丁香酚+4%水溶性氮酮 (ED)、50 mg·L−1MS-222 (MC)、40 mg·L−1 MS-222+7%石菖蒲挥发油 (MS) 和40 mg·L−1 MS-222+4%水溶性氮酮 (MD) 共7个处理组。将幼鱼按照不同组处理后进行复苏,复苏后6 h [ 0~1 h内丁香酚在罗非鱼 (Oreochromis spp. ) 肝脏中的浓度逐渐升高,第1小时达到峰值,之后逐渐降低[21];MS-222在白斑角鲨 (Squalus acanthias) 血液中的半衰期约为1.5~4 h [22],考虑到药物在不同动物中的代谢动力学未必相同,故选择在复苏后6 h取样],采集鳃组织进行抗氧化指标测定。取4 ℃无菌生理盐水先对鳃组织进行冲洗,再用9倍体积的4 ℃无菌生理盐水 (m/V) 将鳃组织匀浆,在低温离心机中以6 000 ×g离心20 min,收集上清液备用。酶活检测所用试剂盒均购自南京建成生物工程研究所,相应操作参照说明书进行。采用考马斯亮蓝法测定总蛋白浓度 (以牛血清为基准);采用黄呤氧化酶法测定超氧化物歧化酶 (SOD) 活性 (组织中每毫克蛋白在0.24 mL反应液中,抑制率达50%时所对应的酶量定为1个酶活力单位);采用比色法测定过氧化物酶 (POD) 活性 (在37 ℃条件下,每毫升样品每分钟催化1 μg底物的酶量定义为1个酶活力单位);采用紫外法测定过氧化氢酶 (CAT) 活性 [组织中每毫克蛋白每秒分解1 μmol的过氧化氢 (H2O2)的量为1个酶活力单位];采用比色法测定谷胱甘肽 (GSH) 浓度 (二硫代二硝基苯甲酸与巯基化合物反应生成黄色化合物,可进行比色定量测定);采用硫代巴比妥酸法测定丙二醛 (MDA) 浓度 (过氧化脂质降解产物中的MDA可与硫代巴比妥酸缩合,形成红色产物,进行定量测定)。
1.2.4 数据处理
所有数据均以“平均数±标准差 (
$\overline X\pm {\rm{SD}}$ )”表示。使用SPSS 22.0软件对实验数据进行统计学分析。在单因素方差分析的基础上,采用Duncan多重比较法进行分析。显著性水平设定为P<0.05。使用ORIGIN 8.0软件对实验数据进行绘图。2. 结果
2.1 PE对丁香酚和MS-222的促透效果
随着2种PE质量分数的增加,幼鱼进入镇静阶段和麻醉阶段的时间呈现先减少后增加的趋势 (质量分数为7%的石菖蒲挥发油和3%的水溶性氮酮达到峰值),完全复苏的时间同样呈现先减少后增加的趋势 (质量分数为7%的石菖蒲挥发油和3%的水溶性氮酮达到峰值,图1)。将幼鱼置于5 mg·L−1石菖蒲挥发油、5 mg·L−1水溶性氮酮溶液,均未观察到镇静与麻醉状态。
图 1 中国花鲈达到镇静、麻醉和完全复苏的时间 (n=10)a. 丁香酚 (E) +石菖蒲挥发油 (S);b. MS-222 (M) +石菖蒲挥发油 (S);c. 丁香酚 (E) +水溶性氮酮 (D);d. MS-222 (M) +水溶性氮酮 (D);组间字母不同者差异显著 (P<0.05)Figure 1. Time to achieve sedation, anesthesia and complete resuscitation of L. maculatus (n=10)a. Eugenol (E) + A. tatarinowii essential oil (S); b. MS-222 (M) + A. tatarinowii essential oil (S); c. Eugenol (E) + water soluble azone (D); d. MS-222 (M) + water soluble azone (D); values with different letters were significantly different (P<0.05).2.2 PE对麻醉剂减量使用效果
向低剂量麻醉剂 (麻醉剂减量20%) 中加入质量分数为7%的石菖蒲挥发油或3%的水溶性氮酮后,可达到与高剂量麻醉剂相似的麻醉效果 (图1)。低剂量麻醉剂加入质量分数为7%的石菖蒲挥发油后,进入麻醉和复苏的时间较其对照组显著降低 (图1-a、b)。低剂量麻醉剂加入质量分数为3%的水溶性氮酮后,复苏时间较其对照组显著降低 (图1-c、d)。低剂量麻醉剂不加PE,仅进入镇静阶段,无法进入麻醉阶段 (图2)。
图 2 中国花鲈达到镇静、麻醉和完全复苏的时间 (n=10)E. 丁香酚;S. 石菖蒲挥发油;D. 水溶性氮酮;M. MS-222;组间字母不同者差异显著 (P<0.05);N. 未观察到Figure 2. Time to achieve sedation, anesthesia and complete resuscitation of L. maculatus (n=10)E. Eugenol; S. A. tatarinowii essential oil; D. Water soluble azone; M. MS-222; values with different letters were significantly different (P<0.05); N. Not observed2.3 鳃组织抗氧化指标测定
对鳃组织抗氧化指标分析发现,EC和MC组较AC组POD、SOD、CAT活性和MDA、GSH浓度显著升高 (P<0.05);EC和MC组分别较ES和MS组POD、SOD、CAT活性和MDA、GSH浓度显著升高 (除EC组的POD活性,图3-a,P<0.05);MC组较MD组SOD活性和MDA浓度显著升高 (P<0.05);ED组较ES组SOD、CAT活性和MDA、GSH浓度出现升高,但不显著 (P>0.05);MD组较MS组CAT活性显著升高 (P<0.05),POD、SOD活性和MDA、GSH浓度也出现升高,但不显著 (P>0.05,图3)。
图 3 鳃组织抗氧化指标 (n=5)a. 过氧化物酶;b. 超氧化物歧化酶;c. 过氧化氢酶;d. 丙二醛;e. 谷胱甘肽;AC. 对照组;EC. 10 mg·L−1丁香酚;ES. 8 mg·L−1丁香酚+7%石菖蒲挥发油;ED. 8 mg·L−1丁香酚+3%水溶性氮酮;MC. 50 mg·L−1 MS-222;MS. 40 mg·L−1 MS-222+7%石菖蒲挥发油;MD. 40 mg·L−1 MS-222+3%水溶性氮酮;组间大写或小写字母不同者差异显著 (P <0.05)Figure 3. Antioxidant indices of gill tissue (n=5)a. POD; b. SOD; c. CAT; d. MDA; e. GSH; AC. Control group; EC. 10 mg·L−1 eugenol; ES. 8 mg·L−1 eugenol + 7% A. tatarinowii essential oil; ED. 8 mg·L−1 eugenol + 3% water soluble azone; MC. 50 mg·L−1 MS-222; MS. 40 mg·L−1 MS-222 + 7% A. tatarinowii essential oil; MD. 40 mg·L−1 MS-222 + 3% water soluble azone; values with different uppercase or lowercase letters were significantly different.3. 讨论
3.1 PE对丁香酚与MS-222促透分析
本研究将幼鱼单独置于2种PE溶液中均未观察到镇静和麻醉状态,排除了该质量分数的石菖蒲挥发油和水溶性氮酮的麻醉效应。研究发现,麻醉剂加入2种PE,均能缩短中国花鲈镇静、麻醉和复苏的时间,这可能是因为石菖蒲挥发油和水溶性氮酮对2种麻醉剂的吸收具有促进作用,或是麻醉剂和PE具有协同作用,亦或是PE对麻醉效果具有加成作用。
质量分数为7%的石菖蒲和3%的氮酮促透效果最佳,且促透倍数相似,该结果与王建新和郭力 [23]、白一岑等[11]的相似。王建新和郭力[23]研究表明,质量分数为3%的水溶性氮酮对如意巴布剂能达到最大促透效果,质量分数过低或过高促透效果均出现减弱,表明PE并非质量分数越大效果越好。本研究中,随着2种PE质量分数增加,麻醉剂的麻醉效果呈先增强后减弱的趋势,这一结果说明PE对2种麻醉剂具有促透效果 (若无促透效果,随PE质量分数增加,麻醉剂的麻醉效果不会出现减弱),但不排除存在协同和加成作用。目前,PE的增效方式暂不清晰,未来可根据麻醉剂在体内的浓度进行更深入的研究。
3.2 PE减少麻醉剂剂量分析
本研究显示,8 mg·L−1的丁香酚和40 mg·L−1的MS-222不能使幼鱼进入麻醉状态,加入质量分数为7%的石菖蒲挥发油或3%的水溶性氮酮后,幼鱼能顺利进入麻醉状态 (图2),并且与10 mg·L−1丁香酚和50 mg·L−1MS-222的麻醉效果相似 (图1),表明通过向麻醉剂加入PE可以减少麻醉剂使用剂量。这可能是由于PE改变了细胞脂质双分子层结构,提高了麻醉剂的通透率和吸收速率,从而降低了对麻醉剂的需求。杨晓春等[3]报道PE能减少给药剂量,这一结论与本研究结果一致。此外,有研究提出PE可增加经皮到达体内药物的有效治疗浓度[24]。因此,通过向麻醉剂中加入PE,降低麻醉剂使用剂量可行。
3.3 鳃组织氧化应激比较分析
鳃是参与呼吸、酸碱平衡、离子平衡、含氮废物排泄、渗透调节等多功能的组织[25-26],也被认为是衡量水质的生物指标[27-28],是鱼体与外界水环境互作的重要通道。此外,鳃也是吸收麻醉剂的重要器官。当细胞受到氧化应激时,SOD和CAT是第一道抗氧化防线。SOD和CAT将超氧化物转化为水 (H2O) 和氧气 (O2),消除了自由基的不利影响[29-30],在此过程中POD调节H2O2的水平[31]。因此,POD、SOD和CAT活性是衡量细胞抗氧化能力的关键标志物[32-33]。Ma等[34]研究发现将鲤 (Cyprinus carpio) 暴露于草甘膦后,鳃组织中的SOD和CAT活性升高;姜会民[35]研究发现,鲤暴露于低剂量氯化汞 (HgCl2) POD活性出现升高。GSH是细胞中重要的抗氧化剂,可以中和自由基或氧化剂,从而对抗过氧化损伤[36-37];MDA是脂质氧化损伤的重要生物标志物[38-39]。Jiao等[26]发现将鲤暴露于毒死婢后,鳃组织中GSH和MDA浓度显著增加。本研究中,2种麻醉对照组较乙醇对照组POD、SOD、CAT活性和MDA、GSH浓度显著升高 (P<0.05),可能是MS-222和丁香酚对幼鱼的鳃细胞造成了氧化应激,导致鳃细胞抗氧化酶活性和抗氧化剂浓度升高。MS-222和丁香酚造成的麻醉应激是否会对鳃细胞造成损伤,还有待进一步研究。此外,2种麻醉对照组较促透麻醉组的抗氧化物显著上升 (P<0.05),这可能是通过使用PE减少了MS-222或丁香酚的使用剂量,降低了鳃组织所接触麻醉剂浓度,进而减轻了MS-222或丁香酚对鳃组织造成的氧化应激。因此,通过PE降低麻醉剂的使用剂量来减少对鳃的氧化应激可行。
石菖蒲挥发油和水溶性氮酮对丁香酚和MS-222有明显的促麻醉效果,并能缩短中国花鲈的入麻时间和复苏时间,但石菖蒲挥发油和水溶性氮酮用于水产品的食品安全性尚有待论证。
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图 1 MaChi3 基因 PCR 扩增产物 (a)、保守域分析 (b) 和部分催化域氨基酸多序列比对 (c)
注:(a) M为DNA分子量标准;(c)图中列出的几丁质酶氨基酸序列来源于B. circulans WL-12 (PDB ID : 1ITX),C. shinanonensis (PDB ID : 6KXL),B. thuringiensis (PDB ID : 6BT9),Paenibacillus sp. FPU-7 (PDB ID : 5GZU) 和L. lactis ssp. Lactis (NCBI登录号AAK06048)。保守基序SxGG和DxDxE在底部用带下划线的红色字母标识,保守的酪氨酸残基在上部用※标识。
Figure 1. PCR products (a), conserved domain analysis (b) and sequence alignment of partial predicted catalytic domain (c) of MaChi3
Note: (a) M is DNA molecular mass standard. (c) The listed sequences include the chitinases from B. circulans WL-12 (PDB ID: 1ITX), C. shinanonensis (PDB ID: 6KXL), B. thuringiensis (PDB ID: 6BT9), Paenibacillus sp. FPU-7 (PDB ID: 5GZU) and L. lactis ssp. Lactis (NCBI accession NO.: AAK06048). The conserved sequence motifs SxGG and DxDxE are showed at the bottom with red color underlined, and the conserved tyrosine residue is indicated by ※.
表 1 化学试剂和金属离子对重组酶 MaChi3 酶活力的影响
Table 1 Effects of metal ions and chemical reagents on activity of recombinant chitinase MaChi3
化学试剂
Chemical reagent终浓度/体积分数
Final concentration/
Volume fraction相对酶活力
Relative activity/
%对照 Control 100 尿素 Urea 200 mmol·L−1 70.9±0.8 乙二胺四乙酸 EDTA 10 mmol·L−1 94.2±1.6 十二烷基硫酸钠 SDS 10 mmol·L−1 58.1±0. 5 吐温20 Tween 20 1% 90.4±0.8 吐温40 Tween 40 1% 104.1±1.6 吐温60 Tween 60 1% 79.6±1.0 吐温80 Tween 80 1% 108.4±0.8 曲拉通 X-100 Triton X-100 1% 101.0±0.9 钾离子 K+ 1 mmol·L−1 86.4±0.7 银离子 Ag+ 1 mmol·L−1 43.3±0.4 镁离子 Mg2+ 1 mmol·L−1 117.7±0.7 亚铁离子 Fe2+ 1 mmol·L−1 65.4±0.7 钙离子 Ca2+ 1 mmol·L−1 107.3±0.4 钴离子 Co2+ 1 mmol·L−1 87.2±0.7 钡离子 Ba2+ 1 mmol·L−1 99.5±1.3 锌离子 Zn2+ 1 mmol·L−1 97.6±1.1 铜离子 Cu2+ 1 mmol·L−1 45.0±0.7 铁离子 Fe3+ 1 mmol·L−1 59.3±1.1 -
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