红树林生境产纤维素酶菌株筛选、鉴定、产酶条件及酶学性质研究
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摘要
纤维素酶是几种可以协同降解纤维素为葡萄糖的酶的总称,包括葡聚糖内切酶(EG)、葡聚糖外切酶(CBH)和β-葡萄糖苷酶(BG)等三种酶,滤纸酶(FPase)活力则代表各种酶协同作用后的总酶活力。纤维素酶在生物能源、食品、纺织、造纸、洗涤、饲料、医药等领域有着重要的应用,是继糖化酶、淀粉酶和蛋白酶之后的第四大工业酶种,甚至在我国完全有可能成为第一大酶种,成为酶制剂工业中的一个新的增长点。因此,筛选纤维素酶高产菌具有重要的研究价值。广西壮族自治区拥有中国面积最大的红树林,由于红树林生境非常特殊,微生物产酶也具有特殊性,在红树林内,凋落物非常丰富,在凋落物分解过程中微生物产生的酶以纤维素分解酶、木质素分解酶为主,来自于红树林产纤维素酶的微生物是目前研究的一个重要方向。本文对红树林高产纤维素酶菌株进行了分离筛选、鉴定和产酶条件优化,并对筛选得到的菌株所产的纤维素酶进行了纯化和酶学性质研究,同时以购买的目前产纤维素酶较高的菌株G1M3.140(购于广东微生物菌种保藏中心)为对照进行产酶对比,以期获得高产纤维素酶的产生菌,为新来源纤维素酶的生产提供理论依据。主要结果如下：
产纤维素酶菌株的分离筛选：从广西北仑河口国家级红树林自然保护区采集的桐花树和木榄的树根、树皮、海水和土壤中进行菌株分离,利用刚果红染色法初步分离筛选,共得到78株具有降解CMC-Na能力的菌株,其中大部分属于真菌,放线菌次之,细菌最少,通过滤纸条崩解实验和酶活性实验最终筛选出一株高产纤维素酶的菌株,命名为GA-44。
GA-44形态及种属鉴定：在PDA平板上28℃培养,GA-44菌株菌落生长快,24h后菌落为白色。菌落表面较粗糙,背面无色,中央有一绿色产孢区。老熟的菌落正面为暗绿色,背面无色。光学显微镜下菌丝分枝,有横隔,孢子呈圆形,分生孢子呈串状排列。提取菌株GA-44的DNA,用PCR法扩增GA-44的18S rRNA基因的V8-V9片段,经北京华大基因公司测序,V8-V9序列长为378bp,其GenBank号为lCl|52991。通过BLAST分析,以及采用Neighbor Joining方法构建系统进化树,发现GA-44的18S rRNA与多种真菌的一致性达到99%,系统树分析结果显示GA-44与微紫青霉(Penicillium janthinellum)亲缘关系较近。表明GA-44为一产纤维素酶的青霉属菌株。
GA-44产酶条件的优化：采用液体培养方式,对其培养条件进行单因素和正交试验,以CMC、FPase和CBH酶活力为检测指标。在单因素试验的基础上,选择廉价易得的底物和对产酶影响较大的发酵时间、产酶温度、起始pH,按照L9(34)正交表进行正交试验,得出在以麸皮为底物、28℃～32℃、pH6.5～7.5的条件下发酵108h是较好的发酵条件,CMC、FPase和CBH的酶活分别达到了1038U/mL、1056U/mL和179U/mL,同等发酵条件下比G1M3.140(购于广东微生物菌种保藏中心)有更高的CMC酶活和FPase酶活,尤其是CMC更是G1M3.140的3倍。滤纸崩解实验显示,菌株GA-44在96h内可以将滤纸条完全崩解,而绿色木霉G1M3.140则需要144h。菌株GA-44的CMC、FPase和CBH在NaCl含量为1%～3%的发酵培养基中均保持较高的活性,并且发现该菌没有表现出嗜盐现象,由此推测该菌株可能是从陆地转移到海洋。
纤维素酶的纯化及酶学性质研究：采用盐析和柱层析方法进行酶的纯化。菌株GA-44经液态发酵,提取纤维素酶粗酶液,经硫酸铵盐析,Sephadex G-25脱盐和Sephadex G-100柱层析,经高效液相色谱检测,结果显示出三个峰。根据标准蛋白质分子量大小和保留时间得出三种酶的相对分子质量分别为56.4KD、52.6KD和49.0KD,初步判断分别是β-葡萄糖苷酶、内切葡聚糖酶EG和外切葡聚糖酶CBH。CMC的纯化倍数和酶活回收率分别为8.5倍和26.5%,CBH的纯化倍数和酶活回收率分别为29.0倍和87.6%,滤纸酶FPase的纯化倍数和酶活回收率分别为16.9倍和57.2%,表明纯化倍数和回收率都处于较高水平。采用不同温度和不同pH进行酶学性质研究,结果表明该纤维素酶系最适反应条件为：CMC为55℃、pH3.2；CBH为45℃、pH3.2；FPase为55℃、pH3.6,其最大酶活分别为：1262U/mL、612U/mL和943U/mL。
Cellulolytic enzymes system involved in the hydrolysis of cellulose consists of endoglucanases (EC3.2.1.4, EG), cellobiohydrolases (EC3.2.1.91, CBH) and beta-glucosidases (EC3.2.1.21, BGL),which work together to hydrolyze cellulose to glucose. Cellulases are the fourth industrially important enzymes following glucoamylase, amylase and protease having application in diverse industries such as bioenergy, food, textile, paper, washing, forage and medicine industry, it even may become the first majority industrially enzymes and a new growth point in enzyme preparation industry in our China. So it is important and necessary to screen strains which yield high cellulase activity. Guangxi Zhuang Autonomous Region has the largest mangrove forest in China. Due to the ecologicalhabitat of mangrove forest is very particular, microbial enzyme also has particularity. In mangrove forest, litter is very abundant, the major enzymes are cellulolytic enzyme and lignin decomposing enzyme during the process of microorganism decompose litter. In this paper we did a job on screen and identify the strains which have high enzyme activity, optimized the conditions of cellulases production, purified the cellulases from the candidate strain and researched the property of the cellulases. At the same time we bought a high yield of cellulose enzyme strain named G1M3.140(purchased from Guangdong culture collection center) as controls on contrast enzyme production, so as to get high yield of cellulose enzyme produce bacteria, provide the theory for new sources of cellulose enzyme production. The main results are as follows:
Screen the strains which have high enzyme activity:The samples include roots and skins of aegiceras and bruguiera,sea water and soil collected from Beilunhekou National Mangrove Nature Reserve, through the isolation and pre-screening, we got78strains which can decompose CMC-Na, most of them belong to the fungus, actinomycetes is the second, and bacteria is the least. Through the experiment of disintegrate filter paper strip and test enzymatic activity, we got a strain which was designed GA-44had a high enzymatic activity.
Identify GA-44:The colony of the strain GA-44could grow rapidly on PDA plate at28℃, the color of the strain was white after24h culturing. The surface of the colony was rough, and the back was colorless, there was a round-shaped green zone for spore production in the center of the colony. Aging colony was filamentous which has the dark green positive contrary to the colorless back, the center of the colony was sagged, and the shape of whole colony was three circles. The hypha with horizontal septum had more branches, and the conidial was circular and arranged by bunch observed under optical microscope. Extracted the DNA of GA-44, and with the extraction as a template, The V8-V9region of gene18S rRNA of378bp was amplified by PCR, and sequenced (query ID:lcl|52991) by BGI-Beijing. By the nucleotide blast program, it had99%Homology to several fungi. The distance tree showed the strain GA-44have a close genetic relationship with Penicillium janthinellum by method of neighbor joining.
Optimization of cellulases production from the strain GA-44:Basic on researched each single factor, we selected cheap and facile of substrate, incubation time, temperature and initial pH, designed each factor levels, through orthogonal test followed by L9(34) of orthogonal table, we obtained the optimum cellulases production of GA-44were:wheat bran as substrate, temperature range between28℃and32℃, pH range between6.5and7.5and108h of incubation time, the enzyme activity of CMC, FPase and CBH reached1038U/mL,1056U/mL and179U/mL respectively, higher CMC and FPase enzyme activity than G1M3.140(purchased from Guangdong culture collection center) under the same condition of fermentation, especially3times CMC enzyme activity of GA-44than G1M3.140. Disintegration of filter paper strip confirmed that G1M3.140needed144h to disintegrate filter paper strip but GA-44only needed96h. The strain of GA-44still had high enzyme activity when the fermentation medium contained sodium chloride which concentration range from1%(w/v) to3%(w/v), no halophilic phenomenon happened, so it maybe came from land.
Study on purification and characterization of GA-44. Cellulase has been extracted from the liquid media fermentation by Penicillium strain GA-44from GuangXi mangrove. The crude cellulase was purified by ammonium sulfate precipitation, Sephadex G-25desalination and Sephadex G-100gel filtration, The purified cellulase showed that the three distinct protein peaks assayed by HPLC. According to the molecular weight and the retention time, showed molecular weights about56.4KD,52.6KD and49.0KD, respectively, the three compositions were assayed as β-1,4-glucosidase, EG and CBH, the purification multiples and enzyme recovery rate of CMC was8.5times and26.5%, the purification multiples and enzyme recovery rate of CBH was29.0times and87.6%, the purification multiples and enzyme recovery rate of FPase was16.9times and57.2%.The properties of the cellulase show that the optimal reactions of the cellulose were: CMC was55℃and pH3.2; CBH was45℃and pH3.2; FPase was55℃and pH3.6, the maximum enzyme of them was1262U/mL、612U/mL and943U/mL.
产纤维素酶菌株的分离筛选：从广西北仑河口国家级红树林自然保护区采集的桐花树和木榄的树根、树皮、海水和土壤中进行菌株分离,利用刚果红染色法初步分离筛选,共得到78株具有降解CMC-Na能力的菌株,其中大部分属于真菌,放线菌次之,细菌最少,通过滤纸条崩解实验和酶活性实验最终筛选出一株高产纤维素酶的菌株,命名为GA-44。
GA-44形态及种属鉴定：在PDA平板上28℃培养,GA-44菌株菌落生长快,24h后菌落为白色。菌落表面较粗糙,背面无色,中央有一绿色产孢区。老熟的菌落正面为暗绿色,背面无色。光学显微镜下菌丝分枝,有横隔,孢子呈圆形,分生孢子呈串状排列。提取菌株GA-44的DNA,用PCR法扩增GA-44的18S rRNA基因的V8-V9片段,经北京华大基因公司测序,V8-V9序列长为378bp,其GenBank号为lCl|52991。通过BLAST分析,以及采用Neighbor Joining方法构建系统进化树,发现GA-44的18S rRNA与多种真菌的一致性达到99%,系统树分析结果显示GA-44与微紫青霉(Penicillium janthinellum)亲缘关系较近。表明GA-44为一产纤维素酶的青霉属菌株。
GA-44产酶条件的优化：采用液体培养方式,对其培养条件进行单因素和正交试验,以CMC、FPase和CBH酶活力为检测指标。在单因素试验的基础上,选择廉价易得的底物和对产酶影响较大的发酵时间、产酶温度、起始pH,按照L9(34)正交表进行正交试验,得出在以麸皮为底物、28℃～32℃、pH6.5～7.5的条件下发酵108h是较好的发酵条件,CMC、FPase和CBH的酶活分别达到了1038U/mL、1056U/mL和179U/mL,同等发酵条件下比G1M3.140(购于广东微生物菌种保藏中心)有更高的CMC酶活和FPase酶活,尤其是CMC更是G1M3.140的3倍。滤纸崩解实验显示,菌株GA-44在96h内可以将滤纸条完全崩解,而绿色木霉G1M3.140则需要144h。菌株GA-44的CMC、FPase和CBH在NaCl含量为1%～3%的发酵培养基中均保持较高的活性,并且发现该菌没有表现出嗜盐现象,由此推测该菌株可能是从陆地转移到海洋。
纤维素酶的纯化及酶学性质研究：采用盐析和柱层析方法进行酶的纯化。菌株GA-44经液态发酵,提取纤维素酶粗酶液,经硫酸铵盐析,Sephadex G-25脱盐和Sephadex G-100柱层析,经高效液相色谱检测,结果显示出三个峰。根据标准蛋白质分子量大小和保留时间得出三种酶的相对分子质量分别为56.4KD、52.6KD和49.0KD,初步判断分别是β-葡萄糖苷酶、内切葡聚糖酶EG和外切葡聚糖酶CBH。CMC的纯化倍数和酶活回收率分别为8.5倍和26.5%,CBH的纯化倍数和酶活回收率分别为29.0倍和87.6%,滤纸酶FPase的纯化倍数和酶活回收率分别为16.9倍和57.2%,表明纯化倍数和回收率都处于较高水平。采用不同温度和不同pH进行酶学性质研究,结果表明该纤维素酶系最适反应条件为：CMC为55℃、pH3.2；CBH为45℃、pH3.2；FPase为55℃、pH3.6,其最大酶活分别为：1262U/mL、612U/mL和943U/mL。
Cellulolytic enzymes system involved in the hydrolysis of cellulose consists of endoglucanases (EC3.2.1.4, EG), cellobiohydrolases (EC3.2.1.91, CBH) and beta-glucosidases (EC3.2.1.21, BGL),which work together to hydrolyze cellulose to glucose. Cellulases are the fourth industrially important enzymes following glucoamylase, amylase and protease having application in diverse industries such as bioenergy, food, textile, paper, washing, forage and medicine industry, it even may become the first majority industrially enzymes and a new growth point in enzyme preparation industry in our China. So it is important and necessary to screen strains which yield high cellulase activity. Guangxi Zhuang Autonomous Region has the largest mangrove forest in China. Due to the ecologicalhabitat of mangrove forest is very particular, microbial enzyme also has particularity. In mangrove forest, litter is very abundant, the major enzymes are cellulolytic enzyme and lignin decomposing enzyme during the process of microorganism decompose litter. In this paper we did a job on screen and identify the strains which have high enzyme activity, optimized the conditions of cellulases production, purified the cellulases from the candidate strain and researched the property of the cellulases. At the same time we bought a high yield of cellulose enzyme strain named G1M3.140(purchased from Guangdong culture collection center) as controls on contrast enzyme production, so as to get high yield of cellulose enzyme produce bacteria, provide the theory for new sources of cellulose enzyme production. The main results are as follows:
Screen the strains which have high enzyme activity:The samples include roots and skins of aegiceras and bruguiera,sea water and soil collected from Beilunhekou National Mangrove Nature Reserve, through the isolation and pre-screening, we got78strains which can decompose CMC-Na, most of them belong to the fungus, actinomycetes is the second, and bacteria is the least. Through the experiment of disintegrate filter paper strip and test enzymatic activity, we got a strain which was designed GA-44had a high enzymatic activity.
Identify GA-44:The colony of the strain GA-44could grow rapidly on PDA plate at28℃, the color of the strain was white after24h culturing. The surface of the colony was rough, and the back was colorless, there was a round-shaped green zone for spore production in the center of the colony. Aging colony was filamentous which has the dark green positive contrary to the colorless back, the center of the colony was sagged, and the shape of whole colony was three circles. The hypha with horizontal septum had more branches, and the conidial was circular and arranged by bunch observed under optical microscope. Extracted the DNA of GA-44, and with the extraction as a template, The V8-V9region of gene18S rRNA of378bp was amplified by PCR, and sequenced (query ID:lcl|52991) by BGI-Beijing. By the nucleotide blast program, it had99%Homology to several fungi. The distance tree showed the strain GA-44have a close genetic relationship with Penicillium janthinellum by method of neighbor joining.
Optimization of cellulases production from the strain GA-44:Basic on researched each single factor, we selected cheap and facile of substrate, incubation time, temperature and initial pH, designed each factor levels, through orthogonal test followed by L9(34) of orthogonal table, we obtained the optimum cellulases production of GA-44were:wheat bran as substrate, temperature range between28℃and32℃, pH range between6.5and7.5and108h of incubation time, the enzyme activity of CMC, FPase and CBH reached1038U/mL,1056U/mL and179U/mL respectively, higher CMC and FPase enzyme activity than G1M3.140(purchased from Guangdong culture collection center) under the same condition of fermentation, especially3times CMC enzyme activity of GA-44than G1M3.140. Disintegration of filter paper strip confirmed that G1M3.140needed144h to disintegrate filter paper strip but GA-44only needed96h. The strain of GA-44still had high enzyme activity when the fermentation medium contained sodium chloride which concentration range from1%(w/v) to3%(w/v), no halophilic phenomenon happened, so it maybe came from land.
Study on purification and characterization of GA-44. Cellulase has been extracted from the liquid media fermentation by Penicillium strain GA-44from GuangXi mangrove. The crude cellulase was purified by ammonium sulfate precipitation, Sephadex G-25desalination and Sephadex G-100gel filtration, The purified cellulase showed that the three distinct protein peaks assayed by HPLC. According to the molecular weight and the retention time, showed molecular weights about56.4KD,52.6KD and49.0KD, respectively, the three compositions were assayed as β-1,4-glucosidase, EG and CBH, the purification multiples and enzyme recovery rate of CMC was8.5times and26.5%, the purification multiples and enzyme recovery rate of CBH was29.0times and87.6%, the purification multiples and enzyme recovery rate of FPase was16.9times and57.2%.The properties of the cellulase show that the optimal reactions of the cellulose were: CMC was55℃and pH3.2; CBH was45℃and pH3.2; FPase was55℃and pH3.6, the maximum enzyme of them was1262U/mL、612U/mL and943U/mL.
引文
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[30]李厚金,林永成,刘晓红,等.红树林内源真菌2524号的肽类成分(Ⅰ)[J].中山大学学报(自然科学版).2002,41(1)：110-112.
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[1]林永成.海洋微生物及其代谢产物[M].北京：化学工业出版社.2003.
[2]洪葵,谢晴宜等.药用微生物资源研究技术[M].北京：中国农业大学出版社,2006.
[3]358996929.第一步分离菌(实验计划书):http://wenku.baidu.com/view/28a22999-51e79b89680226dl.html,2010-10-09.
[4]北京纺织信息网.纤维素酶DNS酶活力测定方法.：1ittp://www.bjtextile.com/pro-duct/xwsmhlcd.htm.
[5]Martin Schulein. Enzymatic properties of cellnlases from Humicola insolens. Journal of Biotechnology [J].1997,57-71.
[6]韩 韫,蔡俊鹏.产纤维素酶海洋菌株的筛选及鉴定[J].现代食品科技,2005,21(3)：36-44.
[7]Ma D B,Gao P J,Wang Z N.Preliminary studies on the mechanism of cellulase formation by trichoderma pseudokoningii S238[J].Enzyme Microb Tech,1990, 12(3):631-635.
[8]朴哲,崔宗均,苏宝琳,路鹏,王伟东.高效稳定纤维素分解菌复合系MC1的酶活特性[J].中国农业大学学报2003,8(1)：59-61.
[9]傅力,丁友昉,张 篪.纤维素酶测定方法的研究[J].新疆农业大学学报,2000,23(2)：45-48.
[10]Ghosh TK (1987) Measurement of cellulase activities[M].Pure Appl Chem 59:257-268.
[11]王文玲,黄雪松.DNS法测定木糖含量时最佳测定波长的选择[J].食品科学,2006,27(4)：196-198.
[12]朱海霞,石瑛,张庆娜等.3,5-二硝基水杨酸(DNS)比色法测定马铃薯还原糖含量的研究[J].中国马铃薯,2005,19(5)：266-269.
[13]赵凯,许鹏举,谷广烨.3,5-二硝基水杨酸比色法测定还原糖含量的研究[J].食品科学,2008,29(8)：534-536.
[14]方尚玲,杨丹丹,钱志伟,李小强,陈茂彬.高产纤维素酶生产菌的筛选及诱变育种[J].食品与发酵科技,2010(1)：13-17.
[15]AMRITKAR N,KAMAT M.LAlI A.Expanded bed affinity purification of bacterial α- amylase and cellulase on composite substrate analogue cellulose matrices[J].Process Biochemistry.2004,39:565-570.
[16]武香玉,陈存社,张京等.绿色木霉固态发酵生产纤维素酶的研究[J].中国酿造,2010,218(5)：93-95.
[17]魏艳红.纤维素酶产生菌的分离鉴定及产酶条件优化[D].武汉：华中师范大学,2009.
[18]顿宝庆,吴薇,王旭静等.一株高纤维素酶活力纤维素分解菌的分离与鉴定[J].中国农业科技导报,2008,10(1)：113-117.
[19]魏景超.真菌鉴定手册[M].上海：上海科技出版社,1979,495-499.
[20]王关林,方宏筠.植物基因工程原理与技术[M].北京：科学出版社,1998.370-376.
[21]刘昌雄,陈士华,吴兴泉.绿色木霉DNA提取方法研究[J].河南工业大学学报(自然科学版),2005,26(6)：33-36.
[22]吴发红,黄东益,黄小龙,周鑫,程文杰.几种真菌DNA提取方法的比较[J].中国农学通报,2009,25(08)：62-64.
[23]萨姆布鲁克,D.W.拉塞尔,著,黄培堂等译.分子克隆实验指南第3版[M]..北京：科学出版社,2002：1713-1726.
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