一株嗜酸真菌的分离鉴定及其胞外糖苷水解酶的产酶分析_英文_

一株嗜酸真菌的分离鉴定及其胞外糖苷水解酶的产酶分析_英文_ Agricultural Science& Technology, 2012, 13(6): 1190-1193 Copyright訫 2012, Information Instituteo f HAAS. All rights reservedAgricultural Biotechnology Isolation and Identification of an Acidophilic Fungus and Analysis on the Secreted Glycoside Hydrolases *Feilong LU, Jiang LI, Yajie LIU, Jianfeng WANG, Xiangkun CAI College of Chemical Biology and Materials Science, East China Institute of Technology, Fuzhou 344000, China glucanase bgl7A; xylanase xylD and Abstract [Objective] This study aimed to isolate an acidophilic fungus and analyze polygalacturonase pga1) were cloned the acidophilic enzymes secreted by this fungus. [Method] A heterotrophic fungus from Bispora sp. MEY-1 and ex- was isolated from the leaching solution of a uranium ore in Jiangxi Province using oligotrophic acid selective medium (pH 2.5), and was named RBS-6. This strain was pressed, and the properties of these then identified according to its colony morphology and molecular indicator rDNA-ITS. recombinant enzymes were also in- Finally, the glycoside hydrolases secreted by RBS-6 were analyzed. [Result] This vestigated; the results showed that fungus RBS-6 was acidophilic, and grew best at pH4.0. Its rDNA-ITS sequence these acidophilic enzymes had a good shared the highest homology (98%) with that of Phialophora sp. CGMCC 3329 (GU [ 3 ] 082377). So it was identified as a fungus of Phialophora sp., and was temporarily application prospect in industries . named as Phialophora sp. RBS-6. It can produce six glycoside hydrolases, in cluding α-galactosidase glucosidase, β-glucosidase, β-mannanase and β-glucanase. All The enzymes secreted by the aci- the dophilic fungus Penidiella sp. HEY-1 enzymes were acidophilic, for which the optimum reaction pH was 3.0 -4.0. Among them, β-glucanase exhibited the highest activity at pH 3.5 and 50 ?; in addition, it which was isolated from a uranium ore was heat-stable as 58% of the enzyme activity was remained after incubation at 50 in Xinjiang were analyzed by He et al., ? for 60 min. [Conclusion] The isolated fungus which was identified as an aci-dophilic member of Phialophora sp., was a new strain producing acidophilic en- and the results reveled that the fungus zymes. This study supplied new data for the research on Phialophora fungi. secreted a variety of glycoside hydro- Key words Acidophilic fungus; Phialophora sp. RBS-6; rDNA-ITS; Glycoside hydro- lases under certain induction condi- lase; Enzyme production analysis tions; in addition, the α-galactosidase and β-glucosidase were purified and their properties were analyzed in this [4]study. The above studies have shown that acidophilic fungi, separated from he acidophilic microorganisms environment, thus overcoming the shortcomings of many industrial en- the extreme acidic environment of the form a large natural group of Tleaching solution of uranium could zymes which are inactive under acidic microorganisms surviving in produce a variety of acidophilic extra- environments. extreme environments. The particular cellular glycoside hydrolases, and Previous researches on aci- genotypes, mechanism and products these enzymes have extensive appli- dophilic microorganisms were mainly of acidophilic microorganisms can help cation prospect and high value in sci- focused on the chemoautotroph used human to solve some major questions entific research. in microbiological metallurgy. In recent (such as the origin and evolution of In the present study, a het-years, more and more acidophilic fungi lives, etc.). They have extensive appli- erotrophic fungus was isolated and pu-have been isolated, and the glycoside cation prospects in the field of biotech-rified from the leaching solution of a hydrolases secreted by them have nology, and have become a focus in uranium ore in Jiangxi Province, and [1]been thoroughly studied. Luo isolated scientific research. the glycoside hydrolases secreted an acidophilic fungus Bispora sp. Acidophilic microorganisms usu- were also analyzed. MEY-1 from the leaching system of a ally produce acidophilic enzymes that uranium ore, and then analyzed the are widely used in feed additives, and glycoside hydrolases secreted by this greatly improve the digestibility of low- fungus, cloned and express a variety Materials and Me thodscost feed to reduce the requirement for [2]of acidophilic glycoside hydrolases . Maea trilsmore expensive feed. Compared with In the study by Yang et al., three gly- Sample The leaching solution of ura-the neutral enzymes, acidophilic en- coside hydrolase genes (β-1,3-1,4-nium was collected from a uranium ore zymes can bio-catalyze in the acidic in Jiangxi Province. Media Enrichment medium con- Supported by Manufacture - Learning - Research Cooperation Project of Education Department of Jiangxi Province(GJJ09008); Nuclear Power Development Projects [CO-tained 3.0 g/L (NH)SO, 0.1 g/L KCl, 424STIND, (2009)1230]. 0.5 g/L KHPO, 0.5 g/L MgSO7HO, ?2442 Corresponding author. E-mail: li66001@163.com*0.01 g/L Ca(NO), 3.0 g/L FeSO7HO ?3242 AReceived: April 20, 2012 Accepted: May 15, 2012 and 2 g/L glucose, with pH 2.0.5. -2 2012 1191 BSYG medium contained 2.0 g/L trophoresis. elecThe mixture of inactivated enzyme and (NH)SO, 0.1 g/L KCl, 0.25 g/L Mg- 424 DNS reagent was used as the blank The rDNA-ITS fragment was puri-SO7HO, 0.01 g/L Ca (NO), 1.0 g/L ?4232tivities control. Finally, the relative acfied and cloned into pGEM-T easy glucose and 0.1 g/L yeast extract, at of these enzymes were calculated. vector and transformed into E. coli. pH value 2.5- 3.0. The recombinant was detected with ? pNPG method . The activities of Acidic PDA medium contained colony PCR using primers T7 and sp6. α-galactosidase, β-glucosidase potato (peeled) 200 g/L, glucose 20 g/L The positive colony was inoculated in- andand agar 20 g/L, at pH 3.0. to LB medium and sequenced by β-glucuroide were determined using Enzyme production medium con- Sangon Biotech (Shanghai) Co., Ltd. pNPG method, namely under the visi- tained 0.5% (NH)SO, 0.1% KHPO, 42424The resulting sequence was submitted ble light of 400 nm, the amount of p-ni-0.05% KCl, 0.05% MgSO?7HO, 4 2 to GenBank and compared by Blast. trophenol produced from three differ-0.001% FeSO?7HO, 0.001% CaCl, 422Finally, a phylogenetic tree was estab- ent substrates pNPGal oNPG and 0.5% konjac flour, 3% wheat bran, 3% lished using Mega 4.0 software. pNPGlu by enzyme hydrolysis was bean pulp, 3% corn cob and 0.5% sol- Effect of initial pH on the fungus respectively measured as following uble starch, at pH 3.0. steps. The reaction system (600 μgrowthl) The spore suspension was inoc- strains Escherichia Vectors and contained 250 μl of pH buffer, 250 μl ulated with the inoculum volume of 2% coli Top10 competent cells and plas- of different substrates and 100 μl of to PDA liquid medium at pH value 1.8, mid pEAEM-Teasy Vector were pur- en- 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0, respec-chased from TaKaRa technology Bio zyme solution. The reaction was car- (Dalian) Co., Ltd. tively, and then was cultured at 30 ? , ried out at 50 ? for 10 min, terminated 160 r/min for 72 h. Three repetitions Methods by adding 1.5 ml of 1 mol/L of NaCO. 23were set for each treatment. The cells Isolation of acidophilic fungus The ODvalue was measured 5 min 400 The leaching solution of uranium was were collected by centrifugation, dried inoculated into the enrichment medi- later, using inactivated enzyme NaCO 23at 70 ? to constant weight and wei- um, cultured at 30 ?, 160 r/min for 3 d. solution as control, to calculate the rel- ghed. The data was processed using Then, the culture medium was inocu- ative activity of the three enzymes. DPS software, analyzed by statistics of lated on BSYG solid medium by re- Effect of pH on enzyme activity single-factor test, and the standard peated streaking for two generations. The crude enzyme solution was mixed deviation was also calculated. Subsequently, a single colony was with the buffer of pH 2.0, 2.5, 3.0, 3.5, Analysis on the glycoside hydro- picked and cultured on acid PDA 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 sep- lases secreted by RBS-6 medium and finally stored for later use. arately to calculate the enzyme activity Preparation of crude enzyme solu- This strain was named RBS-6. using DNS method or pNPG method tion The RBS-6 spore suspension Identification of RBS-6 and to calculate the relative activity of was inoculated into fermentation the enzymes. medium with the inoculation amount of Observation of colony and hypha The optimum temperature and ther- morphology The strain RBS-6 was 2% , cultured at 30 ? , 220 r/min formal stability of β-glucanase Using 120 h. The fermentation broth was cultured at 30 ? to observe its growth D NS method, the activity of β-glu- firstly filtered through nylon mesh and and its spore morphology under a canase to catalyze glucan in the solu- then centrifuged at 4 ? , 10 000 r/min microscope. tion with its optimum reaction pH value for 20 min. The supernatant was the Molecular identification The total was respectively measured at 30, 40, DNA of RBS-6 was extracted with the crude enzyme for the detection of six 50, 60 and 70 ?. [5]method proposed by Sun et al . The different glycoside hydrolases: xy- The β-glucanase solution was in-genomic DNA was served as a tem- lanase, α-galactosidase, β- cubated at 50 and 60 ? for 20, 40 andplate to amplify the ITS1-5.8S rDNA- galactosi-60 min respectively to determine its ITS2 domain, using the universal dase, β-glucosidase, β-mannanase thermal stability. The activity was the and glucanase. primers ITS1 (5’-TCCGTAGGT- enzyme solution without incubation at Determination of enzyme activity -GG-3’) and ITS4 (5’- GAACCTGCResults and Analysis 50 and 60 ? was 100%. ? DNS method. The activities of xy-TCCTCCGCTTATTGATATGC-3’). Colony morphology lanase, β-mannanase and β-The PCR system contained 0.2 μl The RBS-6 strain was cultured on glu-of the acidic PDA medium at 30 ? for 3 d canase were measured by DNS template, 2.0 μl of Taq buffer, 2.0 μl to observe its colony morphology. As method as following steps. The reac- ofshown in Fig.1, the colonies were tion system (600 μl) contained 250 μl dNTP Mix, 0.5 μl of each primer white and round, surrounded by gray of buffer solution, 250 μl of different ITS1 and ITS4 (20 μmol/L) and 0.2 μl mycelia. Under a microscope, we sub-str ates and 100 μl of the enzyme of Taq could clearly see the fungal mycelia solu- DNA polymerase. The amplification like bottlenecks (Fig.2). tion. The reaction was carried out at 50 was started with pre-denaturing at 94 Phylogenetic analysis on the rDNA- ? for 10 min and terminated by adding ITS sequence of RBS-6 strain ? for 5 min; followed by 30 cycles of 1.5 ml of DNS, prior to color develop- The sequencing results showed denaturation at 94 ? for 30 s, anneal- ment by boiling in water for 5 min. After cooled in ice water, the reaction mix- that the sequence of the engineering ing at 58 ? for 45 s, extension at 72 ?tration to determine its value. OD 540ture was diluted to appreciate concen- for 90 s; ended with a final extension at 2012 1192 ts rDNA ITS sequence analysis, the different pH values (Fig.5). The resulstrain obtained in this study was 999 suggested that the α-galactosidase -6 was preliminarily classi- strain RBSbp long. Blast comparison revealed and β-mannanase exhibited higher fied into Phialophora sp., and was ten- that the ITS sequence of RBS-6 tatively named Phialophora sp. RBS-6. activity at pH 3.5 -5.0, and the highest shared homology of 98% with that of activity at pH 4.0. For β-glucosidase, Effect of initial pH on the RBS-6 Phialophora sp. CGMCC 3329 (GU-the optimum pH was 3.0 - 4.0. Beta- growth 082377) homology of 98% . The se-galactosidase exhibited the highest The RSB-6 strain was respective- quences highly related with the ITS activity at pH 4.0. Beta-glucanase and ly inoculated into PDA liquid media sequence of RBS-6 and the homolo- xylanase showed the highest activity with different initial pH values to exam- gous rDNA-ITS sequences of Phial- at pH 3.5 -4.5. In addition, all the ex- ine the influence of pH on its growth. tracellular glycoside hydrolases were ophora sp. were selected and then The results showed that RBS-6 grew compared using acidophilic, as they were active at pH ClustalX1.83. The best in medium with initial pH = 4.0, phylogenetic tree - 2.5. was finally estabsuggesting that the strain RBS-6 was lished using MEGA4 . 0 software Effects of temperature on the en- acidophilic fungus (Fig.4). ( Fig . 3 ) . We could conclude that the zyme activity and thermal stability Extracelluler glycoside hydrolases rDNA-ITS sequence of RBS-6 shared of β-glucanase secreted by Phialophora sp. RBS-6 the closest genetic distance with that The glucan hydrolysis catalyzed Effect of pH on enzyme activity by β-glucanase at different tempera- of Phialophora sp. CGMCC 3329 The relative activity of the enzymes tures revealed that 50 ? was opti- (GU082377) in GenBank. Considering was measured in the buffer systems at mum, and the activity of β-glucanase the morphological characteristics and declined sharply when the tempera- ture was higher than 60 ? (Fig.6). The thermal stability of β-glu-canase was investigated at 50 and 60 ? , using the glucan as the substrate. The results showed that 41% of the β- glucanase activity was remained after incubation at 60 ? for 60 min, and 58% was left after incubation at 50 ? for 60 min, suggesting that β-glu- canase was heat-stable to a certain Fig.1 Colony morphology of RBS-6 on Fig.2 Mycelium morphology of RBS-6 un- degree (Fig.7). acidic PDA medium der a microscope (1 000 ×) Conclusions and Discus- sion Phialophora fungi form a natural group with important economic value. However, related studies were rarely reported. The strain Phialophora sp. p13 was isolated from acid waste wa- [6]ter by Cheng et al. , and a xylanase gene xynllp13 was also cloned from the strain, and expressed in recombi- nant Pichia pastoris. Results showed that the expressed xylanase was 11.8 Fig.3 NJ phylogenetic tree based on rDNA-ITS sequence of RBS-6 U/ml, and showed the highest activity at pH5.5 and temperature 60 ? . A Phialophora sp. fungus was isolated [7] by Zeng et al. from soil sample by preliminarily screening on CMC medi- um plate and rescreening through flask fermentation; in addition, prelimi- nary analysis on the enzymatic proper- ties revealed that the activity of glu- canase produced by this strain was highest at pH 6.0-7.0 and temperature 40 ? , and the enzyme was relatively stable below 50 ?. Fig.4 Effect of initial pH on RBS-6 growth 2012 1193 其功能利用的基础研究 ” 研 究 进 展 )[J]. 微 生 物 学 Acta Microbiologica Sinica ( 报), 2006, 46(2): 336. 罗 会 颖 [2] LUO HY ( ). Secreted enzyme analysis, gene cloning and heterolo- gous expression of glycoside hydro- lases of acidophilic fungus Bispora sp. 极 端 嗜 酸 真 菌 MEY-1( Bispora sp.MEY- 胞外糖苷水解酶类 的产酶分析及其相 1 关基因的克隆与表达)[D]. Beijing: China 北 京 Academy of Agricultural Sciences(: 中国农业科学院), 2008. 杨 君 [3] YANG J ( ). Gene cloning, expres-Fig.5 Effect of pH value on enzyme activity sion and characterization of three gly- coside hydrolases from Bispora sp. 来源于嗜酸真菌 MEY-1 ( Bispora sp. 三种嗜酸糖苷 水解酶的基因克 MEY-1 隆 、 表达与性质研 究)[D]. Beijing: China 北 京 Academy of Agricultural Sciences(: 中国农业科学院), 2010. 何 友 文 [4] HE YW ( ). Isolation identification of a Acidophilus and enzyme properties 一株嗜酸真菌的分 of two glycosidase ( 离鉴定及其两种糖苷酶的酶学性质 )[D]. 南 昌 Nanchang: Nanchang University (: 南 昌 大 学), 2011. 孙 立 夫 张 艳 华 [5] SUN LF( ), ZHANG YH( ), β-glucanase β-glucanase at dif- Fig.6 Effect of temperature on Fig.7 Thermal stability of 裴 克 全 PEI KQ ( ). A rapid extraction of activity ferent temperatures ,一 种 高 效 提 取 genomic DNA from fungiIn the present study, a het- the activity after incubation at 50 ? for 真 菌 总 的 方 法 ,DNA [J]. Mycosystema 菌 物 学 报(), 2009(2): 299-302. 60 min, and 40% of the activity after erotrophic fungus was isolated and pu-程 菲 菲赵 军 旗[6] CHENG FF(), ZHAO JQ(), incubation at 60 ? for 60 min, sug- rified from uranium leaching solution, 石 鹏 君 gesting that it was heat-stable, which SHI PJ ( ), et al. Cloning, enzy- classified into Phialophora sp. accord- mology characteristics analysis of a xy-is an advantage in application that the ing to its colony morphology and lanase gene from Phialophora sp. P13 enzymes produced by other Phialo- molecular indices, and named Phial- 瓶 霉 木 聚 糖 酶 基 ( Phialophora sp. p13 ophora sp. RBS-6. The study showed phora sp. fungi do not have. Thus, the 因的克隆及其酶学性质研究)[J]. Journal that this fungus secreted six glycoside acidophilic fungus Phialophora sp. of Agricultural Science and Technology hydrolases such as α-galactosidase, RBS-6 isolated from the extreme中国农业科技导报 (), 2012(1): 1-5.β-glucosidase, β-mannanase and β- 曾 青 兰黄 小 罗 acidic environment uranium leaching [7] ZENG QL(), HUANG XL(),glucanase. All the enzymes were aci- 洪 玉 枝 HONG YZ ( ). Identification of solution is a new strain which can pro- dophilic as they showed high activity at fungi producing neutral cellulase and duce acidophilic enzymes. pH4.0. Among them, the β-glucanase characterization of its enzymatic reac-was further analyzed, and the result 产中性纤维素酶真菌的 鉴 定 及 其 tions( revealed that 50 ? was best for the 酶 学 性 质 的 初 步 研 究 ) [J]. Journal ofReferences reaction of β-glucanase at pH 3.5. β- 华 中 Huazhong Agricultural University ( 张 敏 东 秀 珠 [1] ZHANG M(), DONG XZ( ). Aglucanase could remain about 60% of 农 业 学 报), 2008, 27(5): 621-624. survey of extromophiles project sup- Responsible editor: Qingqing YIN 项 目 “极 端 微 生 物 ported by 973 (973 及 Responsible proofreader: Xiaoyan WU !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 一株嗜酸真菌的分离鉴定及其胞外糖苷水解酶的产酶分析 *吕飞龙,李 ,江刘 亚洁,王剑锋,蔡向鲲 ,东华理工大学化学生物与材料学院,江西抚州 344000, 摘 要 [目的]分离鉴定 1 株嗜酸真菌,研究其所产嗜酸酶。[方法]利用寡营养嗜酸,pH 2.5,选择性培养基,从江西某铀矿浸铀体系中分离到1 株 异养菌,记为 RBS-6,利用其菌落形态和分子指标 rDNA-ITS 对其进行鉴定,并分析了其胞外糖苷水解。酶[结果]该菌最适生长 pH值 为 4.0,属嗜 酸性真菌。RBS-6 的 rDNA-ITS 序列与瓶霉属真菌 Phialophora sp. CGMCC 3329,G08237U 7,同源性最高,达到 98%,故R BS-6 为瓶霉属真菌 ,Phialophora,,将其暂命名为 Phialophora sp. R BS-6。该菌株可产 α-半乳糖苷酶、β-葡萄糖苷酶、β-甘露聚糖酶和 β-葡聚糖酶等 6 种糖苷水解酶, 这些酶的最适 pH 值在 3.0~4.0,为嗜酸酶,其中的β -葡聚糖酶最适 pH 值为 3.5,最适温度为50 ?,在 50 ?保温 60 min,仍有 58%的酶活力,具 有一定的热稳定性。[结论]该研究鉴定了所得菌株为嗜酸瓶霉属真菌,其为极具产嗜酸酶潜力的新型菌株,该结果丰富了瓶霉属真菌的研究。 关键词 嗜酸真菌,Phaophora sp. R BS-6,rDNA-TS,糖苷水解酶,产酶分析 ilI 基金项目 江西省教育厅产学研合作基金资助项(目GJJ09008),核能开发项目(科工二司〔2009〕1230 号)。 作者简介 吕飞龙 (1986-),男,甘肃天水人,硕士研究生,研究方向,微生物基因工E程-m,ail:lfl8386@126.com。* 通讯作者,教授,硕士生导师 ,从 事微生物基因工程研究E,-mail:li66001@163.com。 收稿日期 2012-04-20 修回日期 2012-05-15