Study on Isolation, Identification, Conditions of Cellulase-producing Strains and Characterization of Cellulose from Mangrove Environment
|School||Guangxi Normal University|
|Course||Biochemistry and Molecular Biology|
|Keywords||cellulase liquid media fermentation screen identify Penicillium purification enzymology characteristic High Performance Liquid Chromatography|
Cellulolytic enzymes system involved in the hydrolysis of cellulose consists of endoglucanases (EC188.8.131.52, EG), cellobiohydrolases (EC184.108.40.206, CBH) and beta-glucosidases (EC220.127.116.11, 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.