Isolation And Identification Of Rhizopus Oryzae From Mulberry With Root Rot And Catalyze Pupa Oil For Biodiesel Production

The types and occurrence conditions of mulberry diseases are various, and thesymptoms of mulberry diseases are different. The mulberry diseases were usuallyclassified into mulberry virus disease, mulberry fungus and mulberry bacterial disease.Moreover, the mulberry root diseases mainly include mulberry fungus disease，mulberry bacterial and mulberry root knot nematode disease. To date, a wide varietyof fungus were found pathogenic to mulberry, including Armillaria, Schizophyllum,Helicobasidium, Rhizopus and Fusarium.In this study，a pathogenic fungal strain, TY.GF1, was isolated from the mulberryroot, and have pathogenicity to the mulberry. The color of single colony in the earlierstage was white, exhibited no regular shape and flocculation obviously. It grew so fastat32℃that it occupied the whole plate between2and three days, and turned blackand gray. The culture in light microscope showed the characters of rhizoid,subglobose sporangium and spores. Meanwhile, the sequence analysis of its18SrRNA gene and ITS gene were used to further identification. The18S rRNA gene andITS gene were amplified by universal primer, and the amplicons were1805bp and627bp. Then the sequences were submitted to GenBank database and used blastnrespectively. The sequences with which it had high homology were selected toconstruct a phylogenetic tree. Bsed on the result of sequence analysis of the18SrDNA and ITS and the morphological character of TY.GF1strain, TY.GF1wasidentified as Rhizopus oryzae.The wild TY.GF1strain could produce lipase by quickly identification offermentation liquid on Rhodamine B plate. Various carbon sources （dextrose, bran,fructose, brown sugar, sucrose, powder of mulberry, amylum, maltose and soybeanoil）, nitrogen sources （peptone, yeast extract, beef extract, corn flour, sodium nitrateand ammonium sulfate）, inducer sources （soybean oil, sesame oil, castor oil, olive oil,canola oil, sunflower seed oil, silkworm chrysalis oil and rap oil）, temperature （25～45℃）, pH value （4～9）, fermentation time （12～96h） surfactants （triton X-100, twain20, twain80, polypropylene glycol and sodium dodecyl sulfate） were screened. Themost important factors influencing lipase production （p <0.05）, as identified by atwo-level Plackett-Burman design with8variables, were maltose, peptone and canolaoil. Steepest ascent method was undertaken to determine the optimal regions of these three significant factors. Central composite design （CCD） and response surfaceanalysis were adopted to further investigate the mutual interactions between thesevariables and to identify their optimal values that would generate maximum lipaseproduction. The predicted results showed that the maximum whole-cell lipase activity（278.154U/g） could be obtained under the optimum conditions of maltose2.08g/L,peptone21.58g/L, canola oil12.73g/L, twain-800.1%（v/v）, NaNO₃1.2g/L,KH₂PO₄1.2g/L, MgSO₄7H₂O0.5g/L at35℃under130rpm.PCR amplification and sequencing to the precursor and mature lipase, andyielded sequence fragments of1101bp and810bp respectively. The sequence ofprecursor lipase was send to GENEBACK, and obtained an accession number:JN689988. The sequence analysis of the precursor and mature lipase shows that therewere no introns in the sequences, and encode366amino acids and269amino acidsrespectively. The conserved domains of mature lipase were SDGGKVVAAT. Thepredicted physic-chemical properties of the mature and precursor lipase showed thatmolecular formula were C₁₃₃₅H₂₀₅₆N_350O396S₇and C₁₇₅₈H_2729N469O547S₁₀, molecularweight were29569.5and39507.4, isoelectric point were8.14and7.15, extinctioncoefficient were1.163～1.175and1.123～1.132, respectively. And they were bothhydrophobic.The optimum condition of biodiesel production catalyzed by TY.GF1whole-cellin tert-butanol system were:6.5g pupa oil，0.8g the whole-cell,3%water content（based on oil’s weight）,1.05g methanol tert-butanol （tert-butanol/oil=1.5:1, v/v）.After being reaction for24h, the methyl yield could reach80%or more. Although thestepwise addition of methanol could avoid the negative influence on the whole-cell,the stability of whole-cell was poor in the reuse process. And the whole-cell lipasewas inactivation in the fifth reuse process. The relative activity of the whole-celllipase could reach67%after the ninth reuse in tert-butanol system.