Studies On Screening Of Lovastatin-producing Fungus

Lovastatin(monacolin K)is an inhibitor of the enzyme hydroxymethylglutaryl coenzyme A(HMG CoA)reductase that catalyzed the reduction of HMG CoA to mevalonate during synthesis of cholesterol.It is a kind of anti-cholesterol drug,it can inhibit the development of atherosclerosis and reduce the risk of myocardial infarction, therefore it is much of significance medically.There is a series of statins developed in USA and Japan,however the investigation to lipid-lowering Monascus falls behind relatively in our country.Reasons that the production of lovastatin is low and operating costs is high in our country are the lack of lovastatin high-production Monascus strains and the lack of qualified industrial fermentation.Therefore it is significant for the selection of high lovastatin-producing fungus.Aspergillus terreus and Monascus spp.are the main lovastatin-producing fungus.Monascus spp not only produces lovastatin,but also produces monacolin J,L,X,which are HMG-CoA reducase inhibitors and lowering cholesterols strongly.As a kind of natural pigment,Monascus pigment are used extensively for brewing rice wine,coloring soy sauce,fish,meat and cake,making Chinese cheese,bean jam,and so forth.Aspergillus terreus is the original strain to produce lovastatin in China,it has many good qualitiy such as the excellent fermentation performance,the high lovastatin-producing capacity and the mature zymotechnique.As one of the contemporary strain-selection technology,protoplast fusion can possibly cause some recessive genes recombined, exposed and expressed or new gene phenotype emerged randomly.Unlike the general reorganization,protoplast fusion technology need no genetic markers and would be more effective to improve the strain's performance.In the experiment,protoplast fusion technology is used to combine the merits of Aspergillus terreus and the Monascus anka for the selection of high lovastatin-producing strains.ObjectiveTo screening high lovastatin-producing fusants,firstly protoplast fusion technology is used to combine the merits of Aspergillus terreus and cause their gene recombination. Secondly fusants are preliminarily screening through proroplast inactivation and final screening by lovastatin determination.Finally the target fusants would be subcultured to ensure the hereditary stability of fusants.MethodsIn this paper,the growth curve and lovastatin-producing curve of Aspergillus terreus and Monascus anka,the pigment-producing curve of Monascus anka were studied;the influences to protoplast preparation and regeneration under different enzymolysis time were investigated,and the time the protoplast inactivation under ultraviolet was studied;to determine lovastatin content,the distinction between ultraviolet spectrophotometry and high efficiency liquid chromatography(HPLC)was discussed;high lovastatin-producing fusants were screened and subcultured to ensure the hereditary stability,also the growth characteristics of fusants were considered.Results1.As to Aspergillus terrers,the 3^rdday is the exponential phase of growth in shaking culture,lovastatin increased rapidly between 7^th-8^thday;as to Monascus anka,the 2^ndday was the exponential phase of growth,lovastatin increased rapidly between 5^th-7^thday.The Monascus pigment did not rise fast until the 10^thday.2.Protoplasts of Aspergillus terreus and Monascus anka respectively formed most quantitatively under 5-hour and 3.5-hour enzymolysis by mixed enzymes,the regeneration rate were high,and the effect would be better in addition to quartz sand.Protoplasts of Aspergillus terreus and Monascus anka could respectively be inactivated totally under 5min and 4min ultraviolet irradiation(30cm,20W).3.Two quantitative analysis methods of lovastatin were established.One was HPLC,the other was ultraviolet spectrophotometry.The results showed that the method exhibited good linearity between the integral area and lovastatin from 0 to 50μg/ml,y=44813x-1330.6,R²=1.There was a linear correlation between lovastatin content and OD value under ultraviolet spectrophotometry,y=0.0952x+0.1093,R²=0.9773.The results did not exhibit precisely,but the method could satisfy the screening purpose in this experiment. 4.Selected from 87 fusants,27 high lovastatin-producing stably strains were obtained. Seven of them gave more than twice the yield of the original Monascus strain.ConclusionIn this study,high lovastatin-producing strains were successfully bred by inactivated protoplast fusion technology,which could provide a theoretical basis for the selection of high-yield strains of lovastatin in industry.