Isolation And Identification Of Monascus And Study On The Characteristics Of Esterase Enzyme

In these study, methods based on the morphology and esterase isozyme analysis, amplified and sequenced ribosomal rDNA ITS,5.8S,18S region, and PCR-DGGE analysis of 18S region. Optimized the conditions of Monascus products esterase, after fractionated by ammonium sulfate, chromatography by Sephadex G25, concentrated by PEG for initial purification and determinate some properties on esterase. For explores esterase basic nature in wine materials, in order to provide theory support to production practice. At the same time, provided important theoretical and practical value on research of Monascus'accuracy, rapid identification. The major findings are as follows:(1) As the 20 strains isolated from Jiang Flavor Daqu, lees, Nong Flavor Daqu and 6 strains from Lab save, a total of 26 Monascus strains were tested strains. According to the investigation made by Zhongqing Li and Guo Fang, based on the morphological characteristic of colony and micro-mophorlogy,26 tested strains were identified as 3 different species primarily, including 11 strains of Monascus fuliginosus,11 strains of Monascus pupureus,1 strain of Monascus ruber.(2) Based on morphological identification of 25 strains, using the optimized NY/T1097-2006 on PAGE analysis of esterase isozyme. The results showed that different species banding pattern of esterase isozymes with diversity, within species is consistent with a model, basically consistent with the morphology. Cluster analysis showed 55% of the similarity Department,25 strains were divided into two groups:Monascus pupureus separated a group of Monascus, Monascus fuliginosus and Monascus ruber belong to a group of Monascus.(3) Base on the morphology and esterase isozyme analysis, selected 13 representative strains of rDNA 5.8S, ITS,18S for PCR amplification and sequencing, and analysis 18S area PCR-DGGE. The results show that, the tested strains were divided into three categories by ITS1 phylogenetic, results are consistent with the morphological identification; the tested strains were divided into two classes by 18S phylogenetic, a class of M. pupureus, M. fuliginosus and M.ruber were in the same type of Monascus; to some extent, ITS2,5.8S area reflect the genetic relationship between strains near and far, not enough room for the classification of Monascus strains; PCR-DGGE analysis base on the location and intensity, divided strains into 4 categories, faster, more accurately reflects the base sequence differences.(4) Comparison of the different sources of esterase activity of Monascus stiains, ester differences in enzyme activity, and optimized the representation of different sources Monascus strains with esterase conditions. the results show that:①strains from different sources produced intracellular enzyme activity higher than the extracellular enzyme; isolated from Jiang flavor Daqu's MY8 intracellular esterase production of extracellular esterase 1% level were significantly higher than other strains, Nong flavor Daqu MY10, MY11 extracellular esterase was significantly higher (P<0.01) than other strains (except MY8 external); Z1 ester of enzyme activity was significantly different (P<0.01) than the rest of the tested strains;②Selected optimal conditions of producing esterase by Monascus: medium initial pH5.0, incubation temperature 32℃, inoculation 2mL, culture Time 5d, the initial medium pH, incubation temperature, inoculum significant effect on the esterase activity, incubation time was not significant.(5) Purificated the representation of different sources of Monascus esterase, and determined some nature of esterase characterization. The results showed that:①the initial and secondary ammonium sulfate precipitation's best saturation was 40%,80%; the sample volume of Sephadex G25 is 0.5mL, flow rate 0.5mL·min-1, isolated two esterase isozyme from strains Q41 intracellular esterase, respectively isolated two esterase isozyme from strains Q5 and MY 10 extracellular esterase. The tested strains'intracellular esterase ourification rate by Sephadex G25 are in 5.4 to 6.5 times, extracellular esterase are in 6.4 to 14.1 times.②Esterase optimum temperature is 35℃, at 50℃in 90min, the enzyme activity kept 40% to 65%, optimum pH is 5.0. The enzyme activity is better preserved in neutral pH environment. In different chemical substances and metal ions, Ca2+ and Mg2+ keep the esterase activity of a catalytic role, the other substances have different degrees of inhibition of esterase.