| A strain which produces pigment was isolated from yellow wine lees in Fujian,then was identified. To obtain a high-yield strain of carotenoid, the mutation breeding and optimization of media ingredients and culture conditions on the strain were studied, and then the isolation and purification of carotenoid and the cleavage from carotenoid into retinol with β-carotene15,15'-monooxygenase were investigated. The main results were as follows:(1) A strain which produces pigment was isolated from yellow wine lees. The pigment was identified as carotenoid by qualitative analysis. The morphology, physiological and biochemical characteristics of the strain were analyzed, the results showed that the cell of the strain was single, oval and budding, and the strain had no ascospore and pseudohypha. Its colony was of regular edge, humid and sticky pink surface in solid medium and deposition in liquid medium. The strain showed negative response to glucose fermentation and positive response to potassium nitrate. It could neither endure hypertonic solution nor produce amyloid, it could grow at37℃. All of these results showed that the strain was a kind of Rhodotorula. The homology of the26S rDNA D1/D2domain sequence from the Rhodotorula showed that the genetic relationship between the strain and Rhodotorula mucilaginosa was closest and they were100%similarity at nucleotide acid sequence. According to its morphological, physiological and biochemical characteristics and molecular identification, the strain was identified as Rhodotorula mucilaginosa, and named RM-1after the strain.(2) In order to obtain a high-yield strain of carotenoid, R.mucilaginosa RM-1was mutated by10keV N+implantation with dose of2.0×1014ion/cm2implantation and then screened by diphenylamine resistance. Mutant RM-127was obtained, its production of carotenoid reached9.31mg/L, increased by66.79%over the5.58mg/L from the original strain. And then mutant RM-213was obtained by UV mutagenesis of RM-127and produced a large quantity of carotenoids (10.59mg/L), which was higher (with89.67%) than in the original strain RM-1. Its hereditary property was stable after ten times of subculture, which suggested a bright prospect of application.(3) Optimization of carotenoid production by RM-213using agricultural waste and by-product as raw matearial were studied. The single-factor method was employed for the optimization of growth and carotenoid production using corn straw hydrolysate (50g/L) as carbon source and corn steep liquor (40g/L) as nitrogen source. The experimental parameters of seven kinds of additives were added by uniform design experiment, the results showed that the optimum additives and quantity were as follows:MgSO4·7H2O0.7g/L, K2HPO40.2g/L, ethanol0.7%, VB21.0g/L, respectively. The corresponding regression equation was:Y=3.94+29.6X1+20.9X2-11.1X3-1.99X6. The optimum culture conditions were obtained as follows by orthogonal test:250mL Erlenmeyer flasks containing50mL fermentation liquid, initial pH5.5,10%inoculum size with agitation rate of170r/min. The temperature was varied during incubating, the first40h incubated at30℃, then kept32h at24℃. Under these conditions, the cell biomass, carotenoid content and carotenoid yield were respectively. Increased by48.19%,54.68%and129.26%over that before optimization.,(4) The carotenoid which were produced by RM-213was purified by macroporous adsorption resins and the optimum conditions of adsorption and desorption were investigated.. The static experiment results showed that X-5resin was the best adsorbent, the adsorption rate was up to71.01%when absorbed1h at25℃, and aether was the best eluent, the desorption rate was up to95.32%when desorbed1h at30℃. The dynamic experiment results showed that the optimum dynamic adsorption and desorption conditions were as follows:the concentration of sample111.82u.g/mL, ratio of column diameter to length1:9, rate of adsorption flow1mL/min, and rate of desorption flow0.5mL/min. Under optimum conditions, the purity of carotenoid was up to33.29%.(5) The conversion of β-carotene produced by R.mucilaginosa in vitro was conducted, the results showed that β-carotene15,15'-monooxygenase from chicken intestinal mucosa can cleaves β-carotene into retinal. The optimum reaction conditions for conversion were123mg/L β-carotene,3.5mmol/L sodium deoxycholate,0.25%(w/v) Tween40, pH8.0and0.5mmol/L d-a-tocopherol, respectively. The enzyme reaction was conducted at37℃for7h under optimum conditions with2.69nmol mg-1h-1enzyme activity,40.1mg/L retinol was obtained by reducing retinal with the NaBH4procedure, the conversion efficiency come up to61.11％(mol/mol).
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