【作者】 何璞；

【导师】 余龙江； 周蓬蓬；

【作者基本信息】 华中科技大学 ， 生物化工， 2005， 硕士

【摘要】 虾青素（Astaxanthin,ASTA）是一种非维生素A原的类胡萝卜素,有极强的抗氧化性能,具有抑制肿瘤发生,增强免疫功能等多种生物活性。因此,虾青素在医药食品领域有着广泛的应用前景。同时虾青素又是一种良好的着色剂,在水产养殖方面具有重要应用价值,故其市场需求日益增加。近年来利用红法夫酵母生物合成虾青素备受关注,但产量低一直是限制其工业化生产的主要因素。本文针对红法夫酵母生物合成虾青素时存在的主要问题,首先通过原生质体诱变进行虾青素高产菌种的筛选,在此基础上采用响应面方法对诱变株进行培养基优化,确定最佳发酵条件,并研究调控NADPH 的产生对虾青素生物合成的影响。得到了如下结论: （1）原生质体制备的条件如脱壁酶浓度、处理温度及渗透压稳定剂对红法夫酵母原生质体的形成与再生有着较大的影响。实验表明,菌龄18h,蜗牛酶浓度1%,30℃处理2h,在以蔗糖为渗透压稳定剂的情况下较有利于原生质体的形成与再生。确定了原生质体UV 照射的致死率情况,并在70%-85%的致死率下筛选到一株高产诱变菌株,虾青素产量较出发菌株提高85.6%。（2）研究不同碳源种类对诱变株合成虾青素的影响。结果表明,菌株利用葡萄糖、蔗糖有利于虾青素积累,二者效果相差不大,本文通过分式析因设计得出基础培养基中对虾青素合成影响最大的因子是蔗糖和酵母粉,并通过最陡爬坡试验和旋转中心组合设计,在较少的试验次数下实现了培养基的优化。优化后培养基组成为:蔗糖49.8g/L,酵母粉9.6 g/L,NH₄Cl₂ g/L,KH₂PO₄ 2g/L,MgSO₄·7H₂O 0.5 g/L,CaCl₂·H₂O 0.3g/L.（3）研究不同培养条件对诱变株合成虾青素的影响,结果表明,摇床转速、培养温度及接种量都对菌体的生长和虾青素的积累影响较为显著。通过正交试验确定了最佳的培养条件为:种龄36h,8%接种量,30mL 装液量,培养温度20℃,摇床转速200rmp,并研究了工艺优化后的发酵动力学,为放大生产提供了实验依据。（4）研究了PPP 中NADPH 产生的关键酶G6PDH 的酶活与红法夫酵母生长及虾青素积累的关系。结果表明,三者之间具有明显的一致性。通过添加0.12g/L 的G6PDH 激活剂谷氨酸能够促进红法夫酵母细胞的生长和虾青素的积累。并探讨了外源谷氨酸对菌体生长及虾青素合成的作用机制,为实现代谢调控提供了新的思路。更多还原

【Abstract】 Astaxanthin（ASTA）, a nonprovitamin A oxygenated carotenoid, has powerful biological activities including antioxidant, antitumor and immuin enhancement effects. Meanwhile ,ASTA is an importment pigment for aquaculter industry, so the market of it is expanding.Recently there is increasing interest in ASTA biosynthesis from Phaffia rhodozymas for commercial production, but low-production of ASTA is a restricted factor of it. Aiming directly at this problem, this paper firstly studied on the breeding of the P.rhodozyma, by UV inducing protoplast, then optimized the medium and feimentation condition, the effect of regulating NADPH on ASTA production was discussed also. The results as follows: （1） Ascertaining the best way to prepare P.rhodozyma protoplasm by enzyma snail solution is that: the inoculum age is 18h, enzyme concentration is 1%, treated under 30℃for 2h, osmotic stabilizer for protoplasm formation and revival is 0.5 mol/L sucrose.By UV inducing the protoplasm, a high ASTA producing mutant was obtatined, compared with the initinal strain, the yield of ASTA was increased 86.5%. （2） Effects of carbon sources on ASTA biosynthesis by mutant was studied. Results showed that glucose and sucrose were advantageous with the similar effects. The influence of medium contents on astaxanthin production was evaluated using a fractional factorial design. Sucrose and Yeast power influenced astaxanthin significantly. The path of steepest ascent was used to approach the optimal region of the medium concentration. The optimal concentrations of sucrose and Yeast power were determined by a central composite design and response surface analysis. The optimized medium was composed of sucrose 49.8g/L, yeast powder 9.6g/L, NH₄Cl 2g/L,KH₂PO₄·7H₂O 0.5g/L,CaCl₂·H₂O 0.3 g/L. （3） The culture conditions suitable for ASTA biosynthese from P.rhodozyma were established. The results of orthogonal designs L₈（2⁷） showed that The optimum temperature and pH for growth were 20℃and 5.5 respectively,and media volume was 30mL,the inoculation concentratons was 8%,with the shaker rotation speed 200 rpm. The ferment dynamic after optimization was studied also, offering an experiment foundation for enlarging produce. （4） The relationship between nicotinamide adenine dinucleotide （NADPH） producing enzyme-glucose-6-phosphate dehyderogenase （G6PDH） and P.rhodozyma growth,ASTA biosynthese was studied.The results showed that G6PDH correlated with the others. Addition of 0.12g/L glutamate accelerated ASTA production. The mechanism of it was discussed , which provided a new thinking for metablic regulation.更多还原