【作者】 胡奇杰；

【导师】 梁新乐；

【作者基本信息】 浙江工商大学 ， 生物化工， 2007， 硕士

【摘要】 阿维拉霉素是一种混合型的低聚糖，作为饲料添加剂应用的抗生素。它通过维持动物肠道内细菌的正常平衡，减少动物正常营养的消耗，从而达到促生长目的。本论文对一株绿色产色链霉菌发酵生产阿维拉霉素进行了初步研究，主要包括菌种鉴定，高产菌株的多重诱变及推理选育，阿维拉霉素发酵培养基及条件优化，以及扩大培养等内容。采用细菌形态学、电镜透射与扫描图象分析，菌株A-05的菌丝较为粗壮，不产生横隔膜，孢子短，卵圆形，表面为长刺所覆盖，孢子丝单分枝，有许多松敞或紧密的左旋螺旋；菌株细胞壁组分含有L，L—二氨基庚二酸，无特征性糖（胞壁I型，糖型C），属于链霉菌属；生理生化特征与与绿色产色链霉菌相似；16S rDNA分析表明此菌株与绿色产色链霉菌同源性100％，因此初步判定菌株A-05为链霉菌属，绿色产色链霉种。该菌株与绿色产色链霉菌在利用棉子糖和纤维素方面存在差异，因此判定该菌株为一株绿色产色链霉菌的变种，并暂命名Streptomyces virichromodenes Var．LH。通过对菌种A-05的代谢产物阿维拉霉素进行定性和定量分析，建立了阿维拉霉素萃取的工艺条件、检测发酵液中阿维拉霉素含量的微生物效价测定法和高效液相色谱法。采用二剂量法回归得到效价计算公式：Log^Y=0.6592R²+0.5717。高效液相色谱法和质谱法分析验证了发酵产物为阿维拉霉素，其中以阿维拉霉素A为主要成分，分子量为1401，保留时间为35.46min。菌株经3000Gy的Co⁶⁰射线诱变、80U／mL阿维拉霉素抗性平板中抗性突变株的筛选、100U／mL链霉素抗性平板中抗性突变株的筛选、0.15％2-脱氧-D-葡萄糖抗性平板中抗性突变株筛选、0.35％α-氨基丁酸抗性平板中抗性突变株的筛选，得到一株阿维拉霉素高产菌株H-15，产量从原来的9.8U／mL提高到68.7U／mL，提高了近580％。通过对碳源、氮源的单因素考察确定了较优碳源为可溶性淀粉，木糖次之，而乳糖产量相对较低；较优氮源为豆粕粉，大豆蛋白胨次之，无机氮硝酸钾则较低。并利用响应面法对发酵培养基的组成进行了优化，得到较适培养基为可溶性淀粉20.12g／L，D-木糖为7.81g／L，豆粕粉为25.23g／L，大豆蛋白胨为5.06g／L，MnCl₂ 0.60g／L，MgSO₄．7H₂O 0.60g／L，pH 7.2～7.4。培养基优化后阿维拉霉素效价比文献报道的初始培养基提高了65％。对摇瓶条件进行了单因素优化试验。斜面菌种保藏时间对种子活力影响不大，低温保藏具有较好的稳定性，保藏1周的斜面生物效价达到最高，斜面相对效价为104％，当保藏三周后，斜面生物效价只有72％；一级摇瓶种龄24h，接种量5％，装液量100mL／500mL三角瓶，pH7.2～7.4，发酵时间48h，阿维拉霉素有较高效价；发酵过程中在38h和46h两次补加1％葡萄糖时，产量比不补料提高了16％左右；乙酸钠在16h补加0.1％时，产量比不补料提高了29％；20h补加0.5％的豆油，产量比不补料提高125％。在5L玻璃罐试验结果表明，菌株发酵需氧量不高，通过转速和溶氧的联合控制，在发酵液中氧分压（pO₂）20％溶氧浓度下菌株阿维拉霉素产量达到160U／mL。放大到50L不锈钢罐，阿维拉霉素产量为164U／mL，豆油和葡萄糖补料，效价分别比分批发酵提高了18％和8％。更多还原

【Abstract】 The avilamycins, which are produced by Streptomyces viridochro-mogenes, are oligosaccharide antibiotics and belong to the orthosomycingroup of antibiotics. Avilamycin is licensed for use as a growth promoterwithin the European Union in the swine and poultry industries but not fortherapeutic use. This paper is to study avilamycin production by the strainA-05.Mycelia of strain A-05, which can produce avillamycin, arecharacterized with relative thick, no diaphragm, short ovate sporules andits surface covered with long thorns, single ramified sporothrix, and manyloose or close left-hand helixes by electronic microscopy transmissionand scanning-image analysis. There was L, L-diaminopimelic acid in thecell wall fraction without diagnostic sugar, indicated strain A-05 wasbelonged toⅠtype of Cell Wall, C type of sugar, Streptomyces sp.Through 16SrDNA analysis, there was 100％similarity in 16SrDNA withthe Streptomyces virichromodenes. However there are some differencesbetween the utilizing ability of raffinose and cellulose from theStreptomyces virichromodenes. Therefore, the strain A-05 is primarily identified as a variety of Streptomyces virichromodenes, and istemporarily named as Streptomyces virichromodenes Var. LH.Technology of extracting avilamycin, microbiological assay toexamine and measure the quality of avilamycin in fermented broth, andHPLC-MS methods were established to qualitative and quantitativedetermination of avilamycin. The potency assay formula was made bydouble-dose regression: LogY=0.6592R2+0.5717. Avilamycin wasverified by HPLC-MS, and avilamycin A is the main component amongavilamycins, whose molecular weight is 1041, retention time is 35.46min.After mutation by 3000Gy Co⁶⁰ and NTG treatment, the mutantH-15 was screened out with a high avilamycin yield at 68.7U/mL, whichwas up to 580％from original 9.8U/mL. Resistant mutants were isolatedfrom 80U/mL avilamycin resistant flat-sheet, 100mg/L streptomycinresistant flat-sheet, 0.15％2-Deoxy- D-glucose resistant flat-sheet, and0.35％α-aminobutyric acidresistant flat-sheet.From the one-way analysis of carbon and nitrogen.starch isconsidered as more optimal carbon, following by xylose, lactose showedthe lowest avilamycin productivity. Soybean meal is considered as moreoptimal nitrogen, following by soybean peptone, Inorganic potassiumnitrate showed the lowest avilamycin productivity. By response surfacemethodology, culture medium components were optimized: starch20.12g/L, D-xylose 7.81g/L, soybean meal 25.23g/L, soybean peptone 5.06g/L, MnCl2 0.6g/L, MgSO₄.7H₂O 0.6g/L, pH 7.2-7.4. Potency ofavilamycin was increased by 65％compared with the original culturemedium reported by literature.Conditions of flask experiment were investigated. The storage timeof slants did not greatly affect seed vigor. Slants stored in lowtemperature （4℃） has the character of good stability, the relative potencyof slants stored for one week reached at the maximum by 104％. Afterthree weeks storage the potency of slants was only 72％. Under theconditions of seed time of flask for 24h, inoculum size 5％,100mL/500mL erlenmeyer flask, pH7.2～7.4, fermentation time course48h, avilamycin output was given out a relatively high potency. Theavilamycin output was increased at 16％by feeding 1％dextrose twice at38h and 46h during the fermentation, while increased at 29％by adding0.1％sodium acetate at 16h, and increased at 125％by adding 0.5％beanoil at 20h.In the 5L fermentor, it was found that oxygen demand of the specieswas relatively low. By the joint control of rotate speed and dissolvedoxygen, avilamycin outputs were up to 160U/mL at 20％dissolvedoxygen concentration. Magnifying in the 50L fermentor further,avilamycin outputs was at 164U/mL similar with ones in the 5Lfermentor150.SU/mL, by feeding bean oil and dextrose in the 50L, thepotency was increased at 18％and 8％by adding bean oil and dextrose respectively.更多还原