紫外诱变原生质体选育赖氨酸高产菌株

以钝齿棒杆菌102S₂-58为出发菌株,在原生质体形成及再生的最佳条件下制备原生质体,并对原生质体进行紫外诱变处理,对大量的再生突变株进行发酵筛选．获得了高产稳定株102－100号,其发酵液经氨基酸自动分析仪测定L－赖氨酸积累量由出发菌株的5O．Omg／ml提高到80．8mg／ml,糖转化率达到63．88％．发酵液中主要副产酸——纈氨酸和蛋氨酸的量明显降低。     

原生质体诱变选育高产异抗坏血酸菌株

以灰黄青霉菌(Penicillium griseofulvum HL)为出发菌株,试验得到灰黄青霉原生质体制备的优化条件为:菌体培养48h,用0.7mol/L的NaC1溶液作为渗透压稳定剂,用0.5％的蜗牛酶+0.5％的纤维素酶,在pH为6,30℃条件下酶解3h,所得原生质体数最多,达到3.14×107/ml.原生质体再生的最佳条件为采用双层平板培养法,在用0.7mol/L的蔗糖溶液配制的改进察氏培养基上其再生率最高,达到24.93％.灰黄青霉原生质体经过紫外线诱变,DES诱变,紫外线-DES诱变复合诱变,紫外线-氯化锂复合诱变选育异抗坏血酸高产菌株,通过对再生平板上长出的诱变菌株进行初筛和摇瓶复筛,最终获得一株异抗坏血酸产量较高的菌株ZD4,其产量为5.28mg/ml,提高到出发菌株产量(1.08 mg/ml)的488.9％,且连续传代6代遗传稳定.     

磷脂酶D高产菌株的原生质体紫外诱变选育

为了获得磷脂酶D高产菌株,由链霉菌野生菌株LD0501出发研究原生质体的制备和再生条件,建立原生质体紫外诱变筛选方案。采用酶解法制备原生质体,用紫外线对原生质体诱变,TLC检测突变株产磷脂酶D活力。原生质体的适宜条件:种子培养基中甘氨酸质量浓度5 g/L,菌龄72 h,用3 mg/m L的溶菌酶在30℃下酶解75min。通过原生质体诱变筛选,得到1株高产菌株,磷脂酶D水解活力达4.29 U/m L,提高幅度为180.4%。该方法有效改善了链霉菌野生菌株原生质体的制备效果,紫外诱变筛选显著提高了磷脂酶D的活力,高产突变株具有较好的稳定性。     

利用原生质体诱变筛选碱性蛋白酶高产菌株

本实验以枯草杆菌ＡＸ—４６为出发菌株,在原生质体形成及再生的最佳条件下制备原生质体,并对原生质体进行紫外诱变处理,对大量的再生突变株进行发酵筛选,获得高产菌株ＡＰ—１２,碱性蛋白酶产量由原来的３２００．８ｕ／ｍｌ提高到４３５３．１ｕ／ｍｌ,提高率达３６％。同时又对ＡＰ—１２菌株进行遗传稳定性考查,考查结果,ＡＰ—１２是高产稳定菌株。     

小麦白粉病生防菌G-1菌株原生质体诱变育种

以链霉菌G-1(Streptomyces sp.G-1)为出发菌株,通过研究菌株G-1原生质体形成与再生的条件,发现该菌株在含0.5%甘氨酸的菌丝体培养基中经过二次培养后,所得菌丝体用2 mg/mL溶菌酶在30℃下处理90 min,可获得大量原生质体,其再生率可达8.2%。菌株G-1的原生质体经紫外诱变和宁南霉素抗性筛选后,得到一高产突变株G-1-125,其有效组分A的产量达到794mg/L,较出发菌株提高了180%。     

氦氖激光、紫外复合诱变天麻素产生菌华根霉LN-A的原生质体

本文通过研究酶组合、酶浓度、酶作用时间和菌龄等因素对天麻素产生菌华根霉(Rhizopus chinensis) LN-A原生质体制备和再生的影响, 总结出了原生质体制备和再生的最佳条件: 选用对数生长期的菌株, 以蜗牛酶5 mg/mL + 纤维素酶5 mg/mL + 溶菌酶2 mg/mL 30°C保温处理2 h, 原生质体形成率达5.8×107, 原生质体再生率为5.7%。在此基础上首次利用He-Ne激光、紫外线复合诱变天麻素合成菌的原生质体, 当选用15 mW的He-Ne激光辐射原生质体20 min, 再用紫外辐照150 s时获得了转化率及天麻素得率都明显提高的突变株, 其天麻素得率比出发菌株提高20%以上。     

羊肚菌原生质体诱变筛选高生物量高氨基酸含量菌株

研究影响尖顶羊肚菌（MOrchellaconicaPers、）CGAC－9506原生质体制备、再生因素基础上,首次诱变羊肚菌原生质体,进行高生物量高氨基酸含量菌株选育。原生质体诱变再生株MconicaCGAC－950637生物量比出发株提高7．3％,总氨基酸比出发株提高38％。     

紫杉醇产生菌Nodulisporium sylviforme原生质体诱变研究

对酶系组成、pH、酶解温度和酶解时间等影响树状多节孢原生质体制备和再生的因素和原生质体诱变进行了研究。结果表明 ,原生质体制备和再生的最佳条件为 :用pH5.5～ 6.0的0.7mol/LNaCl配制由 3%溶壁酶 + 3%蜗牛酶 + 1 %的溶菌酶 + 3%纤维素酶组成的复合酶系 ( 1ml酶液/2 5 0mg湿菌体 ) ,在 30℃恒温水浴条件下酶解 6h ;然后 ,将获得的原生质体过滤洗涤后 ,在含0.7mol/ LNaCl的PDA再生培养基上 ,采用双层平板培养法再生制备到的原生质体。树状多节孢紫杉醇产生菌原生质体诱变的最佳条件为 :30w紫外灯、距离 30cm、照射 5 0s;UV + 0.6%LiCl复合诱变、照射时间 40s,诱变菌株经初筛和复筛 ,选出了两株高产紫杉醇的原生质体诱变菌株———UV_40-19和UL_50-6,其产量从出发菌株紫杉醇的产量 ( 314.07μg /L)分别提高至 376.38μg/L和392.63μg/L。     

紫外线诱变原生质体选育苏氨酸高产菌株

以黄色短杆菌T6-13的苏氨酸产生菌GSR8-25为出发菌株,利用溶菌酶制备其原生质体后进行紫外线(UV)诱变处理,从再生株中筛选出苏氨酸高产菌株。经多次连续诱变处理得到一苏氨酸高产菌株25-20,在含10%葡萄糖的培养基中摇瓶发酵60小时可积累苏氨酸19.5mg/ml。分离纯化后得菌株25-202,其苏氨酸产量可达21.5mg/ml,比出发菌株提高43.3%。     

亚硝酸与紫外线复合诱变原生质体选育产酶菌株

目的：筛选出一株稳定、高产的产中性蛋白酶菌株。方法：以突变株UV_(11)(原生质体紫外诱变得到)为出发菌株,在原生质体形成与再生最佳条件下制备原生质体并进行亚硝酸与紫外线复合诱变。结果：得到突变株Bacillus subtilis UN_(19),产酶活力从最初的378．97U/ml提高到3965．84U/ml。结论：亚硝酸与紫外线复合诱变原生质体是一种很好的诱变方式。     

Studies of protoplast fusion in Streptomyces chrysomallus

Conditions for the regeneration of cells from protoplasts of Streptomyces chrysomallus, a producer of the peptide antibiotic actinomycin, are described. Regeneration of fusion products was most efficient at 27-30 degrees C on regeneration R2 medium (Okanishi et al., 1974) containing 0.25 M-sucrose. The addition of phosphate (150-300 mg 1(-1) to the medium and incubation at 23 degrees C proved to be optimal for the regeneration of individual strains. Highest recombination frequencies after protoplast fusion were obtained by fusing protoplasts in the presence of 45% (w/v) polyethylene glycol 6000. With strains that produce no, or little antibiotic, protoplasts must be present in excess in fusion mixtures in order to overcome inhibition of regeneration by the antibiotic-producing partner.     

银耳原生质体分离与再生条件优化研究*

应用正交设计,研究不同菌株(Tr01、Tr21)、材料(芽孢、菌丝体、子实体)、溶壁酶浓度和酶解温度对原生质体产量的影响。实验结果表明,实验材料对原生质体产量影响最大,以芽孢为材料原生质体产量可达到2.75×107个/ml,而菌丝体和子实体的原生质体产量仅为2.5×106个/ml 和1.0×106个/ml;在35℃下酶解,原生质体产量高;溶壁酶浓度在1%~3%范围内对原生质体产量影响不大;不同菌株原生质体产量差异不显著。本实验还研究了稳渗剂浓度对原生质体再生率的影响,结果表明,0.5 mol/L~0.7 mol/L的KCl 对原生质体再生没有显著差异,再生率最高可达32.3%。     

鹰嘴紫云英甲硫氨酸抗性系原生质体培养及植株再生

本研究建立了鹰嘴紫云英(AstragaluscicerL.)甲硫氨酸抗性系原生质体再生植株的实验体系。以茎切段诱导的松软愈伤组织为材料,通过酶法游离出大量有活力的原生质体。原生质体经培养持续细胞分裂形成了愈伤组织,并分化出再生苗。比较了不同培养基、培养密度对原生质体形成细胞分裂和再生的影响。结果表明,原生质体以2×105个/ml的植板密度,在附加2.0mg/L2,4-二氯苯氧乙酸(2,4-D)、0.2mg/L6-苄氨基嘌呤(6-BA)、200mg/L水解酪蛋白、2%蔗糖和0.3mol/L甘露醇DPD培养基中培养后,其分裂频率达38.3%。原生质体培养形成的愈伤组织仍具有对甲硫氨酸的抗性。转移到附加10mg/LKT、0.5mg/LNAA的MS分化培养基上,获得大量的再生苗。     

麦角菌和雀稗麦角菌原生质体的形成与再生

本文报道了快速制备麦角菌(Claviceps purpurea)和雀稗麦角菌(Claviceps paspali)原生质体的方法及影响原生质体形成的若干因素。用适当方法培养菌丝体,利用商品溶壁酶制剂(10mg/ml),以0.7mol/l KCl为原生质体高渗液,28℃为酶解温度,处理时间仅需1—2h,菌丝体细胞壁即被彻底消化而释放出大量原生质体。在适当的固体再生培养基上,两种麦角菌原生质体的再生频率分别为7.7%和13.2%左右。培养基中添加25mg/l脱氧胆酸钠有利于形成易于计数和分离的小菌落。本文还就CaCl_2,MgCl_2及低温保藏(3—4℃,30天)对于原生质体稳定性与再生的影响作了初步探讨,并证实溶壁酶对原生质体有明显的破坏作用。     

Increase in tylosin production by a commercial strain of Streptomyces fradiae

Conventional mutagenesis (UV irradiation and exposure to nitrosoguanidine) were used to produce and regenerate protoplasts, aiming at increasing the antibiotic activity of a Streptomycesfradiae strain producing tylosin. Variants exceeding the activity of the initial producer strain by 0.5-28.3% were obtained. The most active variants were produced by a combined exposure to UV and nitrosoguanidine, as well as upon regeneration of protoplasts formed from the cells of clones produced by UV irradiation. Unstable inheritance of the trait of increased tylosin production was demonstrated.     

林可链霉菌转化系统的建立

利用基因工程技术改良抗生素生产菌有着广阔的应用前景［１］。值得尝试的是将抗生素生物合成基因片段随机或定向组合导入生产菌中,通过同源重组方式提高生物合成途径中限速步骤相关基因的剂量,以达到提高生产菌发酵单位的目的。此战略被作者称为“基因回转化技术”,并...     

Genetic recombination by protoplast fusion inNocardia asteroides

Summary Fusion and regeneration of protoplasts ofNocardia asteroides strains ATCC 3318, IMRU W3599 and HIK B971 have been used to study genetic recombination in this species. Protoplasts were produced by treatment with lysozyme, following incubation with glycine. Mutants of ATCC 3318 were grown in peptone yeast extract medium at 32°C prior to protoplast production to maximize protoplast frequency, whereas mutants of IMRU W3599 and HIK B971 were grown in trypticase-soy broth. Glycine concentrations favoring protoplast formation varied from 1.5% to 5% depending on strain. For all strains, protoplast formation was complete 1 h after addition of 5 mg/ml lysozyme. Protoplasts were fused by addition of 50% polyethylene glycol-1000. In general, 25% of the protoplasts could be regenerated. The incidence of recombinant recovery was increased up to 750-fold. The distribution of recombinant phenotypes in matings was similar for protoplast fusion and conventional crosses.     

Preparation and characteristics of protoplasts of Streptomyces kanamyceticus

To prepare actively regenerating protoplasts of S. kanamyceticus, the influence of the conditions of the mycelium cultivation, the culture age, lytic conditions, composition of the regeneration medium, the procedure of the culture inoculation to the regeneration medium and other parameters were studied. The study resulted in development of optimal conditions for preparation of S. kanamyceticus protoplasts in a number of 1.10(9) protoplasts per ml. The cultivation on the ST medium with 10 to 15% sucrose and addition of glycine up to 1% for 30 hours (the stationary growth phase) followed by treatment of the culture with lysozyme in an amount of 2 mg/ml for 1 hour at 32 degrees C provided preparation of up to 100% of actively regenerating protoplasts free of mycelium fragments. The size of the protoplasts increased up to 1.5 micron against the usually observed size of 0.7 to 1.0 micron with using modified lyzing buffer with 20% of sucrose according to the method recommended for S. erythreus. However, 50 to 70% of the protoplasts had point of linear regions in the cell walls, which suggested that spheroplasts were mainly forming and the phenomenon was associated with the characteristic properties of the strain cell wall structure.     

蓝色犁头霉原生质体的制备与再生

研究了氢化可的松生产菌蓝色犁头霉原生质体的形成与再生。通过对溶解酶系统的选择,影响原生质体形成的因素如渗透压稳定剂、酶浓度、菌龄、菌丝培养基和培养方式等因素进行考察,发现以0．4mol/L NH4Cl做为稳定剂、2．5mg/mL溶壁酶和5mg/mL纤维素酶组成的混合酶液溶解菌丝,4h后原生质体量可达10^6cell/mL。通过显微镜观察原生质体的形成过程以及在高渗培养基上的再生情况,再生率为15．6％。