用原生质体融合技术选育谷氨酸高产菌

利用原生质体融合技术,在不对亲株做任何诱变的前提下,成功地使天津短杆菌TG-866与钝齿棒杆菌B9的原生质体进行融合,获得兼有两亲株遗传性状——细胞个体大、产酸高的融合子F263及F288。确定了两亲株原生质体制备、再生及融合的最佳条件。在该条件下,其原生质体形成率均在99．9％以上,再生率分别为z 3％及28％,原生质体融合率为3．2×1 0-5在最佳摇瓶发酵条件下,F263及F2 88的谷氨酸产率分别为8．4g／dl及8．03g／dl.通过连续10次摇瓶传代,发现该融合子较稳定,因而具有较高的应用价值。     

通过灭活原生质体融合选育啤酒酵母新菌株

将生产用啤酒酵母(\%Saccharomyces cerevisiae)\% LQ16和QSB7分别进行单倍体化,并筛选LQ16(Ile\+-,Dat\+r)和QSB7(Ala\+-,H\-2S\+-)的遗传标记。经摇振培养后,酶解制备原生质体,对前者进行紫外灭活,而后者进行热灭活,再利用PEG进行融合,在MMR和SMMR培养基上再生,挑选融合子。连续传代稳定后鉴定融合子QSB\|XI\-6。通过细胞体积和生物量的测定,以及遗传型的分析和细胞DNA含量测定等均证实了获得的是融合子。经对啤酒感观评价,双乙酰含量测定与多种成分的气相色谱分析以及啤酒的发酵度,絮凝性、稳定性测定,并经连续多次生产试验证实QSB\|XI\-6是一株口味独特、发酵度高、絮凝性强、遗传性能稳定兼具有多种优良性状的啤酒酵母生产新菌株。     

原生质体融合技术选育分解草酸乳酸菌菌株的研究

将具有草酸分解能力的乳双歧杆菌和具有耐氧特性的嗜酸乳杆菌进行原生质体融合,其最佳条件为:50%的PEG6000,融合温度30℃,融合时间为7 min,CaCl2浓度为0.02 mol/L,MgCl2浓度为0.5 mol/L,在此条件下融合率可达7.6%。从中筛选出同时具有耐氧特性和草酸分解能力的融合子,草酸分解率为13.4%。     

原生质体紫外诱变选育高产抗菌脂肽菌株及其活性物质的研究

本研究以实验室自主分离的枯草芽孢杆菌mutHS-301为出发菌株,通过原生质体紫外诱变选育出高产抗菌脂肽突变菌株,并对其产生的抗菌脂肽提取物进行单组分分离纯化及对黄曲霉抑制作用进行初步研究。结果表明,在溶菌酶浓度为0.5 mg/mL,酶解时间为15 min,酶解温度为37℃条件下,获得原生质体的形成率和再生率效果最佳。采用紫外照射时间60 s进行该原生质体诱变,经筛选获得一株遗传性状稳定的高产抗菌脂肽菌株,命名为mutHS-539。研究表明,该突变株mutHS-539发酵上清液对副溶血性弧菌和金黄色葡萄球菌抑菌直径较原始菌mutHS-301分别提高了21.49%和21.05%,提取得到的抗菌脂肽产量较原始菌提高了40%。利用制备型硅胶板对发酵提取物进行分离纯化得到四种组分,分别为a、b、c和d;进一步检测对黄曲霉的抑菌活性,结果发现只有组分d对黄曲霉具有显著的抑制作用。经RP-HPLC分析及液质联用数据比对,该组分d的主要成分为杆菌霉素D。该抗菌脂肽提取物对黄曲霉抑制作用的研究显示,当抗菌脂肽浓度为0.2 mg/mL时能有效抑制黄曲霉菌丝的生长,抑制率达到了74.22%,且对黄曲霉孢子的致死浓度为0.8 mg/mL。     

香菇漆酶高产菌株筛选及漆酶基因的表达研究

漆酶是一种含铜的多酚氧化酶,其作用底物范围广,在食品、能源和环保等领域具有重要的应用价值,因此筛选高产漆酶菌株对于其工业应用具有重要意义。本实验以78株香菇菌株为实验材料,利用愈创木酚平板法初筛,液体发酵复筛的方法筛选高产漆酶香菇菌株,并通过实时定量PCR分析香菇漆酶同工酶在不同菌株之间转录表达的差异性和特异性。结果表明,不同香菇菌株的生长速度、变色圈大小存在差异,以菌丝圈和变色圈大小及其比例为依据,从78株香菇菌株中筛选出19株潜在的漆酶高产菌株;液体发酵从这19株菌株中复筛出3株高产漆酶的菌株:香240、15和冕宁浅灰,其中香240在培养第3天酶活达到最高(3.583U/m L),香15和冕宁浅灰则在培养第7天酶活达到最高(分别为3.842U/m L和2.806U/m L);实时荧光定量PCR结果显示,10个漆酶同工酶基因在两株香菇菌株中的表达存在差异性和特异性,其中Lelac5和Lelac9只在菌株香240中检测到转录本;Lelac1–4在菌株15中表达量较高,而Lelac8、Lelac10和Lelac11在香240中的表达量较高。研究结果将为香菇漆酶的进一步研究提供基础。     

蛹虫草原生质体紫外诱变选育胞外多糖高产菌株

蛹虫草是重要的药食兼用两用真菌,具有较高的医用及经济价值。本文通过单因素和正交试验的方法研究了不同酶系统、酶解温度、酶解时间、渗透压稳定剂、菌龄对蛹虫草原生质体形成的影响,并对蛹虫草原生质体进行紫外诱变,以生物量和胞外多糖产量为指标选育胞外多糖高产菌株。结果表明：在30℃、1％溶壁酶＋0．5％蜗牛酶＋0．5％纤维素酶条件下,以甘露醇为渗透压稳定剂对4日龄蛹虫草菌丝酶解2h,原生质体产量可达到9．2×10^6个/mL。从150株诱变株中筛选出1株最佳正诱变株,编号为44#,经深层培养其生物量比出发菌株提高10％,胞外多糖产量提高84．3％,继代培养10代后,遗传稳定性良好。     

原生质体融合选育赖氨酸高产菌种的研究

北京棒杆菌1134衍生株与枯草芽孢杆菌151衍生株的原生质体融合得到了一株可以甜菜糖蜜为原料,在摇床与小罐发酵均能稳定产赖氨酸6 50％以上的高产菌株——Q4413。Q441 3菌落形态类似于11 34株,而菌体形态区别于双亲,呈较短的杆状,无鞭毛,但有较稀疏的纤毛。生理生化研究表明：Q4413耐盐性、蔗糖发酵,石蕊牛奶试验产碱状况酷似于151株,而与1134株略有差异;运动性、糊精、柠檬酸盐利用,MR试验、酪素水解、明胶水解、石蕊牛奶还原,又酷似于1134株而区别于151株;04413的最适生长温度、pH值居双亲之间;Qq4413为原养型加三重抗性[Rifr,AECr Nalr];其细胞DNA含量与GC比测定结果均居双亲之中,而且生物量测定结果也均与双亲有一定的差异。     

内生多粘类芽胞杆菌纤溶酶基因PPFE-I在大肠杆菌中融合表达及活性分析

以内生多粘类芽胞杆菌EJS-3基因组DNA为模板,PCR扩增PPFE-I基因,并克隆到pMD19-T载体上,构建克隆载体pMD-PPFE-I,经测序正确后,将PPFE-I基因克隆至表达载体pET-DsbA上构建重组表达质粒pET-DsbA/PPFE-I,将其转化至E. coli BL21(DE3),在IPTG诱导下实现了融合蛋白DsbA-PPFE-I的表达,表达产物酶活性达228 IU/mL。表达产物用SDS-PAGE和Western blotting进行鉴定。SDS-PAGE电泳检测表明融合蛋白主要以可溶形式表达,占菌体总蛋白的18.4％。Western blotting结果表明在相应分子量处有一条特异性条带,证实该蛋白为DsbA-PPFE-I融合蛋白。表达产物通过Ni亲和柱、凝血酶酶切及Sephadex G-100等步骤进行分离纯化,并用 MALDI-TOF 质谱对重组酶进行了鉴定。纯化后的表达产物在纤维蛋白平板上表现出明显的纤溶活性。     

影响枯草芽胞杆菌和荧光假单胞菌原生质体再生的因素

目的：为了提高再生率,对影响革兰阳性菌枯草芽胞杆菌KR株和革兰阴性菌荧光假单胞菌B13株原生质体再生的因素进行研究。方法：研究了酶解时间,再生方式,再生培养基中稳定剂的种类,Ca^2＋、Mg^2＋、琥珀酸钠、L-色氨酸的浓度及培养基的放置时间对KR和B13株原生质体再生的影响。结果：对KR株酶解20min,采用夹层培养,再生培养基中加入0.6mol/L蔗糖、0.03mol/L Ca^2＋、0.02mol/L Mg^2＋、0.3mol/L琥珀酸钠、0.2mol/L L-色氨酸,培养基在37℃放置72h,原生质体再生率可达42.7%;对B13酶解15min,采用夹层培养,培养基中加入0.6mol/L NaCl、0.02mol/L Ca^2＋、0.01mol/L Mg^2＋、0.3mol/L琥珀酸钠、0.1mol/L L-色氨酸,培养基在37℃放置48h,原生质体再生率可达15.3%。结论：影响革兰阳性菌枯草芽胞杆菌KR株和革兰阴性菌荧光假单胞菌B13株原生质体再生的因素是不同的。     

种间双亲灭活原生质体融合选育高产Monacolin K红曲菌

为获得高产MonacolinK的红曲菌菌株,将经农杆菌介导转化获得的携带潮霉素抗性基因且以甘油为原料液态发酵高产MonacolinK的发白红曲菌H2和以大米为原料固态发酵产Monaco-1inK的烟色红曲菌9908作为亲本,对其原生质体分别进行热灭活及紫外灭活,然后对灭活双亲用PEG作融合剂进行原生质体融合。从融合子中选出有潮霉素抗性的突变株,通过发酵与亲本对比,筛选得到一株以大米为原料固态发酵高产MonacolinK的融合株F12．11,其MonacolinK产量达到8．73mg／g;较发白红曲菌H2与烟色红曲菌9908分别提高了100．23％和48．98％;一株以甘油为原料液态发酵高产MonacolinK的融合株F13-2,其MonacolinK的产量达到1752．46mg／L,较发白红曲菌H2与烟色红曲菌9908分别提高了32．98％和1979．33％。     

Interspecific hybridization between Volvariella volvacea and V. bombycina by PEG-induced protoplast fusion

Interspecific hybridization between Volvariella volvacea and V. bombycina was studied using the protoplast fusion technique. The fusion frequency was found to be in the range of 0.032 to 0.333%. Protoplasts from various hybrids were released and regenerated to determine whether they were heterokaryons. In all regenerated colonies, both parental types could not be recovered at the same time. The nuclear DNA contents of hybrids were compared with their parents, and no diploid (parent 1 genome plus parent 2 genome) was found. Some hybrids revealed novel fragments in mitochondrial rDNA PCR profiles, which indicated that rearrangement of mtDNA could have occurred after fusion. Results from arbitrarily-primed polymerase chain reaction (AP-PCR) fingerprints also revealed that the majority of hybrids were similar to one parental type, but heterologous fragments were found in some hybrids.     

Identification of LI-F type antibiotics and di-n-butyl phthalate produced by Paenibacillus polymyxa

Paenibacillus polymyxa strain JSa-9, a soil isolate that displayed antibacterial and antifungal activities in vitro, had been found to produce two types of antimicrobial substances. The two compounds were extracted from the fermentation broth of JSa-9 using ethyl acetate and subsequently purified by high performance liquid chromatography. By means of liquid chromatography-mass spectrometry and tandem mass spectrometry analysis, one of two antagonistic compounds was determined as di-n-butyl phthalate. And another was characterized as a mixture of related peptides of molecular masses of 883, 897, 911, 947, and 961 Da, with the most likely structure of them determined to be a cyclic depsipeptide with an unusual 15-guanidino-3-hydroxypentadecanoic acid moiety bound to a free amino group. These peptides were therefore members of the LI-F group of cyclic depsipeptides.     

Intraspecific protoplast fusion of amylase-producing strains of Candida fennica

The production of -amylase was increased by protoplast fusion of auxotrophic mutants of Candida fennica FTPT-8903. One prototrophic fusant was 90% and 32% more efficient in producing -amylase in semi-solid and liquid fermentation, respectively, than the parental strains. Protoplast fusion did not significantly stimulate the synthesis of glucoamylase in the fusants.     

Genetic analysis of intraspecies recombinant formation by protoplast fusion with three species of Streptomyces

Abstract Protoplast fusion was shown to produce high frequencies of recombinant progeny in intraspecies crosses with auxotrophic mutants of Streptomyces canescens, Streptomyces griseus and Streptomyces limosus . The fused protoplasts were regenerated on non-selective media and the progeny spores subsequently analysed on selective media to allow detection of all possible genotypes. Prototrophic recombinants arose with frequencies of between 1% and 8%. All 4 possible genotypes were recovered in a series of 2-factor crosses and 6 of the 8 possible genotypes were detected in a 3-factor cross. In spite of attempts to equalise the ratios of parental protoplasts in the fusion mixture, there were noticeable deviations from unity in the ratios of parental genotypes in the progeny.     

利用原生质体紫外诱变技术选育耐高温香菇菌株

【目的】运用原生质体紫外诱变技术选育香菇耐高温新菌株。【方法】以香菇菌株18为出发菌株,紫外诱变处理其原生质体,通过47°C热激3 h后菌丝恢复生长的情况来筛选获得耐高温诱变株,测定18及其所有诱变株在木屑培养基中的恒温长速、高温长速以及恢复长速,并进行高温出菇试验。【结果】筛选得到57株耐高温诱变株,其中诱变株N6、N44和N24的综合性状较好。恒温长速、高温长速以及恢复长速与出菇性状具有相关性,恢复长速与出菇产量、单菇性状、耐高温能力呈正相关,可初步作为预测耐高温菌株综合性状的指标。【结论】利用原生质体紫外诱变技术,可初步选育出耐高温香菇新菌株。     

灭活原生质体融合选育木糖、葡萄糖共发酵酿酒酵母工程菌

为了选育高效利用木糖、葡萄糖共发酵,并使乙醇产量有所提高的酿酒酵母工程菌株。以酿酒酵母Saccharomyces cerevisiae W5和休哈塔假丝酵母Candida shehatae 20335为亲本株,确定了双亲株原生质体灭活剂量,并进行原生质体融合获得融合子,用高效液相色谱（HPLC）测定融合子以木糖、葡萄糖单碳源及混合碳源发酵时的乙醇得率。结果表明,获得一株发酵性能优良的融合子HDY2-14,其利用木糖和葡萄糖单碳源发酵的乙醇得率分别为0.213g/g和0.257g/g,混合碳源发酵的乙醇得率为0.310g/g,其中混合碳源乙醇得率比亲本株W5和20335的乙醇得率分别提高了20.2%和15.2%。     

Strain improvement ofArthrobacter simplex by protoplast fusion

Anl-tryptophan auxotroph and milky mutants were derived from an inducible cholesterol oxidase-producing bacterium,Arthrobacter simplex USA18, via UV-mutagenesis. Protoplasts of these mutants and a constitutive cholesterol oxidase producer, strain US3011, were prepared by growing cells in the presence of ampicillin (20g ml^–1) followed by digestion with lysozyme. Protoplast fusion between tested strains with complementary characteristics was achieved in the presence of 20–40% polyethylene glycol 6000. The fusion frequency was about 1.5–1.7×10^–3. The cholesterol oxidase activity of four fusants in a cholesterol-containing medium was 20–60% higher than that of parental strains. This study demonstrated that protoplast fusion is applicable to strain improvement ofArthrobacter strains for enzyme production.     

原生质体融合构建直接利用淀粉产衣康酸菌株

采用衣康酸高产菌株栖土曲霉T-730的原生质体,与葡萄糖淀粉酶产生菌黑曲霉Ni-5k的原生质体进行融合处理.在以30%PEG6000为促融剂,30℃保温20min的融合条件下进行融合,所得异核体经诱导获得3株稳定的融合株(F3,F9,F11).以生淀粉为唯一碳源,对F3,F9,F11的发酵性能测定表明,F3在发酵过程中积累葡萄糖淀粉酶和衣康酸;对F3进行发酵条件初步探讨的结果表明,以10%生淀粉为碳源,连续发酵6d,F3的衣康酸产酸率达40.9mg/mL,对供给淀粉的转化率为40.9%.