用快中子法选育细菌脂肪酶高产菌株*

研究了从土壤中筛选产脂肪酶的菌株，利用紫外线、快中子、快中子和磁场复合、γ射线、γ射线和磁场复合诱变，以酶活为筛子进行诱变育种。结果，出发菌酶活较低的一株得到了一株酶活为３９６．２２Ｕ／ｍＬ的诱变株，此酶活比出发菌株高９２倍，并发现此菌对紫外线和快中子比较敏感；而出发菌酶活较高的一株得到了酶活为４２４．６０Ｕ／ｍＬ发酵液的诱变株，此酶活为出发菌株３．０倍。在此基础上，初步探讨了快中子、γ射线及磁场复合处理在产脂肪酸菌种诱变中的作用，并认为，在产脂肪酸菌株的诱变中快中子诱变更为有效。     

离子注入结合紫外线及~(60)Co-γ射线诱变选育碱性果胶酸裂解酶高产菌株的研究

以碱性果胶酸裂解酶产生菌芽孢杆菌WZ008为出发菌株,经形态鉴定和16S鉴定为类芽孢杆菌,命名为Paenibacillus sp.WZ008,通过N~+注入诱变、紫外线诱变、~(60)Co-γ射线诱变等多次反复诱变,选育得到一株产碱性果胶酸裂解酶性能稳定且酶活明显提高的突变株,其酶活为97.8U/mL,比出发菌株产碱性果胶酸裂解酶能力提高了1.04倍。     

原生质体诱变选育乳糖酶高产菌株

采用紫外线诱变和^60Co-γ射线协同诱变的方法,对出发菌株Uco-3的原生质体进行诱变处理,通过正突变率与诱变剂量的相互关系,确定最佳诱变剂量。采用4min的紫外线照射和剂量为500Gy的γ射线对黑曲霉Uco-3的原生质体进行诱变,软得一株产高温乳糖酶的高产突变株,突变株产乳糖酶能力显著提高,产酶活力达44．37U／mL,是出发菌株Uco-3的2．73倍。     

紫外、~(60)Co复合诱变选育烟色烟管菌漆酶高产突变株

对烟色烟管菌(Bjerkandera fumosa 5.0172)的孢子悬浮液进行紫外诱变筛选得到一株诱变株ZW-7,其产漆酶的活力是原始菌株的1.33倍,继代培养5代后,未见酶活下降。将诱变株ZW-7的孢子悬浮液用60Co-γ射线进行辐射诱变筛选得到一株诱变株Co-11,其产漆酶的活力是ZW-7的1.18倍,为380.5 U/L,较原始菌株的250.6 U/L提高了约58.1%,继代培养5代后,酶活稳定。     

原生质体诱变选育乳糖酶高产菌株*

采用紫外线诱变和⁶⁰Co-γ射线协同诱变的方法,对出发菌株Uco-3的原生质体进行诱变处理,通过正突变率与诱变剂量的相互关系,确定最佳诱变剂量。采用4min的紫外线照射和剂量为500Gy的γ射线对黑曲霉Uco-3的原生质体进行诱变,获得一株产高温乳糖酶的高产突变株,突变株产乳糖酶能力显著提高,产酶活力达44.37U/mL,是出发菌株Uco-3的2.73倍。     

复合诱变在青霉高产菊粉酶菌株选育中的应用

以Penicillium sp．B01为出发菌株,经吖黄素或DES（硫酸二乙酯）分别与^60Co-γ射线对其孢子悬液进行复合诱变。经过初筛和复筛,在30μg／mL吖黄素诱变时间2h,剂量率为4．11Gy／min的^60Co-γ射线辐射使累计剂量为20．55Gy复合诱变的条件下,筛选出一株菊粉酶活比出发菌株高32％的突变菌株B01-A13-Co31。经同工酶电泳验证,变异株与出发菌株相比酶带有所变化。将此菌种连续传代6次进行产酶性能的稳定性测定,表明此菌株具有良好的遗传稳定性。     

采用复合诱变方法选育高产β-半乳糖苷酶菌株

目的:研究复合诱变方法选育高产β-半乳糖苷酶菌株.方法:以马克斯克鲁维酵母为出发菌株,经过紫外线诱变及硫酸二乙酯、亚硝基胍复合诱变,从大量突变株中进行筛选.结果:成功地选育出一株高产、稳定的菌株15D,其产酶活力由出发菌的124.5U/mg提高到172.4U/mg,酶活力提高了约1.4倍.结论:该方法选育β-半乳糖苷酶菌株是有效的.     

UV与DES复合诱变选育高产脂肪酶菌株

以产脂肪酶菌株BaciUus sp CS-4为出发菌株,进行了UV与硫酸二乙酯(DES)复合诱变处理.筛选出一株高酶活的目的菌株,命名为Bacillus spDE-8.其酶活为每毫升14.85U,比出发菌株提高48.2%.传代实验证明,其遗传性能稳定.     

高产耐热脂肪酶嗜热脂肪地芽孢杆菌的选育

采用氮离子注入技术对耐热脂肪酶产生菌嗜热脂肪地芽孢杆菌（Geobacillus stearothermophilus）L4进行诱变,筛选获得酶活力有较大提高且传代稳定的正突变菌株L4-3;再对L4-3进行紫外线诱变,得到脂肪酶活力提高的正突变菌株L4-3-2,其脂肪酶活力达25．71U／mL,较原始菌株M提高511．9％。高产突变株L4-3-2所产脂肪酶的最适作用温度为50℃,70℃保温60min的剩余酶活为82％,最适作用pH为7．0～8．0,为一种耐热碱性脂肪酶。     

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。     

原生质体紫外诱变选育白地霉GXU08脂肪酶高产菌株

目的:初步筛选脂肪酶高产菌株。方法:以白地霉GXU08为出发菌,对其进行原生质体紫外诱变选育。结果:筛选得到6株脂肪酶活力比出发菌株GXU08高的突变株,其中菌株4-39的酶活达14.2U,比GXU08提高了63.2%。突变株经9次传代,3次摇瓶复筛,其脂肪酶酶活性保持稳定,为今后进一步研究不同的育种方法进一步提高脂肪酶的产量打下基础。     

Purification and characterization of a novel organic solvent-tolerant and cold-adapted lipase from Psychrobacter sp. ZY124

By screening 25 different psychrophilic strains isolated from the Arctic habitat, we isolated a strain capable of producing lipase. We identified this strain as Psychrobacter sp. ZY124 based on the amplified 16S rDNA sequence. The lipase, named as Lipase ZC12, produced from the supernatant of Psychrobacter sp. ZY124 cultured at 15 °C was purified to homogeneity by ammonium sulfate precipitation followed by Phenyl Sepharose FF gel hydrophobic chromatography. Based on the obtained amino acid sequence, Lipase ZC12 is classified as a member of the Proteus/psychrophilic subfamily of lipase family I.1; it has a molecular weight of 37.9 kDa. We also determined that the apparent optimum temperature for Lipase ZC12 activity is 40 °C. Lipase ZC12 shows remarkable organic solvent tolerance by remaining more 50% after incubated with 10–90% different organic solvents. In addition, acyl chain esters with C12 or longer were confirmed to be preferable substrates for Lipase ZC12. Lipase ZC12 also shows better stereoselectivity for (R, S)-1-phenylethanol chiral resolution in n-hexane solvent with (S)-1-phenylethanol (ee_p 92%) and conversion rate (39%) by transesterification reactions. These properties may provide potential applications in biocatalysis and biotransformation in non-aqueous media, such as in detergent, transesterification or esterification and chiral resolution.

     

基于ARTP诱变和高通量筛选的绿针假单胞菌GP72育种方法

【目的】绿针假单胞菌GP72是一种植物根围促生细菌,其分泌的次级代谢产物2-羟基-吩嗪(2-OH-PHZ)具有广谱抗真菌活性,但其产量较低,不能满足农业生产的应用需求,因此需对GP72进行改造,从而提高产量。【方法】从GP72的野生株出发,首次将2-OH-PHZ合成途径的限制性因子Phz O用绿色荧光蛋白(GFP)替换,以一种新型的常压室温等离子体技术(Atmospheric and room temperature plasma,ARTP)进行诱变,通过酶标仪测定96孔板中突变株的荧光强度进行高通量筛选;最后将荧光强度高的菌株中绿色荧光蛋白(Green fluorescent protein,GFP)替换为Phz O以获得2-OH-PHZ高产突变株。【结果】经过五轮诱变后,获得一株荧光强度增加1.62倍的突变株,用phz O基因回替后,该突变株在KB培养基中摇瓶培养时2-OH-PHZ的产量为野生型的4.62倍。【结论】基于安全、高效ARTP诱变技术,并以GFP替换限制性因子作为标记进行高通量筛选,可以快速获得高产2-OH-PHZ的GP72突变株,克服了传统诱变育种方法筛选难度大、费时费力的不足,为其它微生物的育种提供了参考。     

Screening the Fusarium graminearum inhibitory mutant strain from Bacillus subtilis by atmospheric-pressure plasma jet

Aims:  The aim of this study was to improve the antagonistic activity of Bacillus subtilis JA towards Fusarium graminearum by screening high-yielding mutant using the atmospheric-pressure plasma jet (APPJ).
Methods and Results:  Atmospheric-pressure plasma jet was applied as mutagenic source for breeding high-yielding mutant strain. Helium was used as APPJ operating gas. The mutation effects of different treatment times of APPJ were studied. The mutant strain designated as B. subtilis B06 was successfully screened out, which showed higher antagonistic activity against F. graminearum in vitro . Its inhibition zone against the indicator fungus increased by 23% compared to the original one. HPLC and ESI (electrospray ionization) mass spectrometry analysis indicated that antifungal compounds produced by the mutant and original strain belonged to the lipopeptide, surfactin and iturin families. The mutant strain showed favourable properties of faster growth in the fermentation process and higher production of antibiotics. The lipopeptide production of the mutant was 2·3-fold as that of the original strain.
Conclusions:  A mutant strain with strong antagonistic activity and high yielding of antibiotics was obtained by APPJ in this study. The mutant could be used as a promising biocontrol agent in agriculture.
Significance and Impact of Study:  This study provides a novel mutagenic source for breeding high-yielding microbial mutant, which would be very useful in the application of some valuable metabolites from micro-organism.     

Production of extracellular lipase by the hexachlorocyclohexane utilizing Pseudomonas strain Ptm+

Extracelluar lipase activity was detected in a culture of Pseudomonas strain Ptm⁺ growing on hexachlorocyclohexane. Lipase activity was associated with growth of the bacterium and reached a peak at early idiophase (22 h). Following the lipase activity, bioemulsifier was produced, with maximum activity at mid-idiophase (32 h). The extracellular lipase is probably involved in the extracellular synthesis of the bioemulsifier in the culture broth.     

Cloning and expression of novel lipase from entomopathogenic fungi,Beauveria bassiana

Lipase, which catalyzes the hydrolysis of triacylglycerol, is one of the enzymes of interest in the bio-industry. In this study, strains of entomopathogenic fungi were screened for their lipase activity and the characteristics of lipase isolated from Beauveria bassiana JEF-351 strain showing high lipase activity were investigated. Lipase gene of B. bassiana JEF-351 strain, BBL351, was introduced into the genome of Autographa californica nucleopolyhedrovirus (AcMNPV) and recombinant lipases were expressed as non-secreted and secreted protein, respectively. Enzyme activity of BBL351 was higher when the protein was expressed as secreted form, demonstrating that post-translational modification such as glycosylation is crucial for enzyme activity. In addition, the enzyme activity of BBL351 was higher than that of CML, which is an sn-1(3) regioselective lipase reported from Cordyceps militaris. These results suggested that the lipase derived from the B. bassiana JEF-351 strain could be useful as biocatalyst in the biotechnological applications.     

高价麻疹单克隆抗体的研制和应用

作者曾报道过,获取高价单克隆抗体(McAb)与免疫方法有关。本文报道获得麻疹高价单克隆抗的途径,以及将其用作捕捉抗体以提高ELISA双抗体夹心法检测麻疹IgG抗体的敏感性。     

高产洛蒙真菌素洛蒙德链霉菌高通量筛选方法的建立与复合育种

[背景]洛蒙德链霉菌S015能生物合成具有广谱抗菌活性的吩嗪类化合物洛蒙真菌素。[目的]因S015菌株的洛蒙真菌素产量较低,将S015菌株经复合诱变育种和基因工程改造,提高洛蒙真菌素产量。[方法]建立洛蒙真菌素产生菌的高通量筛选方法,对出发菌株S0 15进行常压室温等离子体(atmospheric and room temperature plasma,ARTP)技术和紫外复合诱变,筛选得到高产菌株;并在高产菌株上敲除洛蒙真菌素的前体分支酸竟争途径中的关键基因trpE1、trpE2,再过表达全局调控基因afsR。[结果]利用洛蒙真菌素在紫外波长375 nm处的特征吸收峰,以及洛蒙真菌素浓度和375 nm处吸光度值的正相关关系,建立了基于24孔深孔板发酵和酶标仪快速检测的高通量筛选方法。经过6轮ARTP和紫外复合诱变及高通量筛选,从4 320株突变株中筛选得到遗传稳定的高产菌株M6,其洛蒙真菌素的产量为61.33 mg/L,是S015菌株的7.35倍;M6菌株的分支途径基因trpE1、trpE2双敲株的洛蒙真菌素产量为81.89 mg/L,是S015菌株的9.82倍;在该基因工程菌株中过表达全局调控基因afsR,产量为109.53 mg/L,是S015菌株的13.13倍。[结论]建立的高通量筛选方法可以有效筛选高产洛蒙真菌素的突变株,并且操作简单快速。通过ARTP和紫外复合诱变,结合高产株M6的基因工程改造,能进一步提升洛蒙真菌素的产量。     

果胶酶高产菌株选育

从土壤中分离筛选到一株具有较强果胶分解能力的野生菌株Ｎ３２８,经初步鉴定为米曲霉（Ａｓｐｅｒｇｉｌｌｕｓｏｒｙｚａｅ）。此菌株经紫外线、ＣＯ６０－γ射线、硫酸二乙酯连续诱变得到突变株ＺＩ－４７,该突变株的发酵滤液,当以果胶为底物时,酶活力达３８８７ｕ／ｍｌ（ｕ：ｍｇ还原糖／ｍｌ·ｈ）,为出发菌株的１０倍。且其遗传性能稳定,连续传代１２代,４℃冰箱保存１年,酶活基本无降低。其果胶酶活力超过国内已报道的所有菌株。     

球孢白僵菌的紫外诱变及几丁质酶高产菌株的筛选

以球孢白僵菌（Ｂｅａｕｖｅｒｉａｂａｓｓｉａｎａ）１３１６为出发菌株,通过２０ｍｉｎ和３０ｍｉｎ的紫外线交替诱变分生孢子,采用透明圈法初筛和摇瓶培养复筛的方法,得到一突变株ＣＨ－１３１６。和原始菌株相比,该突变株的几丁质酶活力提高了近３倍,经传代培养,该菌株的几丁质酶高产特性能稳定遗传。