Screening for microorganisms producing D-malate from maleate.

More than 300 microorganisms were screened for their ability to convert maleate into D-malate as a result of the action of maleate hydratase. Accumulation of fumarate during incubation of permeabilized cells with maleate was shown to be indicative of one of the two enzymes known to transform maleate. The ratio in which fumarate and malate accumulated could be used to estimate the enantiomeric composition of the malate formed. Many strains (n = 128) were found to be capable of converting maleate to D-malate with an enantiomeric purity of more than 97%. Pseudomonas pseudoalcaligenes NCIMB 9867 was selected for more detailed studies. Although this strain was not able to grow on maleate, permeabilized cells were able to degrade maleate to undetectable levels, with a concomitant formation of D-malate. The D-malate was formed with an enantiomeric purity of more than 99.97%.     

Microbial production of D-malate from maleate.

To produce D-malate from maleate by a microbial reaction, we screened a number of maleate-utilizing microorganisms for enzyme activity by an intact cell system. The strain which showed the best productivity among the 440 strains tested was identified taxonomically as Arthrobacter sp. strain MCI2612. The optical purity of the malate produced by this strain was 100% D type. The culture and reaction conditions for the production were studied for this strain. Addition of amino acids such as L-proline, L-histidine, and L-arginine to the culture medium promoted the formation of reaction activity as well as cell growth. Under optimum conditions, 87 g of D-malate per liter was produced in 20 h. The yield was 72 mol%.     

利用Pimelobacter simplex DM18从马来酸转化制备D-苹果酸

利用PimelobactersimplexDM18从马来酸高效制备D 苹果酸 ,优化了菌株DM18的发酵条件 ,最佳发酵培养基组成为 (g/L) :柠康酸 ,2 5 ;葡萄糖 ,3;磷酸二氢钾 ,10 5 ;硝酸铵 ,2 0 ;硫酸镁 ,0 2 5 ;七水硫酸亚铁 ,0 0 15 ;酵母膏 ,2 8;玉米浆 5 ;pH值 7 2。在最适培养基中 30℃培养 30h ,菌体OD660 可达到 4～ 4 5 ,酶活达到 14g/ (L·h) (反应菌液OD660 为 10 )。采用间歇添加马来酸钠反应 32h ,D 苹果酸质量浓度为 15 3 9g/L ,D 苹果酸的收率达到 96 6 7%。,经离子配位体色谱分析 ,产物D 苹果酸光学纯度为 96 98%。     

Screening method for microorganisms producing L-aminoacids from D,L-5-monosubstituted hydantoins

Summary The ability of microorganisms to produce hydantoinase and L-N-carbamoylase could be established by an overlay assay. Enzyme producing strains form clear areas around their colonies caused by the cleavage of D,L-indolylmethylhydantoin. A second overlayer with a tryptophan-auxotroph yeast enables us to detect microorganisms which are able to produce L-tryptophan from D,L-indolylmethylhydantoin.     

Screening of epoxide hydrolase producing microorganisms for biotransformation of deoxynivalenol

Deoxynivalenol (DON) transformation products from selected time course experiments were analyzed by thin-layer chromatography. With the strainAlternaria alternata f. sp.lycopersici AS27-3, one major metabolite of DON in ethyl acetate was observed. This unidentified metabolite was more polar than DON and has a R_f value of 0.71. Derivatization indicated that this metabolite was probably an unidentified trichothecene. Screening of 29 other microbial isolates (bacteria, yeast, filamentous fungi) for DON transformation did not result in any active organism. Presented at the 26^th Mykotoxin-Workshop in Herrching, Germany, May 17–19, 2004     

土壤中选育产壳聚糖酶菌株的研究

采用酶法降解壳聚糖具有反应条件易于控制、产物安全性高和环境污染少等独特的优越性。因此,筛选出高活力产壳聚糖酶菌株有着重要意义。该研究以不同地区采集土样分离出的1株细菌为出发菌株S,采用紫外线诱变（30W,20cm,5min）,经初筛和复筛及控温培养处理,获得了一株产壳聚糖酶较好的突变菌株,结果表明：所产酶活力达到3．47U/ml,酶活力提高近2倍,并具有较好的遗传稳定性,明显优于出发菌株,为发酵产壳聚糖酶的进一步研究提供了高产菌株。     

Rapid assay for detection of microorganisms producing DNA-damaging metabolites.

Microorganisms producing DNA-damaging metabolites (i.e., fungi and streptomycetes) were detected by the Bacillus subtilis rec assay with agar plugs from plates on which the microorganisms had been grown. This assay allowed rapid identification of aflatoxinogenic fungi and streptomycetes producing strong DNA-damaging metabolites. For screening programs, several media have to be used to grow the microorganisms to be tested.     

Screening for novel lipolytic enzymes from uncultured soil microorganisms

The construction and screening of metagenomic libraries constitute a valuable resource for obtaining novel biocatalysts. In this work, we present the construction of a metagenomic library in Escherichia coli using fosmid and microbial DNA directly isolated from forest topsoil and screened for lipolytic enzymes. The library consisted of 33,700 clones with an average DNA insert size of 35 kb. Eight unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. Subsequently, secondary libraries in a high-copy-number plasmid were generated to select lipolytic subclones and to characterize the individual genes responsible for the lipolytic activity. DNA sequence analysis of six genes revealed that the enzymes encoded by the metagenomic genes for lipolytic activity were novel with 34–48% similarity to known enzymes. They had conserved sequences similar to those in the hormone-sensitive lipase family. Based on their deduced amino acid similarity, the six genes encoding lipolytic enzymes were further divided into three subgroups, the identities among which ranged from 33% to 45%. The six predicted gene products were successfully expressed in E. coli and secreted into the culture broth. Most of the secreted enzymes showed a catalytic activity for hydrolysis of p-nitrophenyl butyrate (C₄) but not p-nitrophenyl palmitate (C₁₆).     

Isolation and screening of glucose oxidase producing microorganisms from natural sources.

Among 1486 mould strains isolated from natural sources (screened for extracellular glucose oxidase) only 119 (Aspergillus and Penicillium) showed this enzyme activity. As the best glucose oxidase producer, A. niger 0-1 was isolated from decaying tree. The dynamics of glucose oxidase synthesis in A. niger 0-1 during its culture by submerged method show that the intracellular activity of this enzyme is 10-times higher than its extracellular level. Some properties of the crude glucose oxidase preparation, isolated from the postculture liquids by lyophilization, were examined.     

Characterization of psychrotrophic microorganisms producing beta-galactosidase activities.

Investigations of psychrotrophic microorganisms have been limited even though the dominant environment of the Earth is cold and enzymes with high activities at low temperatures could have commercial uses. We have isolated and characterized three psychrotrophic strains with beta-galactosidase activities. The isolates, B7, D2, and D5, were gram-positive, catalase-positive, obligate aerobes. Cells observed with a scanning electron microscope appeared as rods during the early stages of growth but became coccoid during the stationary phase. An analysis of the amino acid composition of the cell walls demonstrated the presence of lysine as the predominant diamino acid in all three isolates. The cell cycle morphology and cell wall composition suggest that the three isolates are members of the genus Arthrobacter. The beta-galactosidase activities in whole cells were labile when incubated at 40 degrees C and had temperature optima about 20 degrees C below that of the enzyme encoded by the lacZ gene of Escherichia coli. Electrophoresis of extracts from the isolates in nondenaturing polyacrylamide gels detected at least two protein bands that hydrolyzed 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), suggesting the presence of beta-galactosidase isozymes.     

Screening of microorganisms for improvement of beta-galactosidase production.

The strain of Penicillium notatum 1 most effective for producing beta-galactosidase (see lactase 3.2.1.23), was selected out of 110 moulds belonging to 15 different species, by the test-tube microculture method. The dynamics of beta-galactosidase synthesis was investigated in P. notatum 1 during its culture by submerged method.     

产黄曲霉毒素B1降解酶菌株的筛选鉴定及发酵条件优化

以香豆素为唯一碳源筛选到27株能高效降解黄曲霉毒素B1(AFB1)的微生物菌株.用高效液相色谱检测AFB1含量的方法进行AFB1降解酶活力测定.以不同菌株发酵上清液中AFB1降解酶活力高低为复筛条件,筛选到AFB1降解酶活力最高的一株菌并命名为HSD8.该菌株经形态学、生理生化及系统发育学方法鉴定为Sinomonas sp..筛选所得最优菌株的发酵上清液中酶活达443 U/mL,通过单因素试验对其产酶发酵条件进行优化,以提高酶活.优化所得最佳发酵条件为:装液量50 mL/250 mL,发酵周期48 h,初始pH 5.0,接种量8％,发酵温度37℃,摇床转速160r/min.在最佳发酵条件下,该菌株发酵上清液中酶活可达548 U/mL,比优化前提高23.7％.优选菌株HSD8在生物降解黄曲霉毒素B1方面具有应用潜力,值得进一步研究开发.     

Screening for microorganisms that produce only endo-inulinase

Sixteen fungal strains reported in the literature as endo-inulinase producers and three bacterial strains, isolated from the dahlia rhizosphere, were analysed for endo-inulinase production. From four isolated strains (one fungus and three bacteria) the results were evaluated in terms of substrate consumption, cell growth and production of endo-inulinases. All three bacterial strains were sole endo-inulinase producers and, among these, strain Paenibacillus sp. CDB 003 was the most suitable for endo-inulinase production, as this enzyme produced inulobiose as the principal substrate as well as inulo-oligosaccharides with polymerisation degrees of 3-5.     

阿维菌素高产菌株的选育Ⅰ

目的：全面了解阿维菌产生菌S.avermitilis的生长特性并获得阿维菌素的高产菌株。方法：以S.avermitilis Y20为出发菌株,考察该菌株的菌落特征和发酵特性,分别利用紫外线（UV）、亚硝酸（HNO3）及亚硝基胍（NTG）并结合L－异亮氨酸（L－Ⅱe）诱导等手段对出发菌株Y20进行诱变处理。结果：获得高产阿维菌素变异株H336,发酵单位达到852μg/ml。结论：与出发菌株相比,突变株阿维菌素的产量稳定,发酵单位提高了10倍以上。     

产漆酶的草菇菌株的筛选

以Bavendamn反应和愈创木酚滴定法为初筛方法,从18株草菇中筛选高产漆酶菌株.结果表明,在加入了0.4mmol/L鞣酸的Bavendamn培养基平板上,通过所显颜色深度及显色圈直径选出13株强阳性菌株;以愈创木酚滴定法筛选出5株显色时间较短且显色较深的菌株.最后,通过液体发酵复筛,测得V123菌株酶活最高,培养8d其漆酶达78U/mL,其次是V8菌株活力为65U/mL.V123和V8可作为草菇中漆酶的高产菌株供以后的研究.     

Rapid assay for detection of microorganisms producing DNA-damaging metabolites

Microorganisms producing DNA-damaging metabolites (i.e., fungi and streptomycetes) were detected by the Bacillus subtilis rec assay with agar plugs from plates on which the microorganisms had been grown. This assay allowed rapid identification of aflatoxinogenic fungi and streptomycetes producing strong DNA-damaging metabolites. For screening programs, several media have to be used to grow the microorganisms to be tested.     

Immobilization of microorganisms on latex for producing artificial flocs

Microorganisms belonging to the genera Pseudomonas, Micrococcus, Bacillus and Rhodotorula are flocculated with divinyl styrene latex. The process of flocculation depends on a number of physico-chemical factors but, all in all, is universal for microorganisms with different taxonomy, morphology and physiology. The structure of artificial bioflocs has been studied using optical and scanning electron microscopy.