原生质体融合选育肌苷产生菌产氨短杆菌GMA—2802

以肌苷产生菌产氨短杆菌ＧＭＡ－２７２２（Ａｄｅ－、 Ｇｕａ－、ＶＢ１－、Ｒｉｆｒ）和 ＧＭＡ－２７７６（Ａｄｅ－、 Ｂｉｏｔｉｎ－、ＶＢ１－、Ｓｍｒ）为出发菌株,经原生质体融合,将目的标志加以组合,获得遗传性状稳定、摇瓶发酵６１ｈ,产肌苷２５．４ｇ／Ｌ的融合子 ＧＭＡ－２８０２（Ａｄｅ－、Ｇｕａ－、Ｂｉｏｔｉｎ－、ＶＢ１－、 Ｒｉｆｒ、Ｓｍｒ）。     

Isolation and characterization of bacterial strains with the ability to utilize high concentrations of levulinic acid,a platform chemical from inedible biomass

Nineteen levulinic acid (LA)-utilizing bacteria were isolated from environmental samples. Following examination of the use of 80 g/L LA by some isolated strains, Brevibacterium epidermidis LA39–2 consumed 62.6 g/L LA following 8 days incubation. The strain also utilized both 90 and 100 g/L LA, with consumption ratio of 84.3 and 53.3%, respectively, after 10 days incubation.     

Purification and Characterization of Fumarase from Corynebacterium glutamicum

Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K _eq) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K _eq=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.     

Development of a large scale process for the conversion of polysialogangliosides to monosialotetrahexosylganglioside with a novel strain of <Emphasis Type="Italic">Brevibacterium casei</Emphasis> producing sialidase

A bioconversion process of producing GM1 (monosialotetrahexosylganglioside) on an industrial scale was developed with a novel sialidase-producing strain Brevibacterium casei. The sialidase hydrolyzed polysialogangliosides to produce GM1 but did not act on GM1. When Brevibacterium casei was cultured in a synthetic medium containing crude pig brain gangliosides (10% w/v) at 30°C for 24 h in a 50 l fermenter, most of the polysialogangliosides were converted to GM1. The content of GM1 was increased from 9% in crude gangliosides to 45% with 70% (w/w) yield.     

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

Comparison of the cell surface barrier and enzymatic modification system inBrevibacterium flavum andB. lactofermentum

To investigate impediments to plasmid transformation inBrevibacterium flavum BF4 andB. lactofermentum BL1, cell surface barriers were determined by measuring growth inhibition whilst enzymatic barriers were determined by comparing DNA methylation properties.B. lactofermentum was more sensitive to growth inhibition by glycine thanB. flavum. Release of cellular proteins during sonication was more rapid forB. lactofermentum than forB. flavum. Plasmid DNA (pCSL17) isolated fromB. flavum transformed recipient McrBC⁺ strains ofEscherichia coli with lower efficiency than McrBC⁻.McrBC digestion of this DNA confirmed thatB. flavum contain methylated cytidines in the target sequence ofMcrBC sequences butB. lactofermentum contained a different methylation pattern. DNA derived from theB. lactofermentum transformed recipient EcoKR⁺ strains ofE. coli with lower efficiency than EcoKR⁻, indicating the presence of methylated adenosines in the target sequence of EcoK sequences. The present data describe the differences in the physical and enzymatic barriers between two species of corynebacteria and also provide some insight into the successful foreign gene expression in corynebacteria.     

L-异亮氨酸产生菌XQ-4在连续培养中的动力学特性

以黄色短杆菌（Brevibacterium flavum)ATCC14067诱变选育获得的L-异亮氨酸高产菌XQ－4（AHV^rSuc^gSG^rEth^rα-AB^rIleHx^r）在连续培养中进行动力学特性研究,以葡萄糖为限制性底物时,XQ－4菌株的生长符合Monod方程,其最大比生长速率μmax=0.265h^-1,饱和常数Ka=0.789g/L.XQ-4菌株L-异亮氨酸发酵时菌体最大实际转化率Yx=0.499g/g,产物最大实际转化率Yp=0.379g/g。     

新型肌苷产生菌──产氨短杆菌GMBA—800选育和特性的初步研究

采用多种方法诱变获得一株新型肌苷产生菌──产氨短杆菌（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍａｍｍｏｎｉａｇｅｎｅｓ）ＧＭＢＡ－８００（具有腺嘌呤、生物素双重营养缺陷型和对８－氮杂鸟嘌呤、磺胺胍、６－流基嘌呤有抗性）,对其生长和发酵条件进行初步研究。肌苷产量从５ｇ／Ｌ提高到１８．４１ｇ／Ｌ,发酵周期从８４ｈ缩短为６３ｈ。菌株遗传性状稳定。     

L一精氨酸产生菌的选育及其发酵条件的研究

以黄色短杆菌（Brevibacterium flavum)AT174(AHV^r,TA^r)为诱变出发株,经亚硝基胍（NTG）诱变处理,获得一株能够积累大量L－O精氨酸的菌株AG77（AHV^r,TA^r,SG^r)。在20L发酵罐中,以葡萄为碳源,以硫酸铵等为氮源的培养基中培养4d,产酸率可达31.3g/L。     

产氨短杆菌GMA-2802 1.2L罐肌苷发酵试验

采用诱变得来的产氨短杆菌GMA－2802,在1.2L自控发酵罐上进行了5批发酵肌苷试验,发酵周期54小时,平均产肌苷20.4g/L。结果表明该菌档是一株具有较多优良特性的肌苷产生菌。