ε-聚赖氨酸高产菌株选育及分批发酵的研究

以北里孢菌(Kitasatospora sp.)PL6-3为出发菌株,经硫酸二乙酯诱变,获得遗传性能稳定的AECr突变株MY5-36。MY5-36摇瓶发酵产ε-聚赖氨酸达1.17 g/L,是出发株PL6-3的3倍;5 L发酵罐批式发酵产酸达7.72 g/L,较出发菌株提高了7倍以上。与PL6-3相比,MY5-36菌株形态发生了很大变化,菌丝和孢子颜色都发生了改变。     

Enhanced epsilon-poly-L-lysine production from Streptomyces ahygroscopicus by a combination of cell immobilization and in situ adsorption

Epsilon-poly-L-lysine (epsilon-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of Llysine, which is used as a safe food preservative. The present study investigates the combined use of cell immobilization and in situ adsorption (ISA) to produce epsilon-PL in shaken flasks. Loofah sponge-immobilized Streptomyces ahygroscopicus GIM8 produced slightly more epsilon-PL than those immobilized on synthetic sponge, and sugarcane bagasse. Moreover, loofah sponge supported the maximum biomass. Hence, loofah sponge was chosen for cell immobilization. Meanwhile, the ion-exchange resin D152 was employed for ISA. The loofah sponge-immobilized cells produced 0.54 +/- 0.1 g/l epsilon-PL, which significantly increased to 3.64 +/- 0.32 g/l after combining with ISA through the addition of resin bags. The free cells with ISA using the dispersed resin yielded 2.73 +/- 0.26 g/l of epsilon-PL, an increase from 0.82 +/- 0.08 g/l. These data illustrate that the proposed combination method improved production most significantly compared with either immobilization or ISA only. Moreover, the immobilized cells could be repeatedly used and an epsilon-PL total amount of 8.05 +/- 0.84 g/l was obtained. The proposed combination method offers promising perspectives for epsilon-PL production.     

Biosynthesis of epsilon-poly-L-lysine in a cell-free system of Streptomyces albulus

epsilon-Poly-L-lysine (epsilon-PL) is a homo-poly-amino acid characterized by a peptide bond between carboxyl and epsilon-amino groups of L-lysine. Here we report the cell-free synthesis of epsilon-PL by a sensitive radioisotopic epsilon-PL assay system. In vitro epsilon-PL synthesis depended on ATP and was not affected by ribonuclease, kanamycin, or chloramphenicol. epsilon-PL synthesizing activity was detected in the membrane fraction. The reaction product, epsilon-PL, from L-lysine was identified by MALDI-TOF MS and the number of lysine residues of the epsilon-PL products was apparently 11-34. These results suggest that the biosynthesis of epsilon-PL is nonribosomal peptide synthesis and is catalyzed by membrane bound enzyme(s). The enzyme preparation showing the epsilon-PL synthesizing activity also catalyzed lysine-dependent AMP production and an ATP-PPi exchange reaction, suggesting that L-lysine is adenylated in the first step of epsilon-PL biosynthesis.     

Substantially monodispersed poly(ɛ-<Emphasis Type="SmallCaps">l</Emphasis>-lysine)s frequently occurred in newly isolated strains of <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

The presence of poly(epsilon-L-lysine) (epsilon-PL) was found quite frequently by screening various strains of Streptomyces sp. Most of the ten newly obtained epsilon-PLs, when they were produced from glucose, showed a polydispersity index of Mw/Mn = 1.01 using ion-pair chromatography analysis. The polymers were classified into five groups according to their chain lengths. The average numbers of residues in the five groups were 32, 28, 25, 19, and 16, respectively. The use of glycerol instead of glucose resulted in decreases of 10 to 20% in the Mn and slight increases in the Mw/Mn. These observations indicated the chain length and polydispersity of epsilon-PL were primarily determined by each producer strain. Proton and 13C NMR analysis revealed the signals of glycerol-derived ester at the C terminus of the polymer from several producers including the first discovered S. albulus strain, although the percentages of the ester were low under our culture conditions. These results, coupled with the previous observation that SO4(2-) was essential for the polymer production, led to discussion on the mechanistic aspects of monomer activation, elongation, and termination in the biosynthesis of epsilon-PL.     

Epsilon-poly-L-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase

Epsilon-Poly-L-lysine (epsilon-PL) consists of 25-35 L-lysine residues in isopeptide linkages and is one of only two amino acid homopolymers known in nature. Elucidating the biosynthetic mechanism of epsilon-PL should open new avenues for creating novel classes of biopolymers. Here we report the purification of an epsilon-PL synthetase (Pls; 130 kDa) and the cloning of its gene from an epsilon-PL-producing strain of Streptomyces albulus. Pls was found to be a membrane protein with adenylation and thiolation domains characteristic of the nonribosomal peptide synthetases (NRPSs). It had no traditional condensation or thioesterase domain; instead, it had six transmembrane domains surrounding three tandem soluble domains. These tandem domains iteratively catalyzed L-lysine polymerization using free L-lysine polymer (or monomer in the initial reaction) as acceptor and Pls-bound L-lysine as donor, directly yielding chains of diverse length. Thus, Pls is a new single-module NRPS having an amino acid ligase-like catalytic activity for peptide bond formation.     

Kitasatospora sp. MY5-36产ε-聚赖氨酸分批补料发酵动力学

以北里孢菌(Kitasatospora sp.)MY 5-36为供试菌株,对ε-聚赖氨酸分批补料发酵动力学模型进行研究。建立了该菌株发酵合成ε-聚赖氨酸的菌体生长、产物合成和总糖消耗的动力学模型,并通过Origin 8.1软件对模型参数进行非线性拟合。结果表明:菌体量和聚赖氨酸的产量分别为16.25和13.15 g/L,产物合成与菌体生长的关系为部分耦联型。经验证,预测值与实验值有良好的拟合性,拟合度分别为0.999、0.995和0.992,说明所构建模型能够较好地反映ε-聚赖氨酸分批补料发酵过程。     

Occurrence of epsilon-poly-L-lysine-degrading enzyme in epsilon-poly-L-lysine-tolerant Sphingobacterium multivorum OJ10: purification and characterization

Epsilon-poly-L-lysine (epsilon-PL)-degrading enzyme was found in the epsilon-PL-tolerant strain Sphingobacterium multivorum OJ10 and purified to homogeneity. The purified enzyme has a molecular mass of approximately 80 kDa. The enzyme catalyzed exo-type degradation of epsilon-PL and released L-lysine. The enzyme was a Co2+ or Ca2+ ion-activated aminopeptidase.     

DNA condensation for gene therapy as monitored by atomic force microscopy.

The atomic force microscope (AFM) was used to assay the extent of DNA condensation in approximately 100 different complexes of DNA with polylysine (PL) or PL covalently attached to the glycoproteins asialoorosomucoid (AsOR) or orosomucoid (OR). The best condensation of DNA was obtained with 10 kDa PL covalently attached to AsOR, at a lysine:nucleotide (Lys:nt) ratio of 5:1 or higher. These conditions produce large numbers of toroids and short rods with contour lengths of 300-400 nm. Some DNA condensation into shortened thickened structures was seen with 10 kDa PL attached to AsOR at Lys:nt ratios of 1.6:1 and 3:1. Some DNA condensation was also seen with 4 kDa PL at Lys:nt ratios of 3:1 and higher. Little DNA condensation was seen with PL alone or with PL convalently attached to OR at Lys:nt ratios up to 6:1. AsOR-PL enhanced gene expression in the mouse liver approximately 10- to 50-fold as compared with PL alone.     

海洋放线菌Streptomyces variabilis strain 6-1次级代谢产物的研究

目的:对来自海洋软珊瑚的链霉菌6-1(Streptomyces variabilis strain 6-1)进行次级代谢产物的分离和鉴定,寻找具有生物活性的化合物,为人类健康服务。方法:采用液体培养基对分自海洋软珊瑚Scleronephthya sp中的链霉菌6-1(Streptomyces variabi-lis strain 6-1)进行发酵培养,用乙酸乙酯对发酵液进行萃取;采用半制备高效液相色谱(semi-preparative HPLC)分离方法对乙酸乙酯萃取物进行分离纯化,得到单体化合物;运用电喷雾质谱(ESI-MS)、核磁共氢振(1H NMR)、核磁共振碳谱(13C NMR)和物理性质对所得单体化合物进行结构鉴定。结果:从海洋链霉菌6-1(strain 6-1)发酵液的乙酸乙酯萃取物中分离得到3个单体化合物,分别鉴定为:7,4'-二羟基异黄酮(1)、5,7,4'-三羟基异黄酮(2)和丁烯酸内酯-Ⅰ(3)。结论:丁烯酸内酯-Ⅰ是从链霉菌属首次分离得到,化合物1和2均是从Streptomyces variabilis中首次分离得到;变异链霉菌6-1(Streptomyces variabilis strain 6-1)可以作为活性化合物3(丁烯酸内酯-Ⅰ)的重要来源。     

海洋放线菌Streptomyces variabilisstrain 6-1次级代谢产物的研究

目的：对来自海洋软珊瑚的链霉菌6-1（Streptomyces variabilisstrain6-1）进行次级代谢产物的分离和鉴定,寻找具有生物活性的化合物,为人类健康服务。方法：采用液体培养基对分自海洋软珊瑚Scleronephthya sp中的链霉菌6．1（Streptomyces vafiabilisstrain6-1）进行发酵培养,用乙酸乙酯对发酵液进行萃取;采用半制备高效液相色谱（semi-preparative HPLC）分离方法对乙酸乙酯萃取物进行分离纯化,得到单体化合物;运用电喷雾质谱（ESI—MS）、核磁共氢振（1HNMR）、核磁共振碳谱（13C NMR）和物理性质对所得单体化合物进行结构鉴定。结果：从海洋链霉菌6-1（strain6-1）发酵液的乙酸乙酯萃取物中分离得到3个单体化合物,分别鉴定为：7,4’-二羟基异黄酮（1）、5,7,4’-三羟基异黄酮（2）和丁烯酸内酯-I（3）。结论：丁烯酸内酯．I是从链霉菌属首次分离得到,化合物1和2均是从Streptomyces variabilis中首次分离得到;变异链霉菌6-1（Stmptomyces variabilis strain6-1）可以作为活性化合物3（丁烯酸内酯-I）的重要来源。     

一株北里孢菌株的分离鉴定及其对松材线虫的致病性

【目的】筛选、鉴定出对松材线虫杀灭活性较高的放线菌菌株,并确定生防菌株的毒力因子。【方法】采用平板活性测试及代谢杀虫活性检测方法进行筛选,采用形态学及16S rDNA序列分析等进行鉴定。对发酵液中的活性物质稳定性分析后,利用醇沉、萃取、层析、气相色谱/质谱分析等方法分离纯化出杀虫毒力因子。【结果】从河南南阳宝天曼的腐木及枯枝落叶样品中共分离获得了79株放线菌,从中筛选出对松材线虫有灭活作用的放线菌6株,其中分离株C620菌株对松材线虫的灭活性最高:该菌株的发酵液处理松材线虫48、60 h后线虫的死亡率分别达到60.0%、81.5%。结合该菌株的形态学、生理学特征及16S rDNA序列分析等结果将其归为北里孢菌属中的一个种,菌株编号Kitasatospora sp.strain C620。该菌株的发酵液中杀线虫活性物质的热稳定性、光稳定性及耐储藏性均较强,在中性偏碱性环境较稳定;经pH纸电泳层析初步确定该物质属于碱性水溶性物质。对菌株C620发酵液分离纯化,得到活性化合物为1-苯基-3-(2-吡啶)-5-吡唑啉酮。【结论】获得一株松材线虫高效生防菌Kitasatospora sp.strain C620,其活性物质为1-苯基-3-(2-吡啶)-5-吡唑啉酮。     

Recombinant agrobacterium AgaE-like protein with fructosyl amino acid oxidase activity

Agrobacterium tumefaciens AgaE-like protein had a similar sequence to that of a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1. To characterize the AgaE-like protein, we produced the enzyme in Escherichia coli, and purified it to homogeneity. The molecular mass of recombinant AgaE-like protein was 42 kDa on SDS-PAGE and 85 kDa on gel filtration. The protein acted on N-fructosyl valine and N-fructosyl glycine as substrates, but not on glycated protein or N(epsilon)-fructosyl lysine. Apparent Km for N-fructosyl valine and N-fructosyl glycine were 1.64 and 0.31 mM, respectively. The AgaE-like protein had maximum activity at pH 7.8 and 35 degrees C in 0.1 M potassium phosphate, but more than 80% of its activity was lost at 40 degrees C or more. In contrast to eukaryotic fructosyl amino acid oxidases, the AgaE-like protein contained noncovalently bound FAD as a cofactor and was inactive against N(epsilon)-fructosyl N(alpha)-Z(benzyloxycarbonyl)-lysine. These characteristics were similar to a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1, suggesting that these prokaryotic enzymes comprise a new family of fructosyl amino acid oxidases.     

Engineering photosynthetic production of L-lysine

L-lysine and other amino acids are commonly produced through fermentation using strains of heterotrophic bacteria such as Corynebacterium glutamicum. Given the large amount of sugar this process consumes, direct photosynthetic production is intriguing alternative. In this study, we report the development of a cyanobacterium, Synechococcus sp. strain PCC 7002, capable of producing L-lysine with CO₂ as the sole carbon-source. We found that heterologous expression of a lysine transporter was required to excrete lysine and avoid intracellular accumulation that correlated with poor fitness. Simultaneous expression of a feedback inhibition resistant aspartate kinase and lysine transporter were sufficient for high productivities, but this was also met with a decreased chlorophyll content and reduced growth rates. Increasing the reductant supply by using NH₄⁺, a more reduced nitrogen source relative to NO₃^-, resulted in a two-fold increase in productivity directing 18% of fixed carbon to lysine. Given this advantage, we demonstrated lysine production from media formulated with a municipal wastewater treatment sidestream as a nutrient source for increased economic and environmental sustainability. Based on our results, we project that Synechococcus sp. strain PCC 7002 could produce lysine at areal productivities approaching that of sugar cane to lysine via fermentation using non-agricultural lands and low-cost feedstocks.     

Purification and partial characterization of a lysine-specific protease of Porphyromonas gingivalis

Abstract A lysine-specific protease hydrolysing peptide bonds at the carboxyl side of lysine residues in Porphyromonas gingivalis was purified from culture supernatant by a combination of ion-exchange chromatography, gel filtration, and affinity chromatography. The molecular mass was 48 kDa and the p I value was 7.3. The enzyme hydrolysed the peptide bonds at the carboxyl side of lysine residues in synthetic substrates and natural proteins.     

Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degrees C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.     

Identification of thermostabilizing residues in a Bacillus alkaline cellulase by construction of chimeras from mesophilic and thermostable enzymes and site-directed mutagenesis

An alkaliphilic Bacillus sp. strain, KSM-64, produces a mesophilic alkaline endo-1,4-beta-glucanase that is suitable for use in detergents. The deduced amino acid sequence of the enzyme showed very high homology to that of a thermostable alkaline enzyme from alkaliphilic Bacillus sp. strain KSM-S237. Analysis of chimeric enzymes produced from the genes encoding the mesophilic and thermostable enzymes suggested that the lysine residues at positions 137, 179, and 194 are responsible for their thermal stabilization. Replacing the corresponding Glu137, Asn179, and/or Asp194 with lysine by site-directed mutagenesis made the mesophilic enzyme more thermostable. Analyses of the hydrophilicity of deduced amino acid sequences and isoelectric focusing of the modified enzymes suggested that these three specific lysine residues and their replacements are all located on the surface of the enzyme molecule. This fact further suggested that specific ionic interaction is involved in the thermal stabilization of the enzyme.     

ε-聚赖氨酸生物合成及其产生菌遗传转化研究进展

ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)是由25-35个L-赖氨酸(L-lysine)通过α-ε酰胺键连接的具有很强抗菌活性的聚合物,是自然界中迄今为止仅发现的2种均聚氨基酸(ε-聚赖氨酸和γ-聚谷氨酸)之一。目前,研究发现ε-聚赖氨酸的合成酶是一种非核糖体肽合成酶,它催化前体物质L-lysine经多轮缩合反应合成链长不均一的ε-聚赖氨酸,与I型聚酮合成酶的合成过程相似。ε-聚赖氨酸的合成不受降解酶控制。同时,针对产生菌遗传转化的穿梭质粒载体pLAE001和pLAE003已构建成功,为进一步探索ε-聚赖氨酸生物合成提供了条件。本文主要就ε-聚赖氨酸生物合成及产生菌遗传转化体系进行综述。另外,扼要介绍了作者所在课题组的相关研究工作、取得的进展并提出了相应的见解,论文最后部分对组合生物合成在ε-PL产生菌菌种改造中的应用前景进行了探讨。     

黄瓜枯萎病拮抗放线菌的筛选、鉴定及发酵条件优化

【背景】黄瓜枯萎病是由尖孢镰刀菌(Fusarium oxysporum f. sp. cucumerinum)黄瓜专化型引起的土传真菌性病害,严重制约着黄瓜产业的发展。【目的】从河西走廊敦煌地区盐碱土壤中分离筛选出一株对黄瓜枯萎病病菌有良好拮抗效果的放线菌菌株,探究其分类地位及其最优发酵条件。【方法】采用稀释平板涂布法分离放线菌,平板对峙法、抑制菌丝生长速率法筛选拮抗菌株,通过培养特征、生理生化试验及16SrRNA基因序列分析确定其分类地位,利用单因素试验和正交试验方法确定其最优发酵配方及培养条件。【结果】菌株16-3-10鉴定为链霉菌属(Streptomyces sp.)菌株,最优发酵配方(g/L):小米10.0,乳糖20.0,蛋白胨1.0,NaCl 5.0,CaCO3 6.0,最优发酵条件:培养温度28°C,装瓶量50/250 mL,培养3 d,起始pH 10.0,抑菌率达82.50%,比优化前增加153.43%。【结论】菌株16-3-10对黄瓜枯萎病病菌具有显著的拮抗效果,有较好的应用前景。     

Screening for an oil‐removing microorganism and oil removal from waste silk by pure culture fermentation

The presence of oil is the major limitation to the regeneration of spun silk from waste silk. A pure culture fermentation process was developed to remove oil from waste silk. Fourteen strains were isolated from natural fermentation liquor of waste silk. The strain D3 showed highest lipase activity and oil‐removing ability. This strain was identified as Rhodococcus sp. on the basis of morphological and biochemical characteristics and 16S rRNA sequence analysis. The strain D3 was used to remove oil from waste silk by pure culture fermentation. The effects of various parameters on oil removal were investigated. A pH of 7.0, a temperature of 35–40°C, an incubation time of three days and an inoculum of 10% were optimum conditions for removing oil from waste silk by stain D3. This study shows that pure culture fermentation is a promising process to improve the oil removal of waste silk.