Production and purification of an extracellularly produced K4 polysaccharide from Escherichia coli

Summary The production of the K4 polysaccharide was obtained for the first time extracellularly from a strain of Escherichia coli. The set up of the fermentation conditions led to the maximum fermentation yield, as extracellular K4, after 20 h. Purification and characterization of this K4 resulted in 200 mg/L of highly purified K4.     

Production of capsular polysaccharide from Escherichia coli K4 for biotechnological applications

The production of industrially relevant microbial polysaccharides has recently gained much interest. The capsular polysaccharide of Escherichia coli K4 is almost identical to chondroitin, a commercially valuable biopolymer that is so far obtained from animal tissues entailing complex and expensive extraction procedures. In the present study, the production of capsular polysaccharide by E. coli K4 was investigated taking into consideration a potential industrial application. Strain physiology was first characterized in shake flask experiments to determine the optimal culture conditions for the growth of the microorganism and correlate it to polysaccharide production. Results show that the concentration of carbon source greatly affects polysaccharide production, while the complex nitrogen source is mainly responsible for the build up of biomass. Small-scale batch processes were performed to further evaluate the effect of the initial carbon source concentration and of growth temperatures on polysaccharide production, finally leading to the establishment of the medium to use in following fermentation experiments on a bigger scale. The fed-batch strategy next developed on a 2-L reactor resulted in a maximum cell density of 56 g_cww/L and a titre of capsular polysaccharide equal to 1.4 g/L, approximately ten- and fivefold higher than results obtained in shake flask and 2-L batch experiments, respectively. The release kinetics of K4 polysaccharide into the medium were also explored to gain insight into the mechanisms underlying a complex aspect of the strain physiology.     

Influence of KfoG on capsular polysaccharide structure in Escherichia coli K4 strain

An enzyme KfoG with unknown function is coded by the gene kfoG. Gene kfoG belongs to genes from region 2, which are responsible for structure of capsular polysaccharide. Only two enzymes, KfoG and KfoC, coded by genes from region 2, have a glycosyltransferase motif. KfoC is the bifunctional enzyme, which is able to add both GalNAc and GlcUA on nascent polysaccharide, termed chondroitin polymerase. KfoG was predicted to be a fructosyltransferase. The gene that codes the KfoG enzyme was disrupted using homological recombination and absence of this gene was confirmed on both DNA and RNA levels. After disruption no structural changes have been observed, what indicates that fructose branching of the chondroitin backbone is not caused by enzymes, which are coded by genes from region 2 of the K4 capsular gene cluster.     

Structure of the fructose-containing K52 capsular polysaccharide of uropathogenic Escherichia coli O4:K52:H-

The chemical structure of the K52 antigenic capsular polysaccharide (K52 antigen) of Escherichia coli O4:K52:H- was elucidated by composition, nuclear magnetic resonance spectroscopy, methylation, periodate oxidation before and after graded acid hydrolysis and by oligosaccharide analysis. The polysaccharide consists of a backbone of alpha-galactose units interlinked between C1 and C3 by phosphodiester bridges. This poly(alpha-galactosyl-phosphate) is substituted at C2 of each galactose unit by beta-fructofuranose residues. About 80% of the galactose units are O-acetylated at C4 and about 10% of the fructose units are both O-acetylated and O-propionylated at C1. The K52 polysaccharide has an average molecular mass of 34 kDa, thus consisting of approximately 65 fructosyl-galactosyl-phosphate repeating units.     

KfoE encodes a fructosyltransferase involved in capsular polysaccharide biosynthesis in Escherichia coli K4

Escherichia coli K4 synthesizes a capsular polysaccharide (CPS) consisting of a fructose-branched chondroitin (GalNAc-GlcA(fructose)n), which is a biosynthetic precursor of chondroitin sulfate. Here, the role of kfoE in the modification of the chondroitin backbone was investigated using knock-out and recombinant complementation experiments. kfoE disruption and complementation had no significant effect on cell growth. CPS production was increased by 15 % in the knock-out strain, and decreased by 21 % in the knock-out strain complemented with recombinant kfoE. CPS extracted from the knock-out strain was chondroitin, whereas CPS extracted from the complemented strain was a fructose-branched chondroitin. The results demonstrated that the kfoE gene product altered the fructose group at the C3 position of the GlcA residue during production of K4CPS.     

Structure of the Escherichia coli 0104 polysaccharide and its identity with the capsular K9 polysaccharide

Spontaneous mutants of Rhizobium leguminosarum biovar viciae strain C1204b were selected for their ability to tolerate 0.2 M NaCl, a growth-inhibiting level of salt for the parental strain. Transposon-mediated salt-sensitive mutants of strain C1204b were screened for their inability to grow in 0.08 M NaCl. Quantitation of the free-amino acid pools in the mutants grown in NaCl revealed a dramatic increase in glutamine, serine, glutamate and proline, and to a lesser extent alanine and glycine in the salt-tolerant mutants in comparison with the parental strain exposed to NaCl; but only glutamate and proline increased in the salt-sensitive mutants under NaCl stress. Extracellular polysaccharide levels were quantitated for the salt-tolerant mutants and determined to be approximately two-fold higher than for the parental strain. Although the mutations that occurred in the NaCl-tolerant and NaCl-sensitive strains did not interfere with nodule formation, no nitrogenase activity could be observed in the NaCl tolerant mutants as evaluated by acetylene reduction.     

Primary structure of the Escherichia coli serotype K30 capsular polysaccharide.

Methylation, 1H nuclear magnetic resonance, and bacteriophage degradation results indicate that the Escherichia coli serotype K30 capsular polysaccharide consists of leads to 2)-alpha-D-Manp-(1 leads to 3)-beta-D-Galp-(1 leads to chains carrying beta-D-GlcUAp-(1 leads to 3)-alpha-D-Galp-(1 leads to branches at position 3 of the mannoses.     

The structure of the O-specific polysaccharide of Escherichia coli O117:K98:H4

The primary structure of the O-antigen of Escherichia coli O117 was shown by monosaccharide analysis, methylation analysis, and by 1D and 2D 1H and 13C NMR spectroscopy to be composed of linear pentasaccharide repeating units with the structure: -->3)-alpha-D-GalpNAc-(1-->4)-beta-D-GalpNAc-(1-->3)-alpha-L-Rhap- (1-->4)- alpha-D-Glcp-(1-->4)-beta-D-Galp-(1-->     

Application of high-performance capillary electrophoresis to analysis of carbohydrates, especially those in glycoproteins

Methods for the component monosaccharide analysis and oligosaccharide mapping for glycoprotein research, based on HPCE of reductively pyridylaminated (PA) derivatives, are described. the component monosaccharides released from glycoproteins by acid hydrolysis are converted to PA derivatives and analyzed by HPCE as borate complexes. They can be quantified in the picomole range (introduced amount) with high reproducibility. The oligosaccharides released by hydrazinolysis are similarly converted to PA derivatives. Two-dimensional mapping of the relative mobilities of these derivatives, obtained in an acidic phosphate buffer and an alkaline borate buffer, ensures reliable identification of the oligosaccharides.     

Preliminary characterisation of an Escherichia coli K5 lyase-deficient strain producing the K5 polysaccharide

Escherichia coli K5 polysaccharide has structural analogies with N-acetylheparosan, a non-sulphated precursor of heparin and, for this reason, can be considered an attractive precursor for the production of semi-synthesis heparin analogues. This polysaccharide has two components: a high molecular weight (HMW) one and a low molecular weight (LMW) one, whose ratio varies depending on the action of a lyase enzyme synthesized by the same K5 producer strain. The present paper reports the production of the K5 polysaccharide by a spontaneous E. coli mutant strain lacking the lyase activity. Similar K5 polysaccharide yields, 180 mg l(-1) after 16 h fermentation, were obtained by both the wild and mutant strains, though K5 lyase activity was only observed in the culture filtrates from the wild strain. The time course of the specific filtrate volume (1 m(-2)) and of the specific filtrate flux rate (1 m(-2) h(-1)) during ultrafiltration (UF) of culture filtrates where the lyase enzyme acted on the K5 chain, showed a decrease of UF performance, probably because of membrane fouling by the LMW K5 fraction. In particular, the specific filtrate volume and specific filtrate flux rate of wild strain samples reached respectively 13 l m(-2) and 4 l m(-2) h(-1), compared to 25 l m(-2) and 15 l m(-2) h(-1) obtained from the mutant strain samples. PCR molecular analysis of the DNA region encoding for the lyase enzyme showed that, in the mutant strain, molecular rearrangements occurred in both regulatory and structural regions.     

Detection of 8-hydroxydeoxyguanosine in K562 human hematopoietic cells by high-performance capillary electrophoresis

A method for the detection of 8-hydroxydeoxyguanosine by high-performance capillary electrophoresis (HPCE) was developed. Separations were performed in an uncoated silica capillary (44 cm × 75 μm I.D.) with a P/ACE system with diode-array detector. The separation of purine deoxynucleosides and 8-hydroxydeoxyguanosine was optimized with regard to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.5), 25°C, 25 kV, 20 s load and detection at 254 nm. This method allowed the detection of 8-hydroxydeoxyguanosine in the presence of a 10⁵-fold higher amount of deoxyguanosine. Isolated nuclei from K562 human hematopoietic cells were treated with 15 mM hydrogen peroxide for 2 h. The nuclei were extensively dialyzed and DNA was isolated, enzymatically hydrolyzed to the deoxynucleosides and analyzed by HPCE. DNA from hydrogen peroxide treated nuclei had a 4-fold higher content of 8-hydroxydeoxyguanosine than untreated controls. HPCE analysis of 8-hydroxydeoxyguanosine is fast and simple. Furthermore, it requires a very small sample volume, which makes it useful for biomedical and clinical applications.     

Influence of growth temperature on the development of Escherichia coli polysaccharide K antigens

The established seventy-one Escherichia coli polysaccharide capsular K antigenic test strains were examined for the development of the K antigen after growth at 37 degrees C and 18 degrees C. Twenty-eight K antigens were not detectable after growth of bacteria at 18 degrees C, while the remaining 43 antigens were developed at both temperatures. Most of the temperature-dependent antigens belonged to electrophoretically fast-moving K polysaccharides of rather low molecular weight, characteristically found among the common K antigens from extraintestinal disease isolates. Lipopolysaccharide O antigens were developed at both growth temperatures.     

Cross-sectional analysis of the polysaccharide composition in cellulosic fiber materials by enzymatic peeling/high-performance capillary zone electrophoresis

A combined enzymatic, chemical, and analytical approach was used to determine the cross-sectional carbohydrate composition in cellulosic fibers. The outer surface of cellulosic fibers was enzymatically removed layer-by-layer with precise quantitative control, and the monosaccharides in the peelings were subsequently analyzed by high-performance capillary electrophoresis (HPCE) after precolumn derivatization with a UV label. This method was applied to dissolving pulps and regenerated cellulose fibers, with special emphasis on the cross-sectional distribution of hemicelluloses. Commercially available enzyme solutions were used, resulting in a reproducible peeling. Significant differences were found in the hemicellulose distribution across the fiber of different dissolving pulps, dependent on both natural source (beech or spruce) and preparation process (acidic sulfite cook or prehydrolysis kraft cook). Among the dissolving pulps, beech prehydrolysis kraft pulp showed the highest enrichment of surface xylan. Similar, albeit smaller, differences were noticed between various regenerated fibers (viscose, viscose Modal, and Lyocell): a thin hemicellulose-rich outermost layer was found in all the regenerated fibers studied.     

Amplification and purification of exonuclease I from Escherichia coli K12

Employing the recombinant runaway replication plasmid pDPK13 [sbcB+], an exonuclease I-overproducing derivative of Escherichia coli K12 has been constructed. The strain SK4258 has exonuclease I activity 140-400-fold higher than wild type control levels. A new purification procedure has been developed such that the protein can be purified to near homogeneity and is free of endonuclease and RNase activities. The specific activity of the purified enzyme is 10-fold higher than reported previously (Ray, R.K., Reuben, R., Molineux, I., and Gefter, M. (1974) J. Biol. Chem. 249, 5379-5381). Native exonuclease I is a single polypeptide having Mr = 55,000 with a Stokes radius of 3.12 nm.     

苏云金芽胞杆菌Sigma K在大肠杆菌中的表达与纯化

【目的】在大肠杆菌中表达纯化苏云金芽胞杆菌HD73的转录调控因子Sigma K(σK)。【方法】PCR扩增出苏云金芽胞杆菌HD73中sig K基因的ORF(Open reading frame)装载到带有His标签的表达载体p ET21b上,转入到表达菌株BL21(DE3)中获得重组菌株BL21(p ETsig K),通过SDS-PAGE、镍柱亲和纯化、阴离子交换纯化和凝胶迁移实验(EMSA)等方法对Sigma K蛋白进行提取、纯化和生物活性分析。【结果】正确表达出大小约为27 k D的His-Sigma K蛋白,并获得了纯化的蛋白。EMSA结果表明纯化的His-Sigma K蛋白可以与受其控制的cry1Ac基因启动子结合。【结论】表达和纯化了His-Sigma K蛋白,His-Sigma K具有与受其控制的启动子结合的功能。     

Molecular cloning and expression of the genes encoding the Escherichia coli K4 capsular polysaccharide, a fructose-substituted chondroitin

The majority of capsular polysaccharides (K antigens) are linear molecules and their genes have a common functional organisation encoding common steps in capsule biogenesis. However, the K4 antigen is a substituted polymer composed of a chondroitin backbone with a fructose side chain. In order to determine whether K4 biosynthesis uses these common mechanisms the K4 antigen genes were cloned. DNA probes taken from the two conserved regions of the K1 genes were used to isolate one plasmid, pRD1, homologous to both probes. Immunological analysis was used to show that pRD1 directs the production of the substituted K4 antigen on the cell surface. Southern hybridisation was used to show that the cloned genes are organised in the same way as other K antigen gene clusters. We conclude that the branched K4 antigen is handled by the same post-polymerisation mechanisms as other linear K antigens.     

Application of high-performance tangential flow filtration (HPTFF) to the purification of a human pharmaceutical antibody fragment expressed in Escherichia coli

High-performance tangential flow filtration (HPTFF) is shown to successfully enable concentration, purification and formulation in a single unit operation. This is illustrated with feedstreams comprising recombinant proteins expressed in Escherichia coli (E. coli). Using positively charged cellulosic membranes of 100 kDa molecular weight cut-off and operating under a selected range of buffer pH and ionic strength at a filtrate flux of 100 L m(-2) h(-1), a 10-fold removal of E. coli host cell proteins (HCP) was obtained with an overall process yield of 98%. The HPTFF performance was shown to be robust and reproducible. In addition, the novel charged membrane was regenerated and re-used seven times without loss of selectivity or throughput. When compared with a conventional purification scheme, the proposed process results in the elimination of one chromatographic step, a 12% yield improvement and a significant reduction in purification cost of goods.     

Primary structure of the Escherichia coli serotype K42 capsular polysaccharide and its serological identity with the Klebsiella K63 polysaccharide.

The Escherichia coli K42 capsular polysaccharide consists of leads to 3)-alpha-D-Galp-(1 leads to 3)-alpha-D-GalUAp-(1 leads to 3)-alpha-L-Fucp-(1 leads to repeating units. The E. coli K42 and Klebsiella K63 antigens are serologically identical.