Culture and bioconversion use of plasmid-harboring strain of immobilized E. coli

Summary Growing Escherichia coli BZ18/pTG 201 cells were immobilized in Kappa-carrageenan gel beads. The bacterial growth after immobilization was studied by cellular counting and by morphological observations with electron microscopy. Kinetic studies of the Catechol 2–3 dioxygenase carried by the plasmid pTG 201 were performed with a packed-bed reactor to show the potential of such a system. High cell densities 1.7×10¹¹ cells/ml) were observed in the cavities of the gel. Due to the difference between the cell density in suspension (8x10⁸ cells/ml) and that within the gel cavities, a reduction of the reactor size and investment cost for processes can be predicted.     

An engineered E.coli strain for the production of glycoglycerolipids

The glycolipid synthase MG517 from Mycoplasma genitalium catalyzes the glucosyl transfer from UDPGlc to diacylglycerol producing glycoglycerolipids (GGL) (Andrés et al., 2011). The enzyme was functional in E. coli accumulating GGL in the plasma membrane. A metabolic engineering strategy for GGL production was evaluated using this microorganism. To increase the levels of GGL precursors, UDPGlc and diacylglycerol, GalU and PlsC enzymes involved in their biosynthesis were overexpressed. Seven engineered strains were obtained containing different combinations of the mg517 with galU and plsC genes. Diacylglycerol synthesis showed to be limiting and the strain overexpressing MG517 and PlsC achieved the highest GGL yield. The new lipids were mono, di- and triglucosyldiacylglycerol with different acyl combinations in each compound. It indicates that the successive glucosyl transferase activities of MG517 have different acyl chain specificity for the acceptor substrate. GGL represented up to 6mg per g of dry weight.     

Effects of cultural conditions on protease production by Aeromonas hydrophila.

Production of extracellular proteolytic activity by Aeromonas hydrophila was influenced by temperature, pH, and aeration. Conditions which produced maximal growth also resulted in maximal protease production. Enzyme production appeared to be modulated by an inducer catabolite repression system whereby NH4+ and glucose repressed enzyme production and complex nitrogen and nonglucose, carbon energy sources promoted it. Under nutritional stress, protease production was high, despite poor growth.     

Expression in E. coli of the gene encoding phenylalanine ammonia-lyase from Rhodosporidium toruloides

Summary The active sites of the enzyme phenylalanine ammonia-lyase (Pal) from Rhodosporidium toruloides contains a dehydroalanine residue that is believed to be essential for catalytic activity. Furthermore, the dehydroalanine is believed to be added post-translationally as part of a prosthetic group covalently attached to the enzyme. Perhaps for this reason no attempts to produce Pal in foreign host cells have been reported. We have inserted the entire uninterupted pal gene from R. toruloides into the Escherichia coli expression vector pKK 223-3. E. coli cells containing this vector synthesize a protein of the expected size, and extracts prepared from these cells contain a Pal-like activity. The potential implications of this finding are discussed.Offprint requests to: H. Ørum     

Effects of cultural conditions on protease production by Aeromonas hydrophila

Production of extracellular proteolytic activity by Aeromonas hydrophila was influenced by temperature, pH, and aeration. Conditions which produced maximal growth also resulted in maximal protease production. Enzyme production appeared to be modulated by an inducer catabolite repression system whereby NH4+ and glucose repressed enzyme production and complex nitrogen and nonglucose, carbon energy sources promoted it. Under nutritional stress, protease production was high, despite poor growth.     

Shikimic acid production by a modified strain of E. coli (W3110.shik1) under phosphate-limited and carbon-limited conditions

Shikimic acid is one of several industrially interesting chiral starting materials formed in the aromatic amino acid pathway of plants and microorganisms. In this study, the physiology of a shikimic acid producing strain of Escherichia coli (derived from W3110) deleted in aroL (shikimic acid kinase II gene), was compared to that of a corresponding control strain (W3110) under carbon- and phosphate-limited conditions. For the shikimic acid producing strain (referred to as W3110.shik1), phosphate limitation resulted in a higher yield of shikimic acid (0.059 +/- 0.012 vs. 0.024 +/- 0.005 c-mol/c-mol) and a lower yield of by-products from the shikimate pathway, when compared to carbon-limited condition. The yield of the by-product 3-dehydroshikimic acid (DHS) decreased from 0.076 +/- 0.028 to 0.022 +/- 0.001 c-mol/c-mol. Several other by-products were only detected under carbon-limited conditions. The latter group included 3-dehydroquinic acid (0.021 +/- 0.021 c-mol/c-mol), quinic acid (0.012 +/- 0.005 c-mol/c-mol), and gallic acid (0.002 +/- 0.001 c-mol/c-mol). For both strains, more acetate was produced under phosphate than the carbon-limited case. Considerable cell lysis was found for both strains but was higher for W3110.shik1, and increased for both strains under phosphate limitation. The advantages of the latter condition in terms of an increased shikimic acid yield was thus counteracted by an increased cell lysis, which may make downstream processing more difficult.     

Purification of alpha-hemolysin from an overproducing E. coli strain

Summary The genetic determinant of the -hemolysin encoded by plasmid pHly152 has been cloned in both orientations in plasmid pBR322 giving rise to plasmids pSU157 and pSU158. E. coli strains carrying either of these recombinant Hly plasmids produced about 20 times more hemolysin activity than the parental plasmid pHly152, when grown in minimal medium supplemented with hemoglobin. Thus high hemolytic activity is not lethal to the cells, contrary to previous assumptions.-hemolysin was purified from culture supernatants of strain SU100 (pSU157) by ammonium sulfate precipitation and gel filtration in Sephacryl S-200 in the presence of 6 M urea. When purified -hemolysin preparations were subjected to electrophoretic analysis in denaturing conditions, a single 107 kdal polypeptide was observed. This probably corresponds to the -hemolysin protein, since an isogenic E. coli strain carrying plasmid pSU161, an Hly^- mutant derivative of pSU157, did not synthesize the 107 kdal polypeptide.     

絮凝剂产生菌的筛选、培养及产物性质研究

从活性污泥中筛选出一株高效产絮凝剂菌株,经鉴定为嗜温鞘氨醇杆(Sphingobacterium thalpophilum).采用单因素实验,结果显示该菌产絮凝荆的最适碳源为可溶性淀粉,最适氮源为硫酸铵,最适初始pH值7.0,最适培养温度为30℃.性质研究分析显示,絮凝剂纯品的热稳定性差,其在pH值2.0～8.0的范围内可以保持较高的絮凝活性.     

Cellobiohydrolase from Clostridium thermocellum, synthesized by a recombinant E. coli strain]

Clostridium thermocellum cellobiohydrolase was isolated in preparative amounts from the recombinant strain of E. coli K12 C600 carrying plasmid pCU 304 with a C. thermocellum chromosomal DNA insertion. The isolation procedure included chromatography on Ultrogel AcA 44, ion-exchange chromatography on DEAE-Sepharose CL-6B, rechromatography on Ultrogel and FPLC on Mono Q resulting in a 17.6% yield and 1530-fold purification. According to data from sodium dodecylsulfate polyacrylamide gel electrophoresis performed under nondenaturing conditions and analytical gel isoelectrofocusing, the enzyme preparation contains only one active protein band with Mr 56.2 +/- 1.0 kDa and pI 4.15. The enzyme does not reduce the viscosity of the CM-cellulose solution but forms reducing sugars from this soluble substrate. Cellobiose (93-97%) is the major component produced by the enzyme from crystalline and amorphous cellulose (specific activity 2.3 x 10(-3) and 2.8 x 10(-2) U/mg, respectively). The activity optimum of the enzyme is at pH 5.6, 60 degrees C. The half-inactivation time at 60 degrees C and 65 degrees C is 450 and 15.5 min, respectively. The action pattern of the enzyme on the low molecular fluorogenic cellooligosaccharides suggests that the enzyme pertains to typical cellobiohydrolases.     

Secretion of peptides from E. coli: a production system for the pharmaceutical industry

Peptides have been used as pharmaceuticals ever since 1922, when a diabetic patient at Toronto General Hospital was injected with an extract of canine pancreas. The area of peptide drug therapy is in a state of change. It is expanding from its ‘traditional’ niche of treating hormonal disturbances into the areas of cancer therapy or the treatment of physiological disorders. At the same time, new production processes are providing greater amounts of peptide so that a larger proportion of patients in the traditional areas receive the proper treatment. This review briefly describes the kinds of peptide drugs now or soon to be available and shows one way in which biotechnology is contributing to the development of peptide drug therapy.     

产腈水解酶基因工程菌的产酶诱导条件及培养基优化

本文通过对产酶诱导条件及发酵培养基进行优化,成功提高了产腈水解酶基因工程菌E. coli BL21(DE3)-pETNYNit的产酶水平。研究结果显示,最佳发酵培养基为：葡萄糖0.2%、甘油0.7%(v/v)、蛋白胨1.2%、酵母膏0.8%、NaCl 0.3%、(NH4)2SO40.3%、NH4Cl 0.13%、Na2 HPO4·12H2 O 1.04%、KH2 PO40.39%、MgSO4·7H2 O 0.03%,pH 7.2。最佳产酶诱导条件为：发酵4 h时加入0.5 mmol/L IPTG,然后在28℃、240 r/min下诱导腈水解酶基因表达14 h~16 h。采用优化方案,重组菌产酶水平可提升至0.9~1×105 U,与野生菌株的产酶水平相比,提高幅度超过50%。同时重组菌培养仅需24 h,培养周期缩短超过50 h。     

High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production

A bioengineered heparin, as a replacement for animal-derived heparin, is under development that relies on the fermentative production of heparosan by Escherichia coli K5 and its subsequent chemoenzymatic modification using biosynthetic enzymes. A critical enzyme in this pathway is the mammalian 6-O-sulfotransferase (6-OST-1) which specifically sulfonates the glucosamine residue in a heparin precursor. This mammalian enzyme, previously cloned and expressed in E. coli, is required in kilogram amounts if an industrial process for bioengineered heparin is to be established. In this study, high cell density cultivation techniques were exploited to obtain recombinant 6-OST-1. Physiological studies were performed in shake flasks to establish optimized growth and production conditions. Induction strategies were tested in fed-batch experiments to improve yield and productivity. High cell density cultivation in 7-l culture, together with a coupled inducer strategy using isopropyl β-d-1-thiogalactopyranoside and galactose, afforded 482 mg?l^?1 of enzyme with a biomass yield of 16.2 mg?g_cdw ^?1 and a productivity of 10.5 mg?l^?1?h^?1.     

Protease from a strain of bacteria E. coli A2, specifically cleaving actin

A novel bacterial protease specifically hydrolyzing actin with the formation of a stable fragment with Mr of 36 kDa was obtained. This protease was shown to be synthesized at the stationary phase of bacterial culture growth. The actin hydrolysis by bacterial protease was inhibited by o-phenanthroline, EDTA and p-chloromercuribenzoate but not by N-ethyl-maleimide, phenylmethylsulfonylfluoride, Leu-peptin, pepstatin and other serine proteinase inhibitors. The protease was stable within the pH range of 4.5-8.5 and had an activity optimum at pH 7.0-8.0. The protease activity was maintained for 40 min at 45 degrees C and for 30 min at 50 degrees C; at 65 degrees C the enzyme was fully inactivated by 5 min heating. The protease preparations causing quantitative conversion of actin into a 36 kDa fragment did not hydrolyze casein, albumin, ovalbumin, lysozyme, DNAase I, RNAase, myosin, alpha-actinin, tropomyosin and troponin. It was assumed that the protease under consideration is a neutral metalloprotease specifically hydrolyzing actin.