Overexpression of Serratia marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen

Lipase from Serratia marcescens ECU1010 was cloned and overexpressed in E. coli. After optimization, the maximum lipase activities reached 5000–6000 U/l and this recombinant lipase could enantioselectively hydrolyze (S)-ketoprofen esters into (S)-ketoprofen. Among six alkyl esters of racemic ketoprofen investigated, this lipase showed the best enantioselectivity for the kinetic resolution of ketoprofen ethyl ester, with an ee_p (enantiomeric excess of product) of 91.6% and E-value of 63 obtained at 48.2% conversion. Twelve nonionic surfactants were tested for enhancing the enantioselectivity of this lipase in the bioresolution of ketoprofen ethyl ester. A very high E-value of 1084 was achieved, with an optical purity of >99% ee_p and a yield of 42.6% in the presence of 3% Brij 92V. Further studies showed that the selectivity of the lipase was improved with the increase of Brij 92V concentration. The substrate (ketoprofen ethyl ester) does not inhibit the lipase activity, while the product (S)-ketoprofen inhibits the lipase activity to some extent. These results indicate that the S. marcescens lipase is very useful for biocatalytic production of chiral profens such as (S)-ketoprofen.     

Structure of the acidic microcapsular glycan from the reference strain (C.D.C. 6320-58) for Serratia marcescens serotype O14:K12

The acidic polysaccharide from Serratia marcescens serogroup O14:K12 was analyzed by means of chemical studies and NMR spectroscopy and its repeating unit structure found to be carbohydrate sequence [see text] O-Acetyl groups are proposed to be present in non-stoichiometric amounts on O-6 on one of the hexose residues in the main chain.     

Immobilization in polyelectrolyte complex capsules: Encapsulation of a gluconate-oxidizing Serratia marcescens strain

In previous papers, it was shown that eukaryotic microbial systems can be encapsulated in polyelectrolyte complexes (PEC) prepared from sodium cellulose sulfate and poly(dimethyldiallylammonium chloride) with maintainance of vitality. In the present study, prokaryotic cells were successfully encapsulated in these PEC. Serratia marcescens B345 (IMET 11312) was chosen as a model organism. This strain converts gluconic acid to 2-ketogluconic acid. Since the 2-ketogluconic acid produced has very strong complexing properties, the number of applicable immobilization methods is restricted. Due to the high stability of PEC towards complexing agents, these problems can be overcome by the described method.

As already described in previous papers, a preimmobilization of cells in a PEC coprecipitate prior to capsule formation proved to be advantageous also for encapsulation of bacilli. The mean productivity of the encapsulated S. marcescens cells was 1–4.4 g l⁻¹ h⁻¹ in comparison to 5 g l⁻¹ h⁻¹ for free cells. The productivity was highly dependent on the flow rate of the reactor. The encapsulated cells were used for 1,200 h in a continuous biotransformation process for the production of 2-ketogluconic acid.     

Biochemical properties and potential applications of an organic solvent-tolerant lipase isolated from Serratia marcescens ECU1010

An extracellular lipase was purified to homogeneity with a purification factor of 5.5-fold from a bacterial strain Serratia marcescens ECU1010. The purified lipase is a dimer with two homologous subunits, of which the molecular mass is 65 kDa, and the pI is 4.2. The pH and temperature optima were shown to be pH 8.0 and 45 °C, respectively. Among p-nitrophenyl esters of fatty acids with varied chain length, the lipase showed the maximum activity on p-nitrophenyl myristate (C₁₄). The lipase was activated by some surfactants such as Gum Arabic, polyvinyl alcohol (PVA) and Pg350me, but not by Ca²⁺. The enzyme displayed pretty high stability in many water miscible and immiscible solvents. This is a unique property of the enzyme which makes it extremely suitable for chemo-enzymatic applications in non-aqueous phase organic synthesis including enantiomeric resolution. Several typical chiral compounds were tested for kinetic resolution with this lipase, consequently giving excellent enantioselectivities (E = 83 >100) for glycidyl butyrate (GB), 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one acetate (HMPCA), naproxen methyl ester (NME) and trans-3-(4′-methoxyphenyl) glycidic acid methyl ester (MPGM).     

一株牙鲆源黏质沙雷氏菌YP1的分离鉴定及致病性分析

黏质沙雷氏菌(Serratia marcescens)是引起人类、动物及植物感染的重要条件致病菌,但其作为鱼类致病菌却鲜有报道。【目的】本研究以从患病牙鲆(Paralichthys olivaceus)病灶处分离的一株黏质沙雷氏菌YP1为研究对象,分析黏质沙雷氏菌对鱼类的致病性及对疾控的影响。【方法】利用形态学、分子生物学及生理生化实验综合鉴定菌株YP1;利用菌株YP1进行人工感染实验、组织病理实验及药敏试验,研究其感染症状、组织病理学、毒力和药物敏感性。【结果】分离自患病牙鲆体表溃疡病灶处的菌株YP1鉴定为黏质沙雷氏菌。感染实验结果显示,牙鲆和斑马鱼的半数致死量(LD₅₀)分别为3.44×10⁷CFU/g和6.28×10⁵CFU/g,除牙鲆外菌株YP1对其他鱼类也具有高致病性;菌株YP1主要导致牙鲆腹水,同时伴有呼吸急促、摄食减弱、脱肛、白便、鳃缺血及多脏器膨大出血等症状,并随着感染时间的延长对脏器损伤呈加重趋势。病理组织切片结果显示,菌株YP1对牙鲆鳃、肠、肝、脾、肾、心均造成损伤。药敏试验结果表明,YP1对左氧氟沙星、诺氟沙星等14种药物敏感;但对氨苄西林、头孢拉定等19种药物具有耐药性。【结论】本研究结果证实了黏质沙雷氏菌是能导致牙鲆腹水病的一种病原菌,同时对其他鱼类也具高致病性,为该菌感染鱼类导致疾病的检测、鉴别和防治提供科学依据。     

Structure of an acidic glycan present in the lipopolysaccharide extract from the reference strain for Serratia marcescens serogroup O18.

The lipopolysaccharide extract from the cell wall of the reference strain for Serratia marcescens serogroup O18 contained, in addition to a neutral glycan characterised previously, an acidic glycan. Acidity was contributed both by D-glucuronic acid and by 4-O-[(R)-1-carboxyethyl]-D-glucose (4-O-Lac-D-Glc). By using n.m.r. spectroscopy, methylation analysis, and chemical degradations, the repeating unit of the acidic glycan was identified as a branched hexasaccharide having the structure shown; an O-acetyl group also present was not located. The glycan is believed to define the O18 serogroup, but is probably not an integral component of the lipopolysaccharide. [formula: see text].     

Production of chitin from red crab shell waste by successive fermentation with Lactobacillus paracasei KCTC-3074 and Serratia marcescens FS-3

Successive two-step fermentation was carried out from red crab shell wastes for biological extraction of chitin in combination of the 1st step with a lactic acid bacterium Lactobacillus paracasei subsp. tolerans KCTC-3074 and the 2nd step with a protease producing bacterium Serratia marcescens FS-3, and vice versa. In the 1st step fermentation with KCTC-3074, the pH decreased rapidly from pH 6.90 to 3.31 and TTA increased rapidly to 10.99 for 5 days. At day 7 in the 2nd step fermentation with FS-3, pH further dropped to 2.82 and TTA also dropped to 1.71. In the 1st step fermentation using FS-3, the pH decreased slightly from pH 6.90 to 5.89, and TTA was low as indicated by 1.50 at 5 days. At day 7 in the 2nd step fermentation with KCTC-3074, the pH value was 3.62, and TTA increased to 8.95. The successive fermentation in the combination of FS-3 and KCTC-3074 gave the best result in co-removal of CaCO₃ and proteins from crab shells. In this combination, the rates of demineralization and deproteinization were 94.3% and 68.9%, respectively, at the end of fermentation. To date, this is the 1st report on successive fermentation for biological extraction of chitin from crustacean shells.     

Structural studies of an acidic galactoglucomannan from the O3 reference strain (C.D.C. 863-57) of Serratia marcescens

A partially acetylated acidic galactoglucomannan has been isolated from the lipopolysaccharide of the O3 reference strain (C.D.C. 863-57) of Serratia marcescens. By means of n.m.r. spectroscopy, methylation analysis, and degradative studies, the polymer was found to have the branched pentasaccharide repeating-unit shown. The position(s) of partial acetylation were not determined. Although the polymer is believed to confer O specificity on the parent organism, it is probably not an integral component of the lipopolysaccharide. (Formula: see text).     

Isolation and sequence analysis of a small cryptic plasmid pRK10 from a corrosion inhibitor degrading strain Serratia marcescens ACE2

The nucleotide sequence of a smallest cryptic plasmid pRK10 of Serratia marcescens ACE2 was determined. When compared to the all other plasmids reported so far from S. marcescens in sizes of over 70 kb, pRK10 is only 4241 bp long with 53% G + C content and has five coding sequences representing a coding percentage of 65.41. This small plasmid consists of one Tdh gene, four mobilization genes, mobCABD, and an origin of replication homologous to those of ColE1-type plasmids. Analysis of the five open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization containing sequences homologous to the bom region and mobCABD genes of ColE1 and Tdh from Acinetobacter baumannii str. AYE. Results also indicate that pRK10 does not encode any gene for antibiotic/heavy metal resistance. Copy number and incompatibility of the plasmid with plasmids of ColE1 origin of replication was determined and it is quite stable in its natural host as well as in Escherichia coli DH5α. This relatively small plasmid will be useful for construction of shuttle vectors to facilitate the genetic analysis.     

Structural studies of an acidic galactoglucomannan from the O15 reference strain (C.D.C. 4523-60) of Serratia marcescens

Both neutral and acidic polymers have been isolated from the lipopolysaccharide extract of the reference strain (C.D.C. 4523-60) for Serratia marcescens serogroup O15. By means of n.m.r. spectroscopy, methylation analysis, and studies of degradation products, the acidic polysaccharide was shown to have a branched pentasaccharide repeating-unit with the following structure. (Formula: see text)     

黏质沙雷氏菌AHPC29对松树萎蔫病的防治效果及其增菌条件

【目的】从松材线虫的媒介天牛蛹室及其气管中获得的黏质沙雷氏菌Serratia marcescens AHPC29对松材线虫具有致病能力,本研究旨在探究黏质沙雷氏菌AHPC29对松材线虫引起的松树萎蔫病的防治效果以及该菌株在实验室的增菌条件。【方法】通过对温室内人工感染松材线虫的松树灌溉菌剂,分析黏质沙雷氏菌AHPC29对松树萎蔫病的作用效果;通过单组分筛选和正交实验确定培养基组分和培养条件对其生长的影响,探究黏质沙雷氏菌AHPC29的最佳增菌条件。【结果】对于感染松材线虫的松树,灌溉黏质沙雷氏菌AHPC29菌液的处理组生长状态优于对照组,并且树内松材线虫含量显著降低;黏质沙雷氏菌AHPC29增菌的最佳培养基配比为0.1%乳糖、0.5%复合氨基酸、0.5% KNO₃、1.5% MgSO₄,其中影响最大的组分为氮源;培养时其菌液最佳接种量为7%,最佳装液量为40%,最佳转速为180 r·min^-1,最佳温度为30 ℃,最适培养时长为36 h。【结论】本研究获得了黏质沙雷氏菌AHPC29的最佳增菌条件,并证实该菌具有良好的松树萎蔫病防治应用潜力,为其作为生防菌的应用提供了理论基础。     

Structure of a neutral glycan isolated from the lipopolysaccharide of the reference strain for Serratia marcescens serogroup O22

Both a neutral and an acidic polymer have been isolated from a lipopolysaccharide extract of the reference strain for Serratia marcescens serogroup O22. The neutral polymer has a linear structure with the repeating unit shown. The same tetrasaccharide unit also forms the backbone of the branched neutral polymer isolated from the reference strain for serogroup O10, which cross-reacts strongly with O22. ----2)-alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----3)-alpha-L-+ ++Rhap-(1----3)-alpha- D-GlcpNAc-(1----     

Structure of the O-specific polysaccharide from Enterobacter cloacae strain N.C.T.C. 11579 (serogroup O10)

The O-specific polysaccharide from the reference strain (N.C.T.C. 11579) for Enterobacter cloacae serogroup O10 has been isolated and characterised. By means of n.m.r. spectroscopy and methylation analysis, and by studies of the products obtained by Smith degradation or by N-deacetylation-deamination, the repeating unit of the polysaccharide could be allocated the structure shown. The polysaccharides from two cross-reacting serogroups (O9 and O11) have the same monosaccharide composition. (Formula: see text)     

Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite

Serratia marcescens GEI strain was isolated from the gut of the workers of Chinese honey bee Apis cerana and evaluated in the laboratory for the control of Varroa destructor, a parasite of western honey bee A. mellifera. The supernatant and the collected proteins by ammonium sulfate from the bacterial cultures showed a strong miticidal effect on the female mites, with 100% mite mortality in 5 days. Heat (100 °C for 10 min) and proteinase K treatment of the collected proteins destroyed the miticidal activity. The improved miticial activity of this bacterial strain on chitin medium indicated the involvement of chitinases. The expressed chitinases ChiA, ChiB and ChiC1 from S. marcescens GEI by recombinant Escherichia coli showed pathogenicity against the mites in the laboratory. These chitinases were active in a broad pH range (5-9) and the optimum temperatures were between 60 and 75 °C. Synergistic effects of ChiA and ChiB on the miticidal activity against V. destructor were observed. The workers of both honey bee species were not sensitive to the spraying and feeding chitinases. These results provided alternative control strategies for Varroa mites, by formulating chitinase agents and by constructing transgenetic honey bees.     

Structural studies of a neutral polymeric fraction from the lipopolysaccharide of Serratia marcescens C.D.C. 1783-57 (O14:H9)

A "neutral" polymer of glucose, galactose, and 2-acetamido-2-deoxyglucose (molar ratios 1:1:2) has been isolated from the lipopolysaccharide of Serratia marcescens strain C.D.C. 1783-57 (O14:H9). Degradative and spectroscopic studies established that the polysaccharide has a branched tetrasaccharide repeating-unit of the structure shown. The polymer was absent from other strains of serogroup O14 studied, but a polymer differing only in the configuration of the glucose residue has previously been isolated from a strain of S. marcescens O8. The polymer from strain C.D.C. 1783-57 also shares structural features with the Escherichia coli O18 antigen, which is known to be serologically related to the S. marcescens O8 antigen. (Formula: see text).     

Structure of an acidic polysaccharide from the marine bacterium Pseudoalteromonas flavipulchra NCIMB 2033(T)

An acidic polysaccharide was isolated from Pseudoalteromonas flavipulchra type strain NCIMB 2033(T) and found to consist of 6-deoxy-L-talose (L-6dTal), D-galactose and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The identities of the monosaccharides were ascertained by sugar analysis and 1D 1H and 13C NMR spectroscopy in conjunction with 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments, which enabled determination of the following structure of the trisaccharide repeating unit of the polysaccharide:-->3)-alpha-L-6dTalp4Ac-(1-->3)-beta-D-Galp-(1-->7)-alpha-Kdop-(2-->.     

Neuraminidase gene from the early Asian strain of human influenza virus, A/RI/5-/57 (H2N2)

The complete structure of the neuraminidase gene from the A/RI/5-/57 strain of influenza virus has been determined. It is 1467 nucleotides long and codes for a protein of 469 amino acid residues. Comparison with the gene sequence for the N1 strains A/WSN/33 and A/PR/8/34, the N2 strain A/Udorn/72 and the protein sequence for the N2 strain A/Tokyo/3/67 shows the amino acid sequence changes that have occurred during antigenic shift (60%) and drift (7-9%).     

Purification and properties of lipase from a Bacillus strain for catalytic resolution of (R)-Naproxen

A Bacillus strain was screened for asymmetric resolution of (R)-Naproxen. The optical purity (ee (%)) of (R)-Naproxen was found to be 86.47% and conversion rate was 40–50% in bacterial cells PBS reaction system. The dissolved lipase was clarified from the Bacillus bacterial cells by centrifugation and loaded on a phenyl-Sepharose CL-4B column. After purification by a single hydrophobic chromatography, the activity of lipase was approximately 43 times higher than the crude one. The hydrolytic activity of lipase using Naproxen ethyl ester and p-nitrophenyl acetate (p-NPA) as substrate remained essentially constant during the purification procedure. A Bacillus strain with stereochemical selectivity was obtained.