Production and purification of L-phenylalanine oxidase from Morganella morganii.

An enzyme fraction, acting predominantly on L-phenylalanine has been purified and characterized from Morganella morganii. The total envelope was prepared by disrupting the cells with a French press followed by high speed centrifugation. After solubilization of the particulate fraction with 0.1% Triton X-100 and then centrifugation, the resulting supernatant was layered onto a DEAE-Cellulose column. Active fractions eluted were applied to a Phenyl-Sepharose CL-4B column as the final purification step. The activity of the purified enzyme to various L-amino acids in decreasing order was phenylalanine, methionine, leucine, tryptophan, and to a much lesser extent cysteine and tyrosine. At 4 degrees C in 20 mM phosphate buffer pH 7.5, the partially purified fractions collected from the DEAE-Cellulose column were stable for 120 h. On the other hand, the purified fractions obtained from the Phenyl Sepharose CL-4B column showed a drastic decrease in activity within only 24 h. Mg2+ (up to 40 mM), Mn2+ or Ca2+ (up to 10 mM) stimulated the oxidation of the purified enzyme but increases beyond such levels decreased the enzyme activity. Co2+ (0.05 mM), Cu2+ (0.5 mM) or Zn2+ (0.1 mM) decreased the enzyme activity 37, 33 and 20%, respectively.     

摩氏摩根菌ZJB-09203酯酶的分离纯化及性质

立体选择性水解2-羧乙基-3-氰基-5-甲基己酸乙酯(CNDE)是化学-酶法合成重大疾病治疗药物普瑞巴林的关键步骤。分离纯化了能够高效水解S-型CNDE的摩氏摩根菌ZJB-09203胞内酯水解酶,并进行酶学性质研究。采用硫酸铵分级沉淀、Phenyl Sepharose 6 FF疏水柱层析、DEAE Sephadex A-50阴离子交换和羟基磷灰石Bio-Scale CHT层析,纯化得到电泳纯的酯水解酶。SDS-PAGE和凝胶过滤HPLC分析确定该酶为单亚基蛋白,分子量为68 kDa。不同碳链长度p-硝基苯酚酯特异性酶解结果表明,该酶为酯酶,酶促合成普瑞巴林手性中间体(S)-2-羧乙基-3-氰基-5-甲基己酸的最适pH为9.0,最适温度为45℃,且在pH 7.0-9.0和40℃条件下具有良好的稳定性。Ca2+、Cu2+、Mn2+对酶活有一定的促进作用,Co2+、Fe3+、Ni2+及EDTA对酶活有较强的抑制作用。此外,考察了该酯酶水解p-硝基苯酚酯的动力学参数,及CNDE浓度对转化率的影响。首次报道了能够立体选择性水解CNDE的酯酶,相关酶学性质研究将为该酶催化合成普瑞巴林手性中间体的工业化应用提供重要依据。     

Recognition of Morganella subspecies, with proposal of Morganella morganii subsp. morganii subsp. nov. and Morganella morganii subsp. sibonii subsp. nov.

The genus name Morganella was established within the family Enterobacteriaceae in 1978. Morganella morganii is the only species described thus far within this genus, and the name M. morganii has been accepted by usage in the scientific community for strains previously known as Proteus morganii. M. morganii isolates differ in their abilities to ferment trehalose and exhibit variable lysine and ornithine decarboxylase patterns, emphasizing the phenotypic heterogeneity within this species. Previous genetic studies failed to reveal separate entities within the genus Morganella. We observed some trehalose-fermenting strains with different lysine and ornithine decarboxylase patterns. Two strains were lysine and ornithine positive, 3 were lysine positive and ornithine negative, and 29 were lysine negative and ornithine positive. These strains and 25 non-trehalose-fermenting strains with different lysine and ornithine decarboxylase patterns were investigated. DNA-DNA hybridization studies and phenotypic characterizations revealed that M. morganii can be separated into three DNA relatedness groups and seven biogroups. Strains from DNA relatedness group 1 were trehalose negative, and strains from DNA relatedness groups 2 and 3 were trehalose positive. One biogroup from DNA relatedness group 2 was phenotypically indistinguishable from DNA relatedness group 3. On the basis of these studies, we propose that M. morganii be subdivided into M. morganii subsp. morganii (type strain ATCC 25830) containing biogroups A, B, C, and D (DNA relatedness group 1) and M. morganii subsp. sibonii (type strain 8103-85; = ATCC 49948) containing biogroups E, F, and G (DNA relatedness groups 2 and 3).     

Antimicrobial sensitive of Morganella morganii

The aim of this study was the evaluation of the antimicrobial sensitive of Morganella morganii rods isolated from clinical samples. This study included 50 of M. morganii strains isolated in the Clinical Microbiology Department of dr. A. Jurasz University Hospital in 2008-2009. All of strains were sensitive to carbapenems (imipenem, meropenem, ertapenem, doripenem) and piperacillin/tazobactam and most of them to beta-lactam antibiotics, aminoglycosides and fluorochinolons. Resistance to tetracyclines demonstrated 38,0% strains and to doxycycline - 40,0%. One out of 6 strains isolated from urine samples were sensitive to nitrofurantoin. Extended Spectrum Beta-Lactamases were produced by 5 (10,0%) strains.     

Properties of bacteriocins of Morganella morganii

Some properties of Morganella morganii bacteriocins were determined. For this purpose two strains (115 and 137) which after mitomycin C induction produced bacteriocins in high titer were chosen. The influence of several physical and chemical factors such as: heating and storage at various temperatures, a freezing and thawing, an influence of buffered fluid of different pH, and digestion by trypsin, papain and lysozyme were investigated. Range of bacteriocin activity against various microorganisms and the ability to diffuse in agar were also determined. It was found on the basis of the results obtained that two bacteriocins showed different features. Bacteriocin "115" was thermostable, sensitive to proteolytic enzymes, able to diffuse in 1.5% agar. Bacteriocin designated "137" was thermolabile, intensive to proteolytic digestion, and incapable to diffuse in 1.5% agar. Activity of both bacteriocins was reduced after freezing and thawing. They were both insensitive to lysozyme digestion. Storage at room temperature reduced their activity faster than storage at the temperature of refrigerator. Their activity was completely stopped at pH 3.03, and significantly at pH 5.08 while environment of pH ranged from 7.08 to 11.0 did not influence their activity. Both bacteriocins showed narrow range of activity limited to the growth inhibition of sensitive strains belonging to Morganella morganii genus.     

A bifunctional urease enhances survival of pathogenic Yersinia enterocolitica and Morganella morganii at low pH.

To infect a susceptible host, the gastrointestinal pathogen Yersinia enterocolitica must survive passage through the acid environment of the stomach. In this study, we showed that Y. enterocolitica serotype O8 survives buffered acidic conditions as low as pH 1.5 for long periods of time provided urea is available. Acid tolerance required an unusual cytoplasmically located urease that was activated 780-fold by low-pH conditions. Acid tolerance of Helicobacter species has also been attributed to urease activity, but in that case urease was not specifically activated by low-pH conditions. A ure mutant strain of Y. enterocolitica was constructed which was hypersensitive to acidic conditions when urea was available and, unlike the parental strain, was unable to grow when urea was the sole nitrogen source. Examination of other urease-producing gram-negative bacteria indicated that Morganella morganii survives in acidic conditions but Escherichia coli 1021, Klebsiella pneumoniae, Proteus mirabilis, Providencia stuartii, and Pseudomonas aeruginosa do not. Consistent with these results, biochemical evidence demonstrated that Y. enterocolitica and M. morganii ureases were activated in vitro by low pH with an unusually low activity optimum of pH 5.5. In whole cells activation occurred as medium values decreased below pH 3.0 for Y. enterocolitica and pH 5.5 for M. morganii, suggesting that in vivo activation occurs as a result of cytoplasmic acidification. DNA sequence analysis of portions of the M. morganii ure locus showed that the predicted primary structure of the enzyme structural subunits is most similar to those of Y. enterocolitica urease. One region of similarity between these two ureases located near the active site is distinct from most other ureases but is present in the urease of Lactobacillus fermentum. This region of similarity may be responsible for the unique properties of the Y. enterocolitica and M. morganii ureases since the L. fermentum urease also has been shown to have a low pH optimum for activity.     

New serotypes of Morganella morganii.

On the basis of 8 new O and 11 new H antigens determined in 22 strains, the Morganella morganii antigenic schema was supplemented with 8 serogroups (O35-O42) and 13 serotypes. Four strains belonged to O groups described earlier and 2 strains contained new O antigens in combination with known O antigens. Known H antigens were present in one strain as a single factor and in one strain as combination of two factors. New H antigens were demonstrated in 5 serotypes in combination with known H antigens. Six out of the 22 isolates were classified into O group 35. Two isolates contained different B-type surface antigens; these factors were not related to Escherichia coli B antigens and, unlike the latter, their living suspension gave a higher titre agglutination in OK serum as compared to the boild culture.     

New metalloendopeptidase of Morganella morganii ZM

Proteolytic activity, which is inhibited in the presence of o-phenanthroline, was found in M. morganii ZM. Unlike grimelysin, intracellular proteases of M. morganii ZM actively cleave skeletal muscle actin. Several proteolytic proteins of M. morganii ZM were identified in cells by zymography with gelatin. Individual metalloproteinase (35 kDa) was isolated from the M. morganii ZM cell lysates by ammonium sulphate fractionation and purified by hydrophobic chromatography.     

Haemolytic activity of Morganella morganii strains

Haemolytic activity on solid and liquid media of 103 Morganella morganii strains isolated from clinical sources was investigated. The ability to produce haemolysin was found in 42.7% of strains. All strains capable to produce haemolysin on blood agar media also revealed haemolytic activity in some liquid media. Haemolysins were found in the supernatants and filtrates of the cultures in peptone water but not in Brain Heart Infusion and Trypticase Soy Broth. The maximal titer of haemolysin was observed in the logarithmic phase of growth. Heating and incubation with trypsin led to complete loss of haemolytic activity.     

摩根摩根菌临床分布及耐药性分析

摘要：目的 分析摩根摩根菌的临床分布特点及耐药现状,为抗感染治疗提供参考依据。方法 对医院2013年7月—2015年6月临床分离的80株摩根摩根菌的来源与耐药性进行回顾性分析,并对痰液标本与其他类型标本分离到的摩根摩根菌对抗菌药物的耐药情况进行统计学比较。结果 多种临床标本均可分离到摩根摩根菌,以痰液标本居多,占58.75%;科室分布以ICU为主,占41.25%,其次为急诊内科,占31.25%。20种抗菌药物中,敏感性较高的药物为丁胺卡那霉素（98.75%）、头孢吡肟（91.25%）、氨曲南（87.50%）、亚胺培南（86.25%）;痰液标本与其他标本分离到的摩根摩根菌对头孢哌酮/舒巴坦、头孢他啶、头孢曲松、头孢吡肟、厄他培南、亚胺培南、庆大霉素、环丙沙星、复方新诺明9种抗菌药物有显著性差异（P<0.05）。其他标本分离到的摩根摩根菌对此9种药物的敏感性要显著高于痰液标本分离到的摩根摩根菌。结论 对摩根摩根菌引起的感染应合理使用抗菌药物早期治疗,避免耐药菌株的产生;不同部位的摩根摩根菌感染应选用不同种抗菌药物,以提高治疗效果。     

Structural characterization and MHCII-dependent immunological properties of the zwitterionic O-chain antigen of Morganella morganii

Morganella morganii is a commensal Gram-negative bacterium that has long been known to produce an antigen bearing phosphocholine groups. We determined the structure of this O-chain antigen and found that its repeating unit also contains a free amino group and a second phosphate: This alternating charge character places the M. morganii O-chain polysaccharide into a small family of zwitterionic polysaccharides (ZPSs) known to induce T-cell-dependent immune responses via presentation by class II major histocompatibility complex (MHCII) molecules. In vitro binding assays demonstrate that this O-chain interacts with MHCII in a manner that competes with binding of the prototypical ZPS antigen PSA from Bacteroides fragilis, despite its lack of a helical structure. Cellular studies also showed that the M. morganii polysaccharide induces activation of CD4(+) T-cells. Antibody binding experiments using acid hydrolyzed fragments representing the monomer and higher oligomers of the repeating unit showed that the phosphocholine group was the dominant element of the epitope with an overall affinity (K(D)) of about 5 × 10(-5) M, a typical value for an IgM anti-carbohydrate antibody but much lower than the affinity for phosphocholine itself. These data show that the structure of the M. morganii polysaccharide contains a unique zwitterionic repeating unit which allows for immune recognition by T-cells, making it the first identified T-cell-dependent O-chain antigen.     

摩根摩根菌临床分布及耐药性分析

目的分析摩根摩根菌的临床分布特点及耐药现状,为抗感染治疗提供参考依据。方法对医院2013年7月-2015年6月临床分离的80株摩根摩根菌的来源与耐药性进行回顾性分析,并对痰液标本与其他类型标本分离到的摩根摩根菌对抗菌药物的耐药情况进行统计学比较。结果多种临床标本均可分离到摩根摩根菌,以痰液标本居多,占58.75%;科室分布以ICU为主,占41.25%,其次为急诊内科,占31.25%。20种抗菌药物中,敏感性较高的药物为丁胺卡那霉素(98.75%)、头孢吡肟(91.25%)、氨曲南(87.50%)、亚胺培南(86.25%);痰液标本与其他标本分离到的摩根摩根菌对头孢哌酮/舒巴坦、头孢他啶、头孢曲松、头孢吡肟、厄他培南、亚胺培南、庆大霉素、环丙沙星、复方新诺明等9种抗菌药物有显著性差异(P0.05)。其他标本分离到的摩根摩根菌对此9种药物的敏感性要显著高于痰液标本分离到的摩根摩根菌。结论对摩根摩根菌引起的感染应合理使用抗菌药物早期治疗,避免耐药菌株的产生;不同部位的摩根摩根菌感染应选用不同种抗菌药物,以提高治疗效果。     

摩根摩根菌β-内酰胺酶检测及耐药性分析

目的了解摩根摩根菌临床分离株产超广谱β-内酰胺酶（ESBLs）、头孢菌素酶（AmpC）、金属酶（MBLs）、碳青霉烯酶（KPC）情况,并分析其对17种常见抗菌药物的耐药性。方法 ESBLs和AmpC及MBLs采用三维试验检测,碳青霉烯酶采用改良Hodge试验进行检测,并以K-B法测定17种常见抗菌药物的耐药性。结果 102株摩根摩根菌单产ESBLs 15株,检出率为14.71%;单产AmpC 8株,检出率为7.84%;单产金属β-内酰胺酶（MBLs）3株,检出率为2.94%;所有菌株中未检出碳青霉烯酶（KPC）;同产ESBLs及AmpC 6株,检出率为5.88%;未发现其他双产酶菌株。摩根摩根菌非产酶分离株对17种抗生素的耐药率均低于50.0%;摩根摩根菌产酶分离株对亚胺培南、美罗培南培南的耐药率低于15.0%,与非产酶菌株相比,差异无统计学意义（P〉0.05）;对其余抗生素的耐药率均明显高于非产酶菌株（P〈0.05）。同产ESBLs＋AmpC与耐亚胺培南摩根摩根菌分离株呈多重耐药。结论我院摩根摩根菌分离株产生多种β-内酰胺酶,且对常用抗生素耐药性比较严重,建议临床医师合理使用抗生素,以免耐药菌株的产生。     

Antigenic relationship between Morganella morganii and Yersinia enterocolitica.

Two new O antigens have been described for the Morganella morganii antigenic schema. O antigens of the strains representing the new serotypes (O43 :H2 and O44 : H19, 37) are related to Yersinia enterocolitica O9 and O17, respectively.     

Coconut α-d-galactosidase isoenzymes: isolation,purification and characterization

α-d-Galactosidases (α-d-galactoside galactohydrolase, EC 3.2.1.22) from normal coconut endosperm were isolated and partially purified by a combination of ammonium sulfate fractionation, SP-Sephadex C50–120 ion-exchange chromatography and Sephadex G-200 and G-100 gel filtration. Two molecular forms of the enzyme, designated as A and B, were eluted after SP-Sephadex C50–120 ion-exchange chromatography. α-d-Galactosidase A, which is the major isoenzyme, was partially purified 43-fold on Sephadex G-200 and has a MW of about 23 000 whereas α-d-galactosidase B was partially purified 23-fold on Sephadex G-100 and has a similar MW of about 26 600. Both isoenzymes exhibited optimum activity at pH 7.5. The apparent K_m and V_max of α-d-galactosidase A were obtained at 3.46 × 10^?4M and 1.38 × 10^?3 M p-nitrophenyl α-<d-galactoside, respectively. A distinct substrate inhibition was noted. The enzyme was inhibited strongly by d-galactose and to a lesser extent by myo-inositol, d-glucose-6-phosphate, l-arabinose, melibiose and iodoacetic acid. Similarly, makapuno α-d-galactosidase was localized in the 40–70 % (NH₄)₂SO₄ cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its K_m was obtained at 6.75 × 10^?4 M p-nitrophenyl α-d-galactoside while the V_max was noted at 5.28 × 10^?3 M p-nitrophenyl α-d-galactoside. Based on the above kinetic data, the possible cause(s) of the deficiency of α-d-galactosidase activity in makapuno is discussed.     

Drosophila topoisomerase I: isolation, purification and characterization.

We have purified and characterized topoisomerase I from Drosophila melanogaster. The molecular weight of the enzyme is 135,000; 100,000, 90,000, and 65,000 molecular weight products result from degradation of the enzyme. The enzyme relaxes both positive and negative supercoiled DNA. Mg++ is not absolutely required, but stimulates the enzymatic activity considerably.