Unscrambling the Effect of C-Terminal Tail Deletion on the Stability of a Cold-Adapted,Organic Solvent Stable Lipase from Staphylococcus epidermidis AT2

Terminal moieties of most proteins are long known to be disordered and flexible. To unravel the functional role of these regions on the structural stability and biochemical properties of AT2 lipase, four C-terminal end residues, (Ile–Thr–Arg–Lys) which formed a flexible, short tail-like random-coil segment were targeted for mutation. Swapping of the tail-like region had resulted in an improved crystallizability and anti-aggregation property along with a slight shift of the thermostability profile. The lipolytic activity of mutant (M386) retained by 43 % compared to its wild-type with 18 % of the remaining activity at 45 °C. In silico analysis conducted at 25 and 45 °C was found to be in accordance to the experimental findings in which the RMSD values of M386 were more stable throughout the total trajectory in comparison to its wild-type. Terminal moieties were also observed to exhibit large movement and flexibility as denoted by high RMSF values at both dynamics. Variation in organic solvent stability property was detected in M386 where the lipolytic activity was stimulated in the presence of 25 % (v/v) of DMSO, isopropanol, and diethyl ether. This may be worth due to changes in the surface charge residues at the mutation point which probably involve in protein–solvent interaction.     

A Newly Isolated Organic Solvent Tolerant Staphylococcus saprophyticus M36 Produced Organic Solvent-Stable Lipase

Thirty-eight high lipase activity strains were isolated from soil, seawater, and Brassica napus. Among them, a novel organic solvent tolerant bacterium (strain M36) was isolated from the seawater in Jiangsu, China. Isolate M36 was able to grow at high concentration of benzene or toluene up to 40% (vol/vol), and later identified as Staphylococcus saprophyticus by biochemical test and 16s ribosomal DNA sequence. No work on Staphylococcus producing lipase with organic solvent tolerance has been reported so far. The lipase of strain M36 whose activity in liquid medium was 42 U mL⁻¹ at 24-h incubation time was stable in the presence of 25% (vol/vol) p-xylene, benzene, toluene, and hexane.     

An Organic Solvent-Tolerant Alkaline Lipase from Cold-Adapted Pseudomonas mandelii: Cloning,Expression, and Characterization

Various yeast strains were screened for production of 3-hydroxybutyric acid (3-HBA) from 1,3-butanediol (1,3-BD) by a resting cell system. Many yeasts were found to oxidize 1,3-BD to 3-HBA. Among them, Hansenula anomala IFO 0195 produced (S)-(+)-3-HBA of the highest optical purity. Reaction temperature and addition of glucose were significantly effective on the optical .purity and production of the acid. When resting cells of this strain were incubated at 27°C in an optimal reaction mixture containing 60.0 mg/ml 1,3-BD, 2.0% CaC0₃, and 1.0% glucose, 26.7 mg/ml of 3-HBA were produced with 88% enantiomer excess for 2 days. Dominant accumulation of (S)-(+)-3-HBA might be due to enantioselective degradation of (R)-(-)-3-HBA, though both (S)-(+)- and (R)-(-)-1,3-BD are oxidized by the strain.     

非水相脂肪酶催化有机硅醇的酯化反应

非水相酶催化是酶工程研究热点之一。本文介绍了来自Ｃ．ｃｙｌｉｎｄｒａｃｅａ的脂肪酶催化有机硅醇和脂肪酸的酯化反应。该酶可催化有机硅醇与脂肪酸的酯化反应,并对不同链长的脂肪酸底物、有机溶剂极性及水含量等进行了初步研究。     

Chemical Inactivation of Lipase in Organic Solvent: A Lipase from Pseudomonas aeruginosa TE3285 is More Like a Typical Serine Enzyme in an Organic Solvent than in Aqueous Media

A microbial lipase from Pseudomonas aeruginosa TE3285 was treated in anhydrous diisopropyl ether with three kinds of serine-reactive reagents, ethyl p-nitrophenyl methylphosphonate (ENMP), diisopropyl fluorophosphate (DFP), and phenylmethylsulfonyl fluoride (PMSF) to lose its catalytic activity for both transesterification in an organic solvent and ester hydrolysis in aqueous system. In contrast with the facile inactivation in an organic solvent, no or very slow inactivation was observed in an aqueous solution. The lipase was shown to behave more like a typical serine enzyme in an organic solvent than in aqueous solution with regard to the chemical inactivation by serine-reactive reagents. The unique behavior of the lipase in an organic solvent may be associated with inferfacial activation of the lipase, which is one of the most distinct characteristics of the lipase family, and the activiation of lipase could be induced by a hydrophobic interaction with an organic solvent.     

耐受有机溶剂洋葱伯克霍尔德菌ZYB002全细胞脂肪酶酶学性质

一株对多种有机溶剂具有良好耐受能力的产脂肪酶菌株ZYB002经分子鉴定为洋葱伯克霍尔德菌。其产生的细胞结合脂肪酶最适温度为65°C,最适pH为8.0,在低于70°C和pH3-8.5的范围内,全细胞脂肪酶保持稳定。Ca2+、K+、Na+和NO3-等离子对脂肪酶活性有激活作用,而Zn2+有抑制效应。全细胞脂肪酶对正丁醇有较强的耐受能力,但曲拉通X-100对脂肪酶活性有强烈的抑制效应。洋葱伯克霍尔德菌ZYB002全细胞脂肪酶良好的碱稳定性、热稳定性和有机溶剂耐受性,表明该全细胞脂肪酶具有重要的工业应用潜力。     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.     

The glycerol teichoic acid from walls of Staphylococcus epidermidis I2

1. Walls of Staphylococcus epidermidis I2 contain 30% (w/w) of a glycerol teichoic acid containing phosphate, d-alanine and d-glucose in the molecular proportions 1:0.25:0.50. 2. The teichoic acid was isolated by extraction with trichloroacetic acid and with dilute aqueous NN-dimethylhydrazine at pH7, and was shown to be a (1-->3)-linked poly(glycerol phosphate) containing beta-d-glucopyranosyl and d-alanyl ester substituents. 3. 2-O-beta-d-Glucopyranosylglycerol was isolated and characterized as its crystalline hexa-O-acetate. 4. Unlike that of certain other bacteria, the peptidoglycan component of the wall is not solubilized by NN-dimethylhydrazine. 5. The membrane teichoic acid is also a (1-->3)-linked poly(glycerol phosphate) but contains a smaller proportion of glucosyl substituents.     

Novel Cold-Adapted Alkaline Lipase from an Intertidal Flat Metagenome and Proposal for a New Family of Bacterial Lipases

A new lipase, LipEH166, isolated from an intertidal flat metagenome, showed no amino acid similarity to any known lipolytic enzyme except in the consensus region. This suggested that LipEH166 and its homologues belong to a new family of lipolytic enzymes. Partial characterization indicated that LipEH166 is a novel cold-adapted alkaline lipase.     

Cold-Adapted RTX Lipase from Antarctic Pseudomonas sp. Strain AMS8: Isolation, Molecular Modeling and Heterologous Expression

A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S²⁰⁷, D²⁵⁵ and H³¹³, based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.     

2-Hydroxy-4-methoxybenzaldehyde from Hemidesmus indicus is antagonistic to Staphylococcus epidermidis biofilm formation

Abstract

Staphylococcus epidermidis (SE) is an opportunistic nosocomial pathogen that accounts for recalcitrant device-related infections worldwide. Owing to the growing interest in plants and their secondary metabolites targeting bacterial adhesion, this study was intended to uncover the anti-biofilm potential of Hemidesmus indicus and its major constituent 2-hydroxy-4-methoxybenzaldehyde (HMB) against SE. The minimum biofilm inhibitory concentration (MBIC) of H. indicus root extract and HMB were found to be 500 and 250?µg ml^?1, respectively. The results of time-dependent biofilm inhibition and mature biofilm disruption assays confirmed that HMB targets initial cell adhesion. Furthermore, interference by HMB in the expression of adhesin genes (icaA, aap and bhp) and biofilm components was associated with an increased susceptibility of SE to oxidative stress and antibiotics. To conclude, this study reports for the first time HMB as a potential drug against SE biofilms.     

Fermentation and isolation of epidermin,a lanthionine containing polypeptide antibiotic from Staphylococcus epidermidis

Summary Staphylococcus epidermidis TÜ 3298/DSM 3095 produces epidermin, a basic 21-residue peptide-amide antibiotic active against aerobic and anaerobic Gram-positive bacteria. Fermentations were performed by batch and feeding processes up to the 2001 scale. Highest yields were obtained when the first purification step was integrated into the fermentation process by on-line adsorption of the antibiotic. Isolation and purification by adsorption chromatography and ion exchange chromatography were performed batchwise.     

Drug resistance in Streptococcus faecalis, Streptococcus faecium and Staphylococcus epidermidis isolated from laboratory animals

A total of 1,278 strains of Streptococcus faecalis, 688 of Str. faecium and 796 of Staphylococcus epidermidis were collected from laboratory animals of 20 colonies in 1980, and tested for their resistance to 20 drugs. Thirteen percent of Str. faecalis, 12% of Str. faecium and 25% of Staph. epidermidis were drug resistant. Multiple drug resistant organisms were frequently detected in laboratory animal colonies where antibiotic treatment had been undertaken. TC-resistant streptococci and EM-resistant Staph. epidermidis were distributed in about half of the colonies regardless of the history of antibiotic usage.     

Cloning, expression and purification of extracellular serine protease Esp, a biofilm-degrading enzyme, from Staphylococcus epidermidis

Aims: Staphylococcus epidermidis Esp, an extracellular serine protease, inhibits Staphylococcus aureus biofilm formation and nasal colonization. To further expand the biotechnological applications of Esp, we developed a highly efficient and economic method for the purification of recombinant Esp based on a Brevibacillus choshinensis expression–secretion system. Methods and Results: The esp gene was fused with the N‐terminal Sec‐dependent signal sequence of the B. choshinensis cell wall protein and a C‐terminal hexa‐histidine‐tag gene. The recombinant Esp was expressed and secreted into the optimized medium as an immature form and subsequently activated by thermolysin. The mature Esp was easily purified by a single purification step using nickel affinity chromatography and showed proteolytic activity as well as Staph. aureus biofilm destruction activity. Conclusions: The purification yield of the developed extracellular production system was 5 mg recombinant mature Esp per 20‐ml culture, which was much higher than that of an intracellular production system in Escherichia coli (3 mg recombinant Esp per 1‐l culture). Significance and Impact of the Study: Our findings will be a powerful tool for the production and purification of recombinant Esp and also applicable to a large variety of recombinant proteins used for basic researches and biotechnological applications.     

Genome Sequence of Staphylococcus epidermidis Strain AU12-03, Isolated from an Intravascular Catheter

In recent years, Staphylococcus epidermidis has become a major nosocomial pathogen and the most common cause of intravascular catheter-related bacteremia, which can increase morbidity and mortality and significantly affect patient recovery. We report a draft genome sequence of Staphylococcus epidermidis AU12-03, isolated from an intravascular catheter tip.     

A Cold-Adapted Lipase of an Alaskan Psychrotroph, Pseudomonas sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization

A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas strain. The lipase gene (lipP) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from Antarctic Moraxella TA144 (G. Feller, M. Thiry, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381–388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897–4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinant Escherichia coli C600 cells harboring a plasmid coding for the lipP gene. The enzyme showed a 1,3-positional specificity toward triolein. p-Nitrophenyl esters of fatty acids with short to medium chains (C₄ and C₆) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis of p-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35°C. The enzyme was unstable at temperatures higher than 45°C. The K_m of the enzyme for p-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn²⁺, Cu²⁺, Fe³⁺, and Hg²⁺ but was not affected by phenylmethylsulfonyl fluoride and bis-nitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.     

A transposase-independent mechanism gives rise to precise excision of IS256 from insertion sites in Staphylococcus epidermidis

The mobile element IS256 causes phase variation of biofilm formation in Staphylococcus epidermidis by insertion and precise excision from the icaADBC operon. Precise excision, i.e., removal of the target site duplications (TSDs) and restoration of the original DNA sequence, occurs rarely but independently of functional transposase. Instead, the integrity of the TSDs is crucial for precise excision. Excision increased significantly when the TSDs were brought into closer spatial proximity, suggesting that excision is a host-driven process that might involve most likely illegitimate recombination.     

Gene Cloning and Characterization of a Novel Highly Organic Solvent Tolerant Lipase from Proteus sp. SW1 and its Application for Biodiesel Production

Proteus sp. SW1 was found to produce an extracellular solvent tolerant lipase. The gene, lipA, encoding a bacterial lipase, was cloned from total Proteus sp. SW1 DNA. lipA was predicted to encode a 287 amino acid protein of 31.2?kDa belonging to the Group I proteobacterial lipases. Purified His-tagged LipA exhibited optimal activity at pH 10.0 and 55??C. It was highly stable in organic solvents retaining 112% of its activity in 100% isopropanol after 24?h, and exhibited more than 200% of its initial activity upon exposure to 60% acetone, ethanol, and hexane for 18?h. Biodiesel synthesis reactions, using a single step addition of 13% an acyl acceptor ethanol, showed that LipA was highly effective at converting palm oil into biodiesel.     

Nucleotide sequence of the constitutive macrolide-lincosamide-streptogramin B resistance plasmid pNE131 from Staphylococcus epidermidis and homologies with Staphylococcus aureus plasmids pE194 and pSN2.

The complete nucleotide sequence of the Staphylococcus epidermidis plasmid pNE131 is presented. The plasmid is 2,355 base pairs long and contains two major open reading frames. A comparison of the pNE131 DNA sequence with the published DNA sequences of five Staphylococcus aureus plasmids revealed strong regional homologies with two of them, pE194 and pSN2. The region of pNE131 containing the reading frame which encodes the constitutive ermM gene is almost identical to the inducible ermC gene region of pE194, except for a 107-base-pair deletion which removes the mRNA leader sequence required for inducible expression. A second region of pNE131 contains an open reading frame with homology to the small cryptic plasmid pSN2 and potentially encodes a 162-amino-acid protein.