Improvement of catalytic properties of Escherichia coli penicillin G acylase immobilized on glyoxyl agarose by addition of a six-amino-acid tag

A tag of three lysines alternating with three glycines was added to the C-terminal end of the beta chain of penicillin G acylase (PGA). This modification improved the immobilization efficiency of PGA on glyoxyl agarose and the catalytic properties of the PGA derivative, although it impaired the posttranslational steps of overexpressed protein maturation.     

Microbial production and chemical transformation of poly-γ-glutamate

Poly-γ-glutamate (PGA), a novel polyamide material with industrial applications, possesses a nylon-like backbone, is structurally similar to polyacrylic acid, is biodegradable and is safe for human consumption. PGA is frequently found in the mucilage of natto, a Japanese traditional fermented food. To date, three different types of PGA, namely a homo polymer of d-glutamate (D-PGA), a homo polymer of l-glutamate (L-PGA), and a random copolymer consisting of d- and l-glutamate (DL-PGA), are known. This review will detail the occurrence and physiology of PGA. The proposed reaction mechanism of PGA synthesis including its localization and the structure of the involved enzyme, PGA synthetase, are described. The occurrence of multiple carboxyl residues in PGA likely plays a role in its relative unsuitability for the development of bio-nylon plastics and thus, establishment of an efficient PGA-reforming strategy is of great importance. Aside from the potential applications of PGA proposed to date, a new technique for chemical transformation of PGA is also discussed. Finally, some techniques for PGA and its derivatives in advanced material technology are presented.     

Newly characterized genetic polymorphism of uropepsinogen group A (PGA) using both isoelectric focusing and immunoblotting

Summary Genetic polymorphism of uropepsinogen group A (PGA) was characterized in human urine using a technique involving both polyacrylamide gel isoelectric focusing and immunoblotting with an anti-PGA antibody. PGA was clearly separable into five fractions, termed I to V in order of decreasing anodal mobility. The most slowly migrating fraction V was composed of F (fast) and/or S (slow) band(s). The population frequencies of the three patterns of fraction V (F, FS, and S) and family studies indicated that PGA V is controlled by a pair of alleles, PGA V ^* F and PGA V ^* S, at a single autosomal locus, and that both are codominant. The frequencies of the genes are 0.07 for PGA V ^* F and 0.93 for PGA V ^* S.     

Genomic structure and evolution of the human pepsinogen A multigene family

Summary A human cosmid library was screened with a pepsinogen A (PGA) cDNA probe, yielding 18 clones with (parts of) one, two or three PGA genes. By aligning these cosmids a restriction map of a PGA gene quadruplet was obtained in which the four genes are arranged in a highly ordered fashion in a head-to-tail orientation. Using the length in kilobases of the large polymorphic EcoRI fragment of the PGA genes, this quadruplet can be described as 15.0-12.0-12.0-16.6. An AvaII polymorphism allowed us to identify the two PGA haplotypes of the individual whose DNA had been cloned in the cosmid library to be a gene triplet and a gene quadruplet. By comparing the restriction maps of the central 12.0 genes in these multiplets to those of the flanking 15.0 and 16.6 genes, we postulate that these central genes arose from unequal but homologous crossing over between two 15.0–16.6 gene pairs. This hypothesis provides for the creation of a variety of haplotypes by additional cross overs and mutations. Southern blots of family and population material supports the existance of at least five common PGA haplotypes, including a single-gene haplotype, giving rise to a large number of different EcoRI patterns. The single PGA gene is probably the reciprocal crossing over product. Comparison between the DNA and protein polymorphisms suggests further micro-heterogeneity in the different PGA haplotypes.     

Polyglutamic acid (PGA) production by Bacillus sp. SAB-26: application of Plackett–Burman experimental design to evaluate culture requirements

A locally isolated thermostable Bacillus strain producing polyglutamic acid (PGA) was characterized and identified based on 16S rRNA sequencing. Phylogenetic analysis revealed its closeness to Bacillus licheniformis. To evaluate the effect of different culture conditions on the production of PGA, Plackett–Burman factorial design was carried out. Fifteen variables were examined for their significance on PGA production. Among those variables, K₂HPO₄, KH₂PO₄, (NH₄)₂SO₄ and casein hydrolysate were found to be the most significant variables that encourage PGA production. A correlation between cellular growth, PGA and the produced traces of polysaccharides was illustrated. An inverse relationship practice between cell dry weights and the produced PGA was demonstrated. On the other hand, a direct proportional relation was shown between polysaccharides on one side and cell dry weight and produced PGA on the other. The preoptimized medium, based on statistical analysis, showed a production of 33.5 g/l PGA, which is more than three times the basal medium.     

Molecular cloning of a pair of human pepsinogen A genes which differ by a Glu→Lys mutation in the activation peptide

Summary Three human cosmid clones containing pepsinogen A (PGA) encoding sequences were isolated from a genomic bank derived from a single individual. One cosmid contains two PGA genes in tandem in a head-to-tail orientation, while the other two cosmids each contain a single PGA gene. The three cosmids were characterized by restriction mapping and sequence analysis (exons 1 and 2 and flanking regions). As judged from these data, three of the four PGA genes isolated appear to be nearly identical, but one of the tandem genes is clearly different from the other genes. The first exon of all four genes codes for the same amino acid sequence. However, in the second exon of one of the tandem genes we found a nucleotide substitution giving rise to a GluLys substitution of the 43rd amino acid residue of the activation peptide, leading to a charge difference of the corresponding isozymogens. The presence of two distinct PGA genes in the isolated gene pair conclusively proves the multigene structure of the PGA system. These genes might be responsible for at least part of the electrophoretic polymorphism at the protein level.     

Monoclonal antibody against the poly-γ-d-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge

Background

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT.

Methods

To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K_d values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed.

Results

Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen.

General significance

Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.     

Production and Catabolite Repression of the Constitutive Polygalacturonic Acid trans-Eliminase of Aeromonas liquefaciens

Production of polygalacturonic acid (PGA) trans-eliminase was greatly stimulated under conditions of restricted growth of Aeromonas liquefaciens. This was accomplished either by substrate restriction in a continuous-feeding culture or by restricting divalent cations in a batch culture, with the use of PGA as the sole source of carbon in a chemically defined medium containing inorganic nitrogen. Slow feeding of glucose, glycerol, or PGA to carbon-limited cultures allowed PGA trans-eliminase to be formed at a maximum differential rate 500 times greater than in batch cultures with excess substrate present. The differential rate of enzyme formation obtained by slow feeding of these three substrances or of a mixture of PGA plus glucose was observed to be the same. Therefore, PGA trans-eliminase produced by A. liquefaciens, contrary to the current view, appears to be constitutive. These observations also indicate that production of PGA trans-eliminase is subject to catabolite repression and that limiting the substrate reverses this repression. It was also found that, under conditions of unrestricted growth, any compound which the bacteria can use as a source of carbon and energy repressed constitutive PGA trans-eliminase production. The heritable reversal of catabolite repression of PGA trans-eliminase synthesis was demonstrated by isolation of mutant strain Gc-6 which can readily synthesize the constitutive catabolic enzyme PGA trans-eliminase while growing in the presence of excess substrate.     

In vitro stabilization and minimum active component of polygalacturonic acid synthase involved in pectin biosynthesis

Polygalacturonic acid (PGA) synthase successively transfers galacturonic acid to oligogalacturonic acid by an alpha1,4-linkage to synthesize PGA, the backbone of plant pectic homogalacturonan. PGA synthase has not been purified to date due to its instability in vitro. In this study, we found stable conditions in vitro and separated a minimum active component of the enzymes from pea and azuki bean epicotyls. The PGA synthase lost its activity in 500 mM of sodium chloride or potassium chloride, while it was relatively stable at low salt concentrations. Under low salt concentrations, three peaks bearing PGA synthase activity were separated, by gel filtration and sucrose density gradient centrifugation. The molecular masses of these enzymes solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid were estimated to be 21,000, 5,000, and 590 kDa. The two higher molecular mass PGA synthases converted to smaller PGA synthase proteins when treated with high salt concentrations, while retaining their activity, indicating that PGA synthase has a minimum active component for its activity.     

PGA基因在酿酒酵母中的表达研究

本文根据GenBank 中巨大芽孢杆菌（Bacillus megaterium）的PGA基因序列设计了上下游引物,通过PCR扩增出巨大芽孢杆菌1.1741中的PGA基因。将该基因连接到T7lac启动子控制下的表达载体pYES2(amp+,ura+)上,构建了重组质粒pYES2-PGA。用LiAc/SSDNA/PEG方法将其转化进酿酒酵母(Saccharomyces cerevisiae)H158中表达,在不需要苯乙酸诱导的重组菌株发酵液中检测到了青霉素酰化酶活性,最高酶活达到0.75 U/ml。将该PGA基因测序结果与GenBank中巨大芽孢杆菌L04471.1、U07682.1和Z37542三株的PGA基因序列比对,表现出很高的同源性,分别达到97.1%、99.8% 和99.8%。     

Modulation of penicillin acylase properties via immobilization techniques: one-pot chemoenzymatic synthesis of Cephamandole from Cephalosporin C.

The modulation of penicillin G acylase (PGA) properties via immobilization techniques has been performed studying the acylation of 7-aminocephalosporanic acid with R-mandelic acid methyl ester. PGA from Escherichia coli, immobilized onto agarose activated with glycidol (glyoxyl-agarose), has been used for the design of a novel one-pot synthesis of Cephamandole in aqueous medium and without isolation of intermediates, through three consecutive biotransformations catalyzed by D-amino acid oxidase, glutaryl acylase and PGA.     

Regulation of Poly glutamate Production in Bacillus subtilis (natto): Transformation of High PGA Productivity

Bacillus subtilis (natto) produces a considerable amount of polyglutamate (PGA). The genetic character of high PGA productivity of B. subtilis (natto) was transferred by DNA-mediated transformation to B. subtilis Marburg 168 which cannot produce PGA. The enzyme activity of γ-glutamyltranspeptidase (γ-GTP) of the three transformants, 3F1, F1-9 and M5B4, was 124, 233 and 147 mU/ml, which is about 25, 250 and 100% of that of the donor strains, respectively. However, other enzyme activities such as those of alanine racemase or transaminase among the parental strains and representative transformants were almost the same.

These results suggested that γ-GTP activity might mainly participate in the biosynthesis of PGA in B. subtilis (natto).     

Fumonisin B1 influenced the effects of arachidonic acid, prostaglandins E2 and A2 on cell cycle progression, apoptosis induction, tyrosine- and CDC2-kinase activity in oesophageal cancer cells

In a previous study, we showed that, of a group of lipids including arachidonic acid (AA), prostaglandins E2 (PGE2) and A2 (PGA2), PGA2 had the most marked effect on the inhibition of cell growth, activation of tyrosine kinase activity, lowering of the number of G1-phase cells, and induction of p53 levels in oesophageal carcinoma (WHCO3) cells. No significant effects by the three lipids were seen in normal monkey kidney cells. In the present study, the effects of the inhibitor of ceramide synthesis, fumonisin B1 (FB1), a metabolite of Fusarium verticillioides (= F. moniliforme) which is implicated in the high incidence of oesophageal cancer, were determined on AA, PGE2 and PGA2 WHCO3 treated cells. In the presence of FB1, the lipid-enhanced tyrosine kinase activity was lowered. Flow cytometric and morphological studies showed that FB1 lowered the marked apoptosis induced by especially PGA2. FB1, however, in combination with AA, PGE2 or PGA2 increased the number of G2/M cells. AA>PGE2>PGA2 alone decreased CDC2-kinase activity, but, in the presence of FB1, CDC2-kinase activity was significantly increased. The PGA2- and AA-induced p53 levels were lowered in the presence of FB1. We concluded that FB1 diminished the cytotoxic effects of the lipids on oesophageal tumour cells.     

Relationships between the human pepsinogen DNA and protein polymorphisms.

Pepsinogens (PGA) are the inactive precursors of pepsin, the major acid protease found in the stomach. The PGA gene family exhibits polymorphic variation in human populations that can either be demonstrated by electrophoretic analysis of the proteins or by analysis of the respective genes with cDNA probes. Here, we describe the interrelationships between the most common pepsinogen protein phenotypes and the corresponding pepsinogen haplotypes (A, B, and C) containing different combinations of the PGA3, PGA4, and PGA5 genes. We propose that this unusual genetic variation involving haplotypes that contain three, two, and one genes, respectively, is the result of molecular evolution by gene duplication.     

Chromosomal integration of a synthetic expression control sequence achieves poly‐γ‐glutamate production in a Bacillus subtilis strain

Poly‐γ‐glutamate (γ‐PGA) has applications in food, medical, cosmetic, animal feed, and wastewater industries. Bacillus subtilis DB430, which possesses the γ‐PGA synthesis ywsC‐ywtAB genes in its chromosome, cannot produce γ‐PGA. An efficient synthetic expression control sequence (SECS) was introduced into the upstream region of the ywtABC genes, and this resulted in γ‐PGA‐producing B. subtilis mutant strains. Mutant B. subtilis PGA6‐2 stably produces high levels of γ‐PGA in medium A without supplementation of extra glutamic acid or ammonium chloride. The mutant B. subtilis PGA 6‐2 is not only a γ‐PGA producer, but it is also a candidate for the genetic and metabolic engineering of γ‐PGA production. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Poly-γ-d-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule of Bacillus anthracis in guinea-pigs and rabbits

Anthrax is a lethal infectious disease caused by the spore-forming Bacillus anthracis . The two major virulence factors of B. anthracis are exotoxin and the poly-γ- d -glutamic acid (PGA) capsule. The three components of the exotoxin, protective antigen (PA), lethal factor and edema factor act in a binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of the anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of the anthrax vaccine. In this study, we report that PA–PGA conjugates induce expressions of anti-PA, anti-PGA and toxin-neutralizing antibodies in guinea-pigs and completely protect guinea-pigs against a 50 × LD₅₀ challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA-15mer offer a partial passive protection to guinea-pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role. Our results demonstrate that PA–PGA conjugate vaccines are effective in the guinea-pig model, in addition to the previously reported mouse model.     

Structure mediation in substrate binding and post‐translational processing of penicillin acylases: Information from mutant structures of Kluyvera citrophila penicillin G acylase

Penicillin acylases are industrially important enzymes for the production of 6‐APA, which is used extensively in the synthesis of secondary antibiotics. The enzyme translates into an inactive single chain precursor that subsequently gets processed by the removal of a spacer peptide connecting the chains of the mature active heterodimer. We have cloned the penicillin G acylase from Kluyvera citrophila (KcPGA) and prepared two mutants by site‐directed mutagenesis. Replacement of N‐terminal serine of the β‐subunit with cysteine (Serβ1Cys) resulted in a fully processed but inactive enzyme. The second mutant in which this serine is replaced by glycine (Serβ1Gly) remained in the unprocessed and inactive form. The crystals of both mutants belonged to space group P1 with four molecules in the asymmetric unit. The three‐dimensional structures of these mutants were refined at resolutions 2.8 and 2.5 Å, respectively. Comparison of these structures with similar structures of Escherichia coli PGA (EcPGA) revealed various conformational changes that lead to autocatalytic processing and consequent removal of the spacer peptide. The large displacements of residues such as Arg168 and Arg477 toward the N‐terminal cleavage site of the spacer peptide or the conformational changes of Arg145 and Phe146 near the active site in these structures suggested probable steps in the processing dynamics. A comparison between the structures of the processed Serβ1Cys mutant and that of the processed form of EcPGA showed conformational differences in residues Argα145, Pheα146, and Pheβ24 at the substrate binding pocket. Three conformational transitions of Argα145 and Pheα146 residues were seen when processed and unprocessed forms of KcPGA were compared with the substrate bound structure of EcPGA. Structure mediation in activity difference between KcPGA and EcPGA toward acyl homoserine lactone (AHL) is elucidated.     

微重力条件下心肌细胞在聚羟基乙酸纤维支架上的三维固定化培养

以 1d龄Wistar乳鼠的心室肌组织为心肌细胞的来源 ,采用胰蛋白酶消化及细胞差速贴壁分离心肌细胞 ,以未经修饰和经鼠尾胶原溶液浸泡修饰的聚羟基乙酸 (polyglycolicacid ,PGA)纤维支架作为心肌细胞体外三维 (3D)固定化培养的支架 ,比较心肌细胞在静置培养体系及微重力培养体系下的生长、形态和收缩状况。心肌细胞在未经处理的PGA纤维支架 3D固定化培养时 ,心肌细胞在其上的分布不均匀 ,大部分心肌相互连接形成球状聚集体 ;PGA纤维支架经鼠尾胶原溶液浸泡处理后 ,心肌细胞在其上的分布较为均匀 ,细胞形态多呈梭形或不规则状 ,心肌细胞自律性搏动的幅度加大。在模拟微重力培养条件下 ,心肌细胞在经鼠尾胶原溶液浸泡修饰的PGA纤维支架上的分布更为均匀 ,心肌细胞形成具有自律性同步收缩特性、面积约为 15mm2 的类组织样 3D结构     

Poly (glutamic acid)--an emerging biopolymer of commercial interest

Poly (γ-glutamic acid) (PGA) is water-soluble, anionic, biodegradable, and edible biopolymer produced by Bacillus subtilis. It has multifarious potential applications in foods, pharmaceuticals, healthcare, water treatment and other fields. The production of PGA has already been established on the industrial scale. Various studies regarding the fermentative production, downstream processing and characterization of PGA have been reported in the literature. This review provides updated information on fermentative production of PGA by various bacterial strains and effect of fermentation conditions and media component on production of PGA in submerged as well as solid state fermentation. Information on the application of genetic engineering for enhancement of yield of PGA, kinetic studies for production of PGA in submerged fermentation and recovery and purification of PGA is included. An attempt has also been made to review the current and potential applications of PGA. This review may contribute to further development of this commercially and academically interesting biopolymer.