Preparation of hyaluronan oligosaccharides by a prokaryotic beta-glucuronidase: Characterization of free and immobilized forms of the enzyme.

Popularity of hyaluronan (HA) in the cosmetics and pharmaceutical industries, led to the investigation and development of new HA-based materials, with enzymes playing a key role. Beta-D-glucuronidases catalyze the hydrolysis of a beta-D-glucuronic acid residue from the non-reducing end of various substrates. However, lack of specificity towards HA for most beta-D-glucuronidases, in addition to the high cost and low purity of those active on HA, have prevented their widespread application. In this study, we investigated a recombinant beta-glucuronidase from Bacteroides fragilis (rBfGUS). We demonstrated the rBfGUS's activity on native, modified, and derivatized HA oligosaccharides (oHAs). Using chromogenic beta-glucuronidase substrate and oHAs, we characterized the enzyme's optimal conditions and kinetic parameters. Additionally, we evaluated rBfGUS's activity towards oHAs of various sizes and types. To increase reusability and ensure the preparation of enzyme-free oHA products, rBfGUS was immobilized on two types of magnetic macroporous bead cellulose particles. Both immobilized forms of rBfGUS demonstrated suitable operational and storage stabilities, and their activity parameters were comparable to the free form. Our findings suggest that native and derivatized oHAs can be prepared using this bacterial beta-glucuronidase, and a novel biocatalyst with enhanced operational parameters has been developed with a potential for industrial use.

Hyaluronidases and hyaluronate lyases: From humans to bacteriophages.

Hyaluronan is a non-sulfated negatively-charged linear polymer distributed in most parts of the human body, where it is located around cells in the extracellular matrix of connective tissues and plays an essential role in the organization of tissue architecture. Moreover, hyaluronan is involved in many biological processes and used in many clinical, cosmetic, pharmaceutic, and biotechnological applications worldwide. As interest in hyaluronan applications increases, so does interest in hyaluronidases and hyaluronate lyases, as these enzymes play a major part in hyaluronan degradation. Many hyaluronidases and hyaluronate lyases produced by eukaryotic cells, bacteria, and bacteriophages have so far been described and annotated, and their ability to cleave hyaluronan has been experimentally proven. These enzymes belong to several carbohydrate-active enzyme families, share very low sequence identity, and differ in their cleaving mechanisms and in their structural and functional properties. This review presents a summary of annotated and characterized hyaluronidases and hyaluronate lyases isolated from different sources belonging to distinct protein families, with a main focus on the binding and catalytic residues of the discussed enzymes in the context of their biochemical properties. In addition, the application potential of individual groups of hyaluronidases and hyaluronate lyases is evaluated.

Development of a rapid method for site-directed mutagenesis in Streptococcus zooepidemicus.

This paper describes the development of a straightforward method for site-directed gene mutagenesis in Streptococcus zooepidemicus, inspired by the mechanism of natural competence regulated by ComX in other streptococci. An alternative sigma factor comX gene was overexpressed from a plasmid in S. zooepidemicus and electrocompetent cells were prepared. As proof of concept, a DNA cassette with two targeting regions flanking a kanamycin resistance gene was spliced in an overlap extension PCR and electroporated. The cassette was then integrated in the genomic DNA by homologous recombination. Next, the gene SeseC_00180 (fibrinogen- and Ig-binding protein precursor) was selected as target for markerless gene deletion and the impact of its loss on the resulting hyaluronan production was determined. The new method of site-directed mutagenesis is significant because it is not necessary to clone the DNA cassette in an auxiliary vector, electroporating it in S. zooepidemicus cells is enough, which allows to bypass the problems with hard to clone DNA sequences and speeds up the whole process of mutation generation in S. zooepidemicus.

Production of hyaluronic acid by mutant strains of group C Streptococcus.

This study addresses the influence of upstream region sequence on the strength of has operon promoter in highly encapsulated S. equi subsp. zooepidemicus (SEZ). For this purpose, seven different strains were constructed. Each strain carries a point mutation in one of the following positions upstream of the has promoter: -43, -44, -49, and -50 bp. To facilitate measuring of the recombinant promoter relative strength, ß-glucuronidase gene was used as a reporter gene. Three mutations located in positions -49 and -50: AT, GT, and AG, positively impacted has promoter strength when compared to the wild type sequence GG. Conversely, two other mutations: TG and TT, exhibited a slight inhibitory effect. Further, three different strains carrying chromosomal mutations in the has promoter region were constructed. In two cases, the has operon is under the control of a stronger promoter and in the third strain the has operon is controlled by a weaker promoter. The laboratory fermenter scale cultivations confirmed the increase of hyaluronan yields for SEZPhasAG and SEZPhas2G, resulting 116 and 105 %, respectively. As expected, the yield of the hyaluronic acid of SEZPhas2B strain fell to 41 %.

High level expression of human enteropeptidase light chain in Pichia pastoris.

Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4U/mL of wet matrix.

High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli.

The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter. The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms. Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 microM; for Staphylococcus aureus 2.31 microM, and for Enterococcus faecalis 5.54 microM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of beta-galactosidase activity in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate quantities of active human antimicrobial peptide LL-37.