Stanisław Bielecki

A cold-adapted extracellular serine proteinase of the yeast Leucosporidium antarcticum.

An extracellular serine proteinase, lap2, from the psychrophilic antarctic yeast Leucosporidium antarcticum 171 was purified to homogeneity and characterized. The enzyme is a glycoprotein with a molecular mass of 34.4xa0kDa and an isoelectric point of pHxa05.62. The proteinase is halotolerant, and its activity and stability are dependent neither on Ca2+ nor on other metal ions. Lap2 is a true psychrophilic enzyme because of low optimal temperature (25°C), poor thermal stability, relatively small values of free energy, enthalpy and entropy of activation, and high catalytic efficiency at 0–25°C. The 35xa0N-terminal amino acid residues of lap2 have homology with subtilases of the proteinase K subfamily (clan SB, family S8, subfamily C). The proteinase lap2 is the first psychrophilic subtilase in this family.

New methods Modified bacterial cellulose tubes for regeneration of damaged peripheral nerves

Introduction The subject of the experiment was bacterial nanocellulose, a natural polymer produced by bacteria – Gluconacetobacter xylinus. Following a specific modification process a cartilage-like material for restoration of damaged tissues may be produced. The obtained implants with excellent biocompatibility, mouldability, biophysical and chemical properties perfectly fit the needs of reconstructive surgery. The goal of the experiment was to develop and analyze cellulosic guidance channels in vivo for the reconstruction of damaged peripheral nerves. Material and methods The experiments were conducted on Wistar rats, femoral nerve. Cellulose was produced according to a self-patented method. In the experimental group tubulization was applied, whereas in the control traditional end-to-end connection was used. Observation time was 30, 60, 90, and 180 days. Results evaluation included histological analysis and postoperative observation of motor recovery. Results The overgrowth of connective tissue and disorganisation of neural structures was evident in 86.67% of control specimens, while for cellulosic group it was only 35% (p = 0.0022). Tubulization prevented the excessive proliferation of connective tissue and isolated from penetration with scar tissue. Autocannibalism, being probably an evidence of neurotrophic factors amassment, was observed in cellulosic group but not in the control one. Motor recovery did not differ significantly (p > 0.05). Biocompatibility of implants was affirmed by very small level of tissue response and susceptibility to vascularisation. Conclusions Cellulosic neurotubes effectively prevent the formation of neuromas. They are of very good biocompatibility and allow the accumulation of neurotrophic factors inside, thus facilitating the process of nerve regeneration.

Diversity of laccase-coding genes in Fusarium oxysporum genomes

Multiple studies confirm laccase role in fungal pathogenicity and lignocellulose degradation. In spite of broad genomic research, laccases from plant wilt pathogen Fusarium oxysporum are still not characterized. The study aimed to identify F. oxysporum genes that may encode laccases sensu stricto and to characterize the proteins in silico in order to facilitate further research on their impact on the mentioned processes. Twelve sequenced F. oxysporum genomes available on Broad Institute of Harvard and MIT (2015) website were analyzed and three genes that may encode laccases sensu stricto were found. Their amino acid sequences possess all features essential for their catalytic activity, moreover, the homology models proved the characteristic 3D laccase structures. The study shades light on F. oxysporum as a new source of multicopper oxidases, enzymes with possible high redox potential and broad perspective in biotechnological applications.

The influence of liquid systems for shoot multiplication, secondary metabolite production and plant regeneration of Scutellaria alpina

To establish an effective system for shoot multiplication and bioactive metabolite (baicalin, wogonoside, luteolin, luteolin-7-glucoside and verbascoside) production, shoot tips of Scutellaria alpina were grown in various culture systems: 0.35 and 0.7% agar solidified medium, liquid stationary culture, and 5xa0L sprinkle bioreactor. S. alpina appeared very sensitive to immersion in the culture medium, and support was needed to achieve shoot proliferation in the liquid stationary culture. For this purpose, bacterial nanocellulose and polyurethane foam or steel mesh in bioreactor system as support were used. The liquid nanocellulose-supported culture system gave the greatest shoot proliferation rates (36.1u2009±u20094.9 within 5xa0weeks). Shoots multiplied in the liquid supported media had greater fresh and dry weights and accumulated higher levels of metabolites. The most evident differences in metabolite content were observed when liquid supported cultures were compared with culture on 0.7% agar solidified medium. The study presents for the first time the use of the liquid culture systems together with bioreactor culture for in vitro S. alpina shoot multiplication and production of therapeutically valuable metabolites.

Medical Devices Regulation

Bacterial nanocellulose (BNC), thanks to its properties, can be used for the production of a wide range of medical devices. Medical devices made from pure bacterial nanocellulose or in association with other substances or materials must meet certain legal requirements while placed onto the market. The legislation in force concerning medical devices aims to eliminate undesirable products and to ensure that only such products are on the market and in use that meet all of the European Union requirements. This chapter provides the necessary definition and classification of a medical device, essential requirements, conformity assessment procedures, obligation of manufacturers (authorized representatives), and other information related to these issues.

Medical and Cosmetic Applications of Bacterial NanoCellulose

Abstract Bacterial nanocellulose, a natural, chemically pure biopolymer produced by microorganisms, is being well recognized as a highly biocompatible material. It has been already successfully applied as wet wound dressing and cosmetic facial mask, but its internal uses as artificial vessels, heart valves, and hernia meshes have been also conducted. Specific modifications of BNC make possible production of cartilage-like substitutes of meniscus, auricular, and nasal concha or even tubes for nerves regeneration. The final products are similar to natural tissues, with biocompatibility, moldability, biophysical, and chemical properties fitting the needs of regenerative medicine. Some of these biotechnological products have been already subjected to intensive in vivo investigation; some others, such as porous scaffolds for tissue engineering, are still under development. Recently, a lot of attention has been put into drug delivery systems production based on bio-cellulose. Continually, microbial cellulose offers a large field for systematic research on its new biomedical applications.

Bacterial NanoCellulose Characterization

Abstract This section summarizes analytical techniques that are used to characterize Bacterial NanoCellulose (BNC) and presents common physical, chemical methods enabling for detailed description of properties of the native and modified BNC. To characterize structure (determination of length and width of cellulose fibers and crosslinking of the fibers), most often used methods are electron microscopy (EM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM), and CRM Confocal (RAMAN microscopy). Other important parameters that characterize BNC are crystallinity and degree of polymerization (DP). Techniques of cellulose crystallinity measurement involve XRD techniques, solid state 13C NMR, IR infrared spectroscopy, and Raman spectroscopy. Degree of polymerization can be determined by membrane osmometry, cryoscopy, size-exclusion chromatography, ebullioscopy, and determination of reducing-ends concentration. FTIR spectroscopy, GC/MS method, colorimetric analysis (e.g., the phenol-sulfuric method), SEC chromatography, or HPAEC (high performance anion exchange chromatography) can be used for the quantification of carbohydrate component of BNC. Mechanical properties of native and modified BNC usually involve measurement of Young’s modulus, per cent elongation at break, and tensile strength.

Taxonomic Review and Microbial Ecology in Bacterial NanoCellulose Fermentation

Abstract Acetic acid bacteria (AAB) have a long history of use in several fermentation processes. Their exploitation gradually emerged in biotechnologic applications, especially in the biosynthesis of useful chemicals and processes for the manufacture of several fermented food products. Taxonomic studies, from traditional to polyphasic approaches, have gradually allowed the proper classification of several ABB into distinct genera and species, among them, the bacterial nanocellulose (BNC) producers, notably Komagataeibacter xylinus. Despite the advantages in using specific (isolated) strains for biotechnologic processes toward controlling the kinetics and process yield, mixed culture fermentations may provide an interesting approach to tailoring the properties of BNC and to increase the product yield when aiming at industrial scale. Microbial population dynamics may play a synergistic role in the coordinative substrate consumption and metabolites’ production, especially if using complex media (as is the case with low cost substrates, eg, residues from other processes). This chapter will first review the main historic steps involved in the taxonomic classification of AAB. It will then address the lying potential behind mixed microbial fermentations, from kombucha to nata de coco, both sharing in common, the contribution of cellulose-producing bacteria for the fermentation process.

Molecular Control Over BNC Biosynthesis

Abstract This short chapter will deal with molecular regulatory issues important for Ga. xylinus as a species living in large communities, organized inside cellulosic pellicle. The role of cyclic nucleotide second messengers will be discussed to the most extent. Present knowledge about cellular signaling and gene expression control mechanisms, active in Ga. xylinus, is limited. Therefore, the aim of the chapter is to draw directions for the future genetic research, which should provide better understanding of molecular control over Bacterial NanoCellulose (BNC) biosynthesis and secretion. Progress in this field of research should ensure the availability of molecular tools for BNC-producing strains, modifications of which will be more powerful than those used so far (see Chapterxa02).

Bacterial NanoCellulose Synthesis, Recent Findings

Abstract In this chapter we attempted to summarize recent reports on molecular biology and metabolism of bacteria belonging to the genus Gluconacetobacter with focus on its application for cellulose synthesis. The following issues were addressed: (1) the taxonomy of cellulose-producing Gluconacetobacter species; (2) cellulose synthase operons and flanking sequences from BNC producers, their role in biosynthesis of cellulose, and differences with other microorganisms; (3) recent advances in studies on three-dimensional structure of cellulose synthase subunits and their impact on Bacterial NanoCellulose (BNC) biosynthesis; (4) conditions of BNC production by Gluconacetobacter species from various substrates, including a variety of agro-industrial wastes; (5) metabolic pathways operating in Gluconacetobacter species verified as crucial for BNC synthesis by metabolic flux analysis; (6) the state of genome sequences data availability, concerning BNC-producing strains and their closest relatives; (7) genetic modifications of Ga. xylinus and Ga. hansenii and their effect on biosynthesis of cellulose with emphasis on methods and vectors used for these modifications.