Synthesis and characterization of poly(d,l-lactic acid) via enzymatic ring opening polymerization by using free and immobilized lipase

Abstract

Polylactic acid is an interesting biodegradable and bioabsorbable material, and is produced from lactic acid, either by the direct polycondensation of lactic acid or via the ring-opening polymerization (ROP) of lactide. A future target of it is to improve some of the polyester properties for specific biomedical applications. The biocatalytic ROP of lactide is attractive as a route to polymer synthesis due to its lack of toxic reactants, mild reaction requirements, and recyclability of immobilized enzyme. Therefore, the use of immobilized enzymes is also being investigated.

The aim of this work was to develop a methodology to synthesize high molecular weight polylactic acid via enzymatic ROP method using free enzyme and Candida antarctica lipase B (CALB) immobilized onto chitin and chitosan. The efficiency of the two approaches has been compared, with polymerization kinetics and resulting products fully characterized by FT-IR, NMR, DSC, XRD, and TGA analyses.     

Chitinases: in agriculture and human healthcare

Abstract

Biological control of phytopathogenic fungi and insects continues to inspire the research and development of environmentally friendly bioactive alternatives. Potentially lytic enzymes, chitinases can act as a biocontrol agent against agriculturally important fungi and insects. The cell wall in fungi and protective covers, i.e. cuticle in insects shares a key structural polymer, chitin, a β-1,4-linked N-acetylglucosamine polymer. Therefore, it is advantageous to develop a common biocontrol agent against both of these groups. As chitin is absent in plants and mammals, targeting its metabolism will signify an eco-friendly strategy for the control of agriculturally important fungi and insects but is innocuous to mammals, plants, beneficial insects and other organisms. In addition, development of chitinase transgenic plant varieties probably holds the most promising method for augmenting agricultural crop protection and productivity, when properly integrated into traditional systems. Recently, human proteins with chitinase activity and chitinase-like proteins were identified and established as biomarkers for human diseases. This review covers the recent advances of chitinases as a biocontrol agent and its various applications including preparation of medically important chitooligosaccharides, bioconversion of chitin as well as in implementing chitinases as diagnostic and prognostic markers for numerous diseases and the prospect of their future utilization.     

Synthesis, characterization, cytotoxicity and antibacterial studies of chitosan, O-carboxymethyl and N,O-carboxymethyl chitosan nanoparticles

Chitosan (CS) is a naturally occurring biopolymer. It has important biological properties such as biocompatibility, antifungal and antibacterial activity, wound healing ability, anticancerous property, anticholesteremic properties, and immunoenhancing effect. Recently, CS nanoparticles have been used for biomedical applications. However, due to the limited solubility of CS in water its water-soluble derivatives are preferred for the above said applications. In this work, the nanoparticles of CS and its water-soluble derivatives such as O-carboxymethyl chitosan (O-CMC) and N,O-carboxymethyl chitosan (N,O-CMC) was synthesized and characterized. In addition, cytotoxicity and antibacterial activity of the prepared nanoparticles was also evaluated for biomedical applications.     

The chitobiose transporter, <Emphasis Type="Italic">chbC</Emphasis>, is required for chitin utilization in <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

Background  

The bacterium Borrelia burgdorferi, the causative agent of Lyme disease, is a limited-genome organism that must obtain many of its biochemical building blocks, including N-acetylglucosamine (GlcNAc), from its tick or vertebrate host. GlcNAc can be imported into the cell as a monomer or dimer (chitobiose), and the annotation for several B. burgdorferi genes suggests that this organism may be able to degrade and utilize chitin, a polymer of GlcNAc. We investigated the ability of B. burgdorferi to utilize chitin in the absence of free GlcNAc, and we attempted to identify genes involved in the process. We also examined the role of RpoS, one of two alternative sigma factors present in B. burgdorferi, in the regulation of chitin utilization.     

Bacterial Chitinases: Properties and Potential

ABSTRACT

Chitin is among the most abundant biomass present on Earth. Chitinase plays an important role in the decomposition of chitin and potentially in the utilization of chitin as a renewable resource. During the previous decade, chitinases have received increased attention because of their wide range of applications. Chito-oligomers produced by enzymatic hydrolysis of chitin have been of interest in recent years due to their broad applications in medical, agricultural, and industrial applications, including antibacterial, antifungal, hypocholesterolemic, and antihypertensive activity, and as a food quality enhancer. Microorganisms, particularly bacteria, form one of the major sources of chitinase. In this article, we have reviewed some of the chitinases produced by bacterial systems that have gained worldwide research interest for their diverse properties and potential industrial uses.     

An easy,inexpensive, and sensitive method for the quantification of chitin in insect peritrophic membrane by image processing

ABSTRACT

Chitin, poly (β-(1→4)-N-acetyl-d-glucosamine), is an important biopolymer for insects that is utilized as a major component of peritrophic membrane. The chitin content in peritrophic membrane is of expedient interest from a pest control perspective, although it is hard to quantify chitin. In this study, we establish a facile method for the quantification of chitin in peritrophic membrane by image processing. In this method, chitin was indirectly quantified using chitosan–I₃^? complex, which exhibited a specific red-purple color. A calibration curve using a chitosan solution showed good linearity in a concentration range of 0.05–0.5 μg/μL. We quantified the amount of chitin in peritrophic membrane of Spodoptera litura (Lepidoptera: Noctuidae) larvae using this method. Throughout the study, only common inexpensive regents and easily attainable apparatuses were employed. This method can be easily applied to the sensitive quantification of the amounts of chitin and chitosan in materials by wide range of researchers.

Abbreviations: LOD: limit of detection; LOQ: limit of quantification; ROI: region of interest; RSD: relative standard deviation.     

The cell factory approach toward biotechnological production of high-value chitosan oligomers and their derivatives: an update

Chitin is one of the most abundant renewable resources, and chitosans, the partially deacetylated derivatives of chitin, are among the most promising functional biopolymers, with superior material properties and versatile biological functionalities. Elucidating molecular structure–function relationships and cellular modes of action of chitosans, however, it is challenging due to the micro-heterogeneity and structural complexity of polysaccharides. Lately, it has become apparent that many of the biological activities of chitosan polymers, such as in agricultural plant disease protection or in mediating scar-free wound healing, may be attributed to oligomeric break-down products generated by the action of chitosanolytic hydrolases present in the target tissues, such as human chitotriosidase. Consequently, the focus of current research is shifting toward chitosan oligomers so that the availability of well-defined chitosan oligosaccharides (COS) becomes a bottleneck. Well-known ways of producing COS use physical and/or chemical means for the partial depolymerization of chitosan polymers, typically leading to broad mixtures of COS varying in their degrees of polymerization (DP) and acetylation (DA), and with more or less random patterns of acetylation (PAs). Even after chromatographic separation according to DP and DA, such mixtures are of limited value to elucidate structure–function relationships and modes of action. More recently, enzymatic means using chitinases and/or chitosanases, and sometimes chitin deacetylases, have been proposed as these can be more tightly controlled and yield slightly better defined mixtures of COS. An alternative would be chemical synthesis of COS which in principle would allow for full structural control, but protocols for it are lengthy, costly, and not yet well developed, and yields are low. Synthetic biology now allows to develop today’s in vitro bio-refinery approaches into in vivo cell factory approaches for the biotechnological production of defined COS using recombinant microbial strains expressing chitin oligomer synthases and chitin oligomer deacetylases. In this review, we will describe the state-of-the-art of this cell factory approach, as a basis for upcoming developments. We will briefly describe traditional chemical protocols and enzymatic production of COS as a background to the more detailed presentation of what has been achieved through in vivo biosynthesis. We will only briefly describe those as a background to the more detailed presentation of what has been achieved through in vivo biosynthesis. We will also touch on the production of COS derivatives that has been achieved in this way, as these oligomers open up another plethora of potential applications when used as building blocks for defined biomaterials.     

Green synthesis of gold nanoparticles with Zingiber officinale extract: Characterization and blood compatibility

Biosynthesis of gold nanoparticles with small size and biostability is very important and used in various biomedical applications. There are lot of reports for the synthesis of gold nanoparticles by the addition of reducing agent and stabilizing agent. In the present study we have synthesized gold nanoparticles, with a particle size ranging from 5 to 15 nm, using Zingiber officinale extract which acts both as reducing and stabilizing agent. Z. officinale extract is reported to be a more potent anti-platelet agent than aspirin. Therefore, green synthesis of gold nanoparticles with Z. officinale extract, as an alternative to chemical synthesis, is beneficial from its biological and medical applications point of view, because of its good blood biocompatibility and physiological stability. The formation and size distribution of gold nanoparticles were confirmed by dynamic light scattering (DLS), UV–vis spectrophotometer and transmission electron microscopy (TEM). Gold nanoparticles synthesized using citrate and Z. officinale extract demonstrated very low protein adsorption. Both nanoparticles were non platelet activating and non complement activating on contact with whole human blood. They also did not aggregate other blood cells, however, nanoparticles synthesised with Z. officinale extract was highly stable at physiological condition compared to citrate capped nanoparticles, which aggregated. Thus the usage of nanoparticles, synthesized with Z. officinale extract, as vectors for the applications in drug delivery, gene delivery or as biosensors, where a direct contact with blood occurs is justified.     

Determination of chitin in fungi and mycorrhizal roots by an improved HPLC analysis of glucosamine

A method to measure chitin content in fungi and ectomycorrhizal roots with high-performance liquid chromatography (HPLC) was developed. Measurements of fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine were made on acid hydrolysates of pure chitin, chitin-root mixtures and fungal-root mixtures. The method was applied on 5 isolates of ectomycorrhizal fungi, and ectomycorrhizal and non-mycorrhizal Pinus sylvestris roots. Interference from amino acids was removed by pre-treatment of samples with 0.2 N NaOH. This pre-treatment did not reduce the recovery of chitin, nor did plant material affect the recovery of chitin. The HPLC method was compared with a colorimetric chitin-method by measurements on root-fungal mixtures, with known fungal content. The HPLC method gave estimates of fungal biomass which were equal to the expected while the colorimetric method showed values significantly (p<0.001) lower than the expected. The present chitin method offers a sensitive and specific tool for the quantification of chitin in fungi and in ectomycorrhizal roots.     

Preparative methods of phosphorylated chitin and chitosan--an overview

Biomaterials such as chitin, chitosan and their derivatives have a significant and rapid development in recent years. Chitin and chitosan have become cynosure of all party because of an unusual combination of biological activities plus mechanical and physical properties. However, the applications of chitin and chitosan are limited due to its insolubility in most of the solvents. The chemical modification of chitin and chitosan are keen interest because of these modifications would not change the fundamental skeleton of chitin and chitosan but would keep the original physicochemical and biochemical properties. They would also bring new or improved properties. The chemical modification of chitin and chitosan by phosphorylation is expected to be biocompatible and is able to promote tissue regeneration. In view of rapidly growing interest in chitin and chitosan and their chemical modified derivatives, we are here focusing the recent developments on preparation of phosphorylated chitin and chitosan in different methods.     

Isolation and characterization of the gene encoding a chitin synthase with a myosin motor-like domain from the edible basidiomycetous mushroom, <Emphasis Type="Italic">Lentinula edodes</Emphasis>, and its expression in the course of fruit-body formation

We isolated and characterized the genomic and complementary DNAs encoding a chitin synthase from an edible basidiomycetous mushroom, Lentinula edodes. The gene (which we designated Lechs1) contains a large open reading frame encoding a polypeptide of 1937 amino acid residues. The open reading frame is interrupted by 14 small introns (49–116 bp). The gene product (LeChs1) consists of a myosin motor-like domain in its N-terminal half and a chitin synthase domain in its C-terminal half, analogous to the class V and VI chitin synthases of other filamentous fungi. Phylogenetic analysis demonstrated that LeChs1 is classified into class VI chitin synthases. Southern blot analysis indicated that Lechs1 is a single-copy gene per haploid genome and that L. edodes has no other highly homologous chitin synthase genes. Northern blot analysis revealed that Lechs1 is expressed throughout the whole stages of fruit-body formation of L. edodes, but its expression level gradually declines in a fruit body-maturation-dependent manner with highest expression in vegetative mycelia and fruit body at the early stage of maturation (immature fruit body). This is the first report on the isolation and characterization of the gene encoding a chitin synthase with a myosin motor-like domain from basidiomycetes.     

Studies on the Mode of Action of Polyoxin D

In the previous papers we reported that the antibiotic Polyoxin D induced the characteristic swelling of the mycelia of fungi,^1,2) and strongly inhibited the incorporation of ¹⁴C-glucosamine into the fungal cell wall chitin.³⁾ The present work has been conducted to further investigate the influence of this antibiotic on the fungal cell wall biosynthesis.

Polyoxin D did not inhibit the incorporation of ¹⁴C-glucose, ¹⁴C-amino acids and ¹⁴C-sodium acetate into the cell wall. In addition, UDP-N-acetylglucosamine, a precurcor of chitin biosynthesis of cell wall, was accumulated in the Polyoxin D-treated mycelia of Cochliobolus miyabeanus more than 150 to 160% of that accumulated in the untreated one.

Chitin synthetase prepared from Piricularia oryzae which is not treated with Polyoxin D was completely inhibited by the addition of 0.1 ppm of Polyoxin D. The fungitoxicity of Polyoxins A to G was positively related to their inhibition of ¹⁴C-glucosamine incorporation into the cell wall chitin of C. miyabeanus. From above results, it became evident that the antibiotic Polyoxin complex inhibited the biosynthesis of fungal cell wall chitin.     

Chitin and chitosan biopolymer production from the Iranian medicinal fungus Ganoderma lucidum: Optimization and characterization

Abstract

Chitin and chitosan with unique properties and numerous applications can be produced from fungus. The production of chitin and chitosan from the mycelia of an Iranian Ganoderma lucidum was studied to improve cell growth and chitin productivity. Inoculum size and initial pH as two effective variables on the growth of G. lucidum and chitin production were optimized using response surface method (RSM) by central composite design (CCD). The results verified the significant effect of these two variables on the cell growth and chitin production. In optimum conditions, including pH?=?5.7 and inoculum size of 7.4%, the cell dry weight was 5.91?g/L and the amount of chitin production was 1.08?g/L with the productivity of 0.083?g/(L day). The produced chitin and chitosan were characterized using XRD and FTIR. Moreover, the antibacterial activity of the produced chitosan was investigated and compared with the commercial chitosan. The results showed that the produced chitin and chitosan had suitable quality and the Iranian G. lucidum would be a great source for safe and high-quality chitin and chitosan production.     

Novel chitin/nanosilica composite scaffolds for bone tissue engineering applications

Biopolymers like chitin are widely investigated as scaffolds in bone tissue engineering. Its properties like biocompatibility, biodegradability, non-toxicity, wound healing ability, antibacterial activity, hemostatic property, etc., are widely known. However, these materials are not much bioactive. Addition of material like silica can improve the bioactivity and biocompatibility of chitin. In this work, chitin composite scaffolds containing nanosilica were prepared using chitin hydrogel and their bioactivity, swelling ability and cytotoxicity was analyzed in vitro. These scaffolds were found to be bioactive in simulated body fluid (SBF) and biocompatible when tested with MG 63 cell line. These results suggest that chitin/nanosilica composite scaffolds can be useful for bone tissue engineering applications.     

Novel chitin and chitosan nanofibers in biomedical applications

Chitin and its deacetylated derivative, chitosan, are non-toxic, antibacterial, biodegradable and biocompatible biopolymers. Due to these properties, they are widely used for biomedical applications such as tissue engineering scaffolds, drug delivery, wound dressings, separation membranes and antibacterial coatings, stent coatings, and sensors. In the recent years, electrospinning has been found to be a novel technique to produce chitin and chitosan nanofibers. These nanofibers find novel applications in biomedical fields due to their high surface area and porosity. This article reviews the recent reports on the preparation, properties and biomedical applications of chitin and chitosan based nanofibers in detail.     

Pentadecanal inspired molecules as new anti-biofilm agents against Staphylococcus epidermidis

Abstract

Staphylococcus epidermidis, a harmless human skin colonizer, is a significant nosocomial pathogen in predisposed hosts because of its capability to form a biofilm on indwelling medical devices. In a recent paper, the purification and identification of the pentadecanal produced by the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125, able to impair S. epidermidis biofilm formation, were reported. Here the authors report on the chemical synthesis of pentadecanal derivatives, their anti-biofilm activity on S. epidermidis, and their action in combination with antibiotics. The results clearly indicate that the pentadecanal derivatives were able to prevent, to a different extent, biofilm formation and that pentadecanoic acid positively modulated the antimicrobial activity of the vancomycin. The cytotoxicity of these new anti-biofilm molecules was tested on two different immortalized eukaryotic cell lines in view of their potential applications.     

Evaluation of biocompatibility and mechanical behavior of chitin-based polyurethane elastomers. Part-II: Effect of diisocyanate structure

Chitin-based polyurethane elastomers having potential for biomedical applications with tunable mechanical properties were synthesized by step growth polymerization techniques using poly(?-caprolactone) (PCL) with different diisocyanates. The prepolymer was extended using chitin and/or 1,4-butane diol (BDO). The structures of the resulted polymers were determined by Fourier transform infrared (FTIR), ¹H NMR and ¹³C NMR spectroscopic techniques. The effect of structure of diisocyanates and chain extenders on mechanical properties and in vitro biocompatibility were investigated. The results revealed that the final polymers extended with chitin are preferred candidates for surgical threads with on going investigations into their in vitro biocompatibility and non-toxicity.     

Characterization of Biosynthesized Silver Nanoparticles Using Lactobacillus rhamnosus GG and its In Vitro Assessment Against Colorectal Cancer Cells

Silver nanoparticles are the most desirable nanoparticles broadly used in diverse fields. This study intends to investigate the anticancer properties of synthesized silver/Lactobacillus rhamnosus GG nanoparticles (Ag-LNPs) as a reducing and stabilizing agent in the synthesis process. To prepare silver/Lactobacillus rhamnosus GG nanoparticles, 1 mg/ml cell lysate of Lactobacillus rhamnosus GG and 1 mM silver nitrate solution were mixed and incubated for 72 h. XRD, FTIR, and TEM methods were used for nanoparticle characterization. MTT assay and annexin/PI staining were employed to analyze the toxicity and apoptotic cells levels of Ag-LNPs, respectively. TEM showed that these nanoparticles are spherical shaped about 233 nm in size. FTIR spectroscopy demonstrated that Ag-LNPs were functionalized with biomolecules. XRD pattern showed high purity and face-centered crystal structure of Ag-LNPs. MTT assay revealed that the percentages of HT-29 live cells significantly reduced in the high concentration of Ag-LNPs. Annexin/PI staining showed that these nanoparticles could lead HT-29 cells to apoptosis. This study showed the new Ag-LNP-synthesizing method using Lactobacillus rhamnosus GG as a cost-effective and efficient approach. Also, it showed that these nanoparticles can be considered as a potential active agent for biomedical applications and drug delivery due to their anticancer activities.