Homogeneous modification of chitin and chitosan based on alkali/urea soluble system and their applications in biomedical engineering

Resource shortage and environmental degradation have forced people to pay attention to the development and application of environmentally friendly production method. Alkali/urea is a green water-based solvent for chitin and...

Off-target glycans encountered along the synthetic biology route towards humanized N-glycans in Pichia pastoris

ABSTRACTBackgroundThe glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized.ResultsWhen expressing mouse interleukin-22 (mIL-22) in a Pichia pastoris (syn. Komagataella phaffi) GlycoSwitchM5 strain which had been optimized to produce Man5GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5GlcNAc2 and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis and NMR, revealed that this novel glycan was Man5GlcNAc2 modified with a Glcα-1,2-Manβ-1,2-Manβ-1,3-Glcα-1,3-R tetra-saccharide. Also the biosynthetic intermediates of this off-target modification were detected. Expression of a Golgi-targeted GlcNAc Transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5GlcNAc2 structure. We have also observed the off-target glycan on other glycoproteins produced in the GlycoSwitchM5 strain. This illustrates the intricacies of Golgi glycosylation pathways and cautions that the use of glyco-engineered expression host cells should always be accompanied by detailed glycan analysis of the particular therapeutic proteins being produced.ConclusionsOur findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in Pichia pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.

Homogeneous Modification of Sugarcane Bagasse by Graft Copolymerization in Ionic Liquid for Oil Absorption Application

Sugarcane bagasse, lignocellulosic residue from the sugar industry, is an abundant and renewable bioresource on the earth. The application of ionic liquids in sugarcane bagasse biorefinery is gaining increasing interest. The homogeneous modification of sugarcane bagasse by free radical initiated graft copolymerization of acrylate monomers using 1-allyl-3-methylimidazolium chloride as solvent was performed. A variety of sugarcane bagasse graft copolymers with different weight percent gain were prepared via adjusting the monomer dosage. FT-IR studies confirmed the success in attaching the poly(acrylate) side chains onto sugarcane bagasse. Oil absorbency studies suggested that the sugarcane bagasse graft copolymers were potential biobased materials for effective treatment of ester-based oils. SEM studies showed that the sugarcane bagasse graft copolymers displayed a dense morphology structure. Thermogravimetric analysis demonstrated that the thermal stability of sugarcane bagasse decreased after the homogeneous modification by the graft copolymerization. The present study provides an alternative strategy to convert sugarcane bagasse into a value-added functional biobased material.

Dissolution Condition Effect on Cellulose in Ionic Liquid and the Recycling of Ionic Liquid

Recently, room temperature ionic liquids (RTILs) have been widely used in dissolution, homogeneous modification of cellulose. Cellulose only could be dissolved in RTILs at elevated temperature. Herein, effect of dissolution condition on microcrystalline cellulose in 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) ionic liquid was studied. The results indicated that the degree of polymerization (DP) of regenerated cellulose decreased from 161 to 83 with the increasing of dissolution temperature in the range of 70 °C to 110 °C, whereas the DP was affected by dissolution time slightly. Moreover, after dissolution [Bmim]Cl could be recycled by distillation and vacuum drying. The recycled [Bmim]Cl also had good dissolving capability of cellulose, and its FTIR spectra was similar to that of original [Bmim]Cl.

Homogeneous Derivatization of Bagasse in Different Solvents

The homogeneous modification of bagasse was investigated without additional catalysts in three solvents with MA and SA as derivatizing reagents. The results indicated that derivatization of bagasse in [C4mim]Cl was achieved at relatively high temperature, while that in DMSO/LiCl and DMSO/NMI was successfully carried out at room temperature. Surprisingly, the higher WPG of bagasse yielded in DMSO/NMI and DMSO/LiCl than in [C4mim]Cl because NMI and LiCl acted as solvent, base, and catalyst. FT-IR and CP/MAS 13C-NMR analyses provided more evidences of derivatization.

Homogeneous Modification of Sugarcane Bagasse with Succinic Anhydride in Novel Solvent Systems without Additional Catalysts

Homogeneous modification of sugarcane bagasse with succinic anhydride (SA) was investigated in novel solvent systems ionic liquid 1-butyl-3-methylimidazolium chloride/dimethyl sulfoxide ([C4mim]Cl/DMSO) and dimethyl sulfoxide/N-methylimidazole (DMSO/NMI), respectively. The results showed that succinoylation of SCB with SA was accomplished in [C4mim]Cl/DMSO at high temperature and in DMSO/NMI at room temperature without any additional catalysts. Increasing SA/SCB mass ratio from 2:1 to 3:1 and 4:1 resulted in an improvement of WPG from 21.1 to 29.6 and 30.4 in [C4mim]Cl/DMSO at 120oC, and from 97.3 to 102.5 and 118.4 in DMSO/NMI at room temperature, respectively. The high succinoylation efficiency in DMSO/NMI at room temperature was probably due to the special role of NMI as a solvent, a base and a catalyst in this solvent system. FT-IR and CP/MAS 13C-NMR analyses also provided evidences of succinoylation reaction of SCB with additional catalysts in [C4mim]Cl/DMSO and in DMSO/NMI. The results indicated that the succinoylation of lignin and carbohydrates all occurred.

Homogeneous Modification of Sugarcane Bagasse Cellulose with 2-(Dimethylamino)ethylmethacrylate in Ionic Liquid under Microwave Irradiation

A cellulose grafted copolymer was synthesized by homogeneous grafting 2-(Dimethylamino)ethylmethacrylate (DMAEMA) onto the sugarcane bagasse cellulose under microwave irradiation. Cellulose was dissolved in 1-butyl-3-methylinidazolium chloride ionic liquid ([Bmim]Cl) to form a homogeneous solution. The grafting polymerization was then initiated by potassium persulfate in the presence of N,Nˊ-methylenebisacrylamide as a crosslinker. Compared with the traditional thermal heating method, microwave irradiation had a drastic reduction of reaction time and an increased grafted efficiency. The characterization of the graft copolymer was confirmed by Fourier transform infrared spectroscopy (FT-IR), Scanning electron microscope (SEM), and Thermogravimetric analysis (TGA). Moreover, the homogenous reaction media applied can be carried out in completely recycled ionic liquid, and the grafted cellulose products have a potential application in wastewater anion absorption.