Endry Nugroho Prasetyo

Antimicrobial enzymes: An emerging strategy to fight microbes and microbial biofilms

With the increasing prevalence of antibiotic resistance, antimicrobial enzymes aimed at the disruption of bacterial cellular machinery and biofilm formation are under intense investigation. Several enzyme‐based products have already been commercialized for application in the healthcare, food and biomedical industries. Successful removal of complex biofilms requires the use of multi‐enzyme formulations that contain enzymes capable of degrading microbial DNA, polysaccharides, proteins and quorum‐sensing molecules. The inclusion of anti‐quorum sensing enzymes prevents biofilm reformation. The development of effective complex enzyme formulations is urgently needed to deal with the problems associated with biofilm formation in manufacturing, environmental protection and healthcare settings. Nevertheless, advances in synthetic biology, enzyme engineering and whole DNA‐Sequencing technologies show great potential to facilitate the development of more effective antimicrobial and anti‐biofilm enzymes.

Polymerization of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility

The ability of laccases from Trametes villosa (TvL), Myceliophthora thermophila (MtL), Trametes hirsuta (ThL) and Bacillus subtilis (BsL) to improve the dispersion properties of calcium lignosulfonates 398 in the presence of HBT as a mediator was investigated. Size exclusion chromatography showed an extensive increase in molecular weight of the samples incubated with TvL and ThL by 107% and 572% from 28400 Da after 17h of incubation, respectively. Interestingly, FTIR spectroscopy, (13)C NMR and Py-GC/MS analysis of the treated samples suggested no substantial changes in the aromatic signal of the lignosulfonates, a good indication of the ability of TvL/ThL-HBT systems to limit their effect on functional groups without degrading the lignin backbone. Further, the enzymatic treatments led to a general increase in the dispersion properties, indeed a welcome development for its application in polymer blends.

LACCASE CATALYZED COVALENT COUPLING OF FLUOROPHENOLS INCREASES LIGNOCELLULOSE SURFACE HYDROPHOBICITY

This work presents for the first time the mechanistic evidence of a laccase-catalyzed method of covalently grafting hydrophobicity enhancing fluorophenols onto Fagus sylvatica veneers. Coupling of fluorophenols onto complex lignin model compounds guaiacylglycerol beta-guaiacyl ether and syringylglycerol beta-guaiacyl ether was demonstrated by LC-MS and NMR. Laccase-mediated coupling increased binding of 4-[4-(trifluoromethyl)phenoxy]phenol (4,4-F3MPP) and 4-(trifluoromethoxy)phenol (4-F3MP) to veneers by 77.1% and 39.2%, respectively. XPS studies showed that laccase-catalyzed grafting of fluorophenols resulted in a fluorine content of 6.39% for 4,4-F3MPP, 3.01% for 4-F3MP and 0.26% for 4-fluoro-2-methylphenol (4,2-FMP). Grafting of the fluorophenols 4,2-FMP, 4-F3MP and 4,4-F3MPP led to a 9.6%, 28.6% and 65.5% increase in hydrophobicity, respectively, when compared to treatments with the respective fluorophenols in the absence of laccase, in good agreement with XPS data.

REACTIVITY OF LONG CHAIN ALKYLAMINES TO LIGNIN MOIETIES: IMPLICATIONS ON HYDROPHOBICITY OF LIGNOCELLULOSE MATERIALS

Enzymatic processes provide new perspectives for modification of lignocellulose materials. In the current study, laccase catalyzed coupling of long chain alkylamines to lignin model molecules and lignocellulose was investigated. Up to two molecules of dodecylamine (DA) and dihexylamine (DHA) were successfully coupled with lignin monomers (guaiacol, catechol and ferulic acid) while coupling onto complex lignin model compounds (syringylglycerol beta-guaiacyl ether, guaiacylglycerol beta-guaiacyl ether and dibenzodioxocin) yielded 1:1 coupling products. Surface analysis of beech veneers enzymatically grafted with DA showed an increase in nitrogen content of 3.18% compared to 0.71% in laccase only treated controls while the O/C ratio decreased from 0.52 to 0.46. Concomitantly the grafting of DHA or DA onto beech veneers resulted in a 53.8% and 84.2% increase in hydrophobicity, respectively when compared to simple adsorption. Therefore, laccase-mediated grafting of long chain alkylamines onto lignocellulose materials can be potentially exploited for improving their hydrophobicity.

Cellulose oxidation and bleaching processes based on recombinant Myriococcum thermophilum cellobiose dehydrogenase.

Myriococcum thermophilum cellobiose dehydrogenase (MtCDH) was expressed in Pichia pastoris using the pPICZαA expression vector under the control of methanol inducible AOX promoter. The purified recombinant MtCDH with a specific activity of 3.1 Umg(-1) was characterized to obtain kinetic constants for various carbohydrate substrates. Additionally, the C1 oxidation of the reducing ends of cellobiose, cellotetraose and maltotriose by MtCDH was verified by HPLC-MS. MtCDH was employed to oxidize several different cellulose-based materials by production of hydrogen peroxide. Based on the obtained results a one-pot enzymatic scouring/bleaching process for cotton fabrics was developed using pectinases as scouring agent and MtCDH to produce H(2)O(2) for bleaching. An average increase in whiteness (Berger) ΔE of 26 and an average 95% increase in wettability were observed in all MtCDH treated fabrics. In addition, MtCDH oxidized typical colored cotton flavonoids (morin, rutin, isoquercitrin).

ANTIOXIDANT ACTIVITY ASSAY BASED ON LACCASE GENERATED RADICALS

A novel antioxidant activity assay was developed using laccase-oxidized phenolics. In a three-step approach, phenolic compounds were first oxidized by laccase. Laccase was then inhibited using 80% (v/v) methanol which also stabilized the oxidized phenolics which were then used to measure antioxidant activities of ascorbic acid and Trolox. From a number of laccase-oxidized phenolics screened for potential use in the measurement of antioxidant activities, syringaldazine emerged the best, giving results comparable to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, which is currently used in conventional methods. Like DPPH radicals, two moles of stoichiometric oxidized syringaldazine were reduced by one mole of either ascorbic acid or Trolox. For the first time we show that antioxidant activity can be correlated to oxygen consumption by laccase. Reduction of one molecule of oxygen corresponded to oxidation of four molecules of syringaldazine which in turn is reduced by two molecules of Trolox or ascorbic acid. This study therefore demonstrates the great potential of using laccase-oxidized syringaldazine for the measurement of antioxidant activity.

An antioxidant regenerating system for continuous quenching of free radicals in chronic wounds.

A novel antioxidant regenerating system consisting of cellobiose dehydrogenase (CDH), cellobiose, and phenolic antioxidants with potential application for continuous quenching of free radical species in chronic wounds was developed. This antioxidant regenerating system, continuously quenched in situ produced ·NO, O(2)(·-) and OH· radicals and the produced oxidized phenolic antioxidants were regenerated back to their original parent compounds by CDH using cellobiose as electron donor. This system therefore prevented the accumulation of oxidized phenolic antioxidants. Interestingly, this study also challenges the relevance of using total antioxidant capacities values of plant crude extracts obtained using biologically none relevant radical species like (2,2-diphenyl-1-picrylhydrazyl (DPPH)), Trolox Equivalent Antioxidant Capacity (TEAC), etc. when applied as medicinal remedies. This is because methoxylated phenolic antioxidants like sinapic acid, ferulic acid; 2,6-dimethoxyphenol readily donate their electrons to these radicals (DPPH, TEAC, etc.), thereby greatly influencing the total antioxidant values although this study showed that they are not at all effective in quenching O(2)(·-) radicals and again are not the most effective quenchers of NO and OH radicals as demonstrated during this study.

Enzymatic Polymer Functionalisation: Advances in Laccase and Peroxidase Derived Lignocellulose Functional Polymers

Enzymatic polymer functionalisation has entered its most fascinating period with development in this field largely at the basic research level and pilot scale applications. Development of enzymatic processes for the development of lignocellulose-based functional polymers has not been spared, ranging from textile fibres with novel properties (antimicrobials properties, hydrophobic properties, attractive shed colours, etc.) to fibreboards. Enzymatic processes are also being actively pursued aimed at developing functional polymers from lignin (a major by product of the pulp and process).

Mechanistic insights into laccase-mediated functionalisation of lignocellulose material.

Abstract Recent emerging studies on the grafting mechanisms of functional molecules onto complex lignocellulose moieties have shown useful insights and possibilities in opening new frontiers in the enzymatic development of multifunctional polymers. Thanks to these studies which have demonstrated in principle the ability of laccases to mediate the coupling of antimicrobial compounds, hydrophobic molecules, including application processes for the development of fibreboards, particle boards, laminates etc. Further, laccase mediated grafting strategies developed using small reactive molecules e.g. phenolic amines which impart reactive properties to an inert polymer demonstrates the remarkable opportunities of enzyme meditated functionalization of polymers. Therefore recent studies focusing on understanding the mechanistic basis of the coupling mechanisms in order to make meaningful contribution to the development of new processes and products are a welcome development.

Bamboo fibre processing: insights into hemicellulase and cellulase substrate accessibility

A biological approach for degumming bamboo substrates has been assessed. The ability of various commercially available enzymes, including cellulase, xylanase, pectinase and mannanase, to hydrolyze bamboo powders was investigated. In addition, a commercial cellulase preparation was applied onto bamboo fibre bundles obtained by natural retting. It was found that almost all enzymes applied can use bamboo material as a substrate. Mild autoclave pre-treatment can enhance reducing sugar yield from different enzyme treatments. A most pronounced effect was observed with cellulase treatment in which the hydrolysis degree increased 1.7 fold as measured by reducing sugars for autoclave pre-treated bamboo powders versus non-treated powders after only a short period of incubation. The combined treatment of hemicellulase preparations showed no effect on the hydrolysis of bamboo substrates. The effect of autoclave pre-treatment on cellulase-treated samples was confirmed by the increase of sugar yield, protein absorption as well as by the enhancement of surface modification and enzyme penetration observed by CLSM (confocal laser scanning microscopy). This work establishes a base for future studies to develop enzymatic hydrolysis of bamboo materials, making them suitable for textile processing.