N-Acetylglucosamine Production by Repeated-Batch Fermentation Using Immobilized Semi-Purified Chitinase Enzyme on Agar

Chitinolytic mold, such as Mucor circinelloidesis can be utilized to produce chitinase enzyme for shrimp shell’s chitin hydrolysis into N-acetylglucosamine (NAG). For that purpose, entrapment of chitinase on agar as a carrier could be an alternative way to improve NAG production. This study aimed to investigate the stability of immobilized semi-purified chitinase on agar for multiple cycles fermentation to produce NAG. In this study, 0.6 mL of semi-purified chitinase enzyme was immobilized into 3% of agar matrices and tested for four fermentation cycles to obtain highest NAG concentration and good enzyme activity. The results indicate that the immobilized chitinase could be used for 6 hours fermentation or three fermentation cycles. The NAG concentration produced after three cycle were 1042.22 ± 16.20 ppm. Besides, the immobilized enzyme was considerably stable up to the third cycles with activity value of about 4.74 U/mL.Keywords: agar; immobilized;NAG; repeated fermentation

Enzymatic Synthesis and Characterization of Different Families of Chitooligosaccharides and Their Bioactive Properties

Chitooligosaccharides (COS) are homo- or hetero-oligomers of D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) that can be obtained by chitosan or chitin hydrolysis. Their enzymatic production is preferred over other methodologies (physical, chemical, etc.) due to the mild conditions required, the fewer amounts of waste and its efficiency to control product composition. By properly selecting the enzyme (chitinase, chitosanase or nonspecific enzymes) and the substrate properties (degree of deacetylation, molecular weight, etc.), it is possible to direct the synthesis towards any of the three COS types: fully acetylated (faCOS), partially acetylated (paCOS) and fully deacetylated (fdCOS). In this article, we review the main strategies to steer the COS production towards a specific group. The chemical characterization of COS by advanced techniques, e.g., high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry, is critical for structure–function studies. The scaling of processes to synthesize specific COS mixtures is difficult due to the low solubility of chitin/chitosan, the heterogeneity of the reaction mixtures, and high amounts of salts. Enzyme immobilization can help to minimize such hurdles. The main bioactive properties of COS are herein reviewed. Finally, the anti-inflammatory activity of three COS mixtures was assayed in murine macrophages after stimulation with lipopolysaccharides.

Label-free monitoring of crystalline chitin hydrolysis by chitinase based on Raman spectroscopy

We demonstrate a method for label-free monitoring of hydrolytic activity of crystalline-chitin-degrading enzyme, chitinase, by means of Raman spectroscopy. We found that crystalline chitin exhibited a characteristic Raman peak at...

EKSPLORASI DAN KARAKTERISASI CENDAWAN TANAH PADA RIZOSFER SAWI

ABSTRACTSoil fungis are one of the soil microbes that can be beneficial or detrimental to plants, so they play an important rolein agriculture. This study aims to determine the abundance and characterization of soil fungi in the rhizosphere ofmustard greens. Isolation and identification of soil fungus was carried out in the Pest and Disease Laboratory of theFaculty of Agriculture, University of Borneo Tarakan. The fungus was characterized macroscopically andmicroscopically, and the pathogenicity test, phosphate dissolution activity test, proteolytic activity test and chitinhydrolysis test were carried out. The results of the study obtained 34 fungus colonies. Based on the phenotypiccharacteristics there were 9 fungi isolates with different characteristics, 4 isolates from 9 isolates were non-pathogenic.In non-pathogenic fungi isolates, 2 genera were obtained, namely genus Phialophora sp. and Paecilomyces sp. Thereare non-pathogenic fungus isolates that have proteolytic activity and chitin hydrolysis so that it has the potential as abiological fertilizer and biological agentKeywords: Soil fungis, rhizosphere, characterization ABSTRAK Cendawan tanah merupakan salah satu mikrobia tanah yang dapat bersifat menguntungkan atau merugikan bagitanaman, sehingga berperan penting dalam bidang pertanian. Penelitian ini bertujuan untuk mengetahui kelimpahandan karakterisasi cendawan tanah di daerah rizosfer sawi. Isolasi dan identifikasi cendawan tanah dilakukan diLaboratorum Hama dan Penyakit Fakultas Pertanian Universitas Borneo Tarakan. Cendawan tanah dikarakterisasisecara makroskopis dan mikroskopis, dan dilakukan uji patogenesitas, uji aktivitas melarutkan fosfat, uji aktivitasproteolitik dan uji hidrolisis kitin. Hasil penelitian diperoleh 34 koloni cendawan. Berdasarkan karakteristik fenotipterdapat 9 isolat cendawan dengan karakteristik yang berbeda, 4 isolat dari 9 isolat bersifat non patogen. Pada isolatcendawan yang non patogenik, diperoleh 2 genus yaitu genus Phialophora sp. dan Paecilomyces sp. Isolat cendawannon patogenik yang diperoleh ada yang memiliki aktivitas proteolitik dan hidrolisis kitin sehingga berpotensi sebagaipupuk hayati dan agens hayati. Kata kunci: Cendawan tanah, rizosfer, karakterisasi

Chitin hydrolysis in acidified molten salt hydrates

At present, there is a lack of green protocols to hydrolyze chitin into its monomer using a chemical approach.

Utilization of Crude Intracellular Chitinase Enzyme from Providencia stuartii for Glucosamine Production from Shrimp Shells

Chitin hydrolysis using enzyme is one of the methods to produce glucosamine in shorter time compared to using microbial cells, but the ability to produce glucosamine at enzyme’s optimum condition is influenced by substrate concentration and fermentation time. The objective of this research was to determine the optimum substrate concentration and fermentation time of shrimp shells’ chitin to produce glucosamine at the optimum pH and temperature of crude intracellular chitinase enzyme from Providencia stuartii. Method used was experimental method, started by extraction of intracellular enzyme from P. stuartii, followed by determination of optimum pH and temperature of enzyme. The optimum condition was used for experiment of shrimp shells’ chitin fermentation with treatments of chitin substrate concentration (0.5; 1.0; 1.5; 2.0%) and fermentation time (2, 4, 6 and 24 hours). Results showed that optimum enzyme activity occurred at pH of 5.0 and temperature of 40oC, which was about 6.03 U/ml. Concentration of chitin substrate and fermentation time influenced the amount of glucosamine obtained. Fermentation of shrimp shells’ chitin using crude intracellular enzyme was optimum at 1.0% substrate concentration and 6 hours fermentation time, which produced glucosamine about 1680.06±58.49 ppm. Keywords: intracellular chitinase enzyme, glucosamine, shrimp shells’ chitin, P. stuartii

Paracoccin overexpression in Paracoccidioides brasiliensis reveals the influence of chitin hydrolysis on fungal virulence and host immune response

ABSTRACTParacoccidioides brasiliensis and P. lutzii, etiological agents of paracoccidioidomycosis (PCM), develop as mycelia at 25-30 °C and as yeast at 35-37 °C. Only a few Paracoccidioides spp. proteins are well characterized. Thus, we studied paracoccin (PCN) from P. brasiliensis, its role in the fungus biology, and its relationship with the host innate immune cells. Cloning and heterologous expression analysis revealed its lectin, enzymatic, and immunomodulatory properties. Recently, we employed a system based on Agrobacterium tumefaciens-mediated transformation to manipulate P. brasiliensis yeast genes to obtain clones knocked-down for PCN, which after all, are unable to transit from yeast to mycelium forms, causing a mild pulmonary disease. Herein, we generate P. brasiliensis overexpressing PCN (ov-PCN). To date, it was not explored the overexpressing of endogenous components in Paracoccidioides spp. Therefore, we investigate the role of PCN in fungal biology and pathogenesis. Augmented levels of PCN mRNA and protein, and N-acetylglucosaminidase activity confirmed PCN overexpression in ov-PCN of P. brasiliensis yeasts. Interestingly, PCN overexpression did not affect the yeasts’ growth or viability and favored cell separation. The ov-PCN clones transitioned faster to the mycelium form than the wt-PCN yeasts. Concerning infection, while most of mice infected with the wt-yeasts (90%) survive at least until the 70th day, all mice infected with ov-PCN yeasts were already died at the 35th day post-infection. In vitro assays showed that ov-PCN were more susceptible to phagocytosis by macrophages. Finally, it was verified that the chitin particles isolated from the ov-PCN cells were smaller than those obtained from the wt-PCN yeasts. Macrophages stimulated with the chitin isolated from ov-PCN produce IL-10, whereas the particles with a wider size range harvested from wt-PCN yeasts induced TNF-α and IL-1β secretion. The anti-inflammatory microenvironment from macrophage stimulation with small chitin particles hampers the development of a protective immune response against the fungus. We postulated that the high grade of chitin cleavage, as the results of augmented PCN expression, favors pathogenesis following P. brasiliensis infection. Thus, PCN is a relevant virulence fungal factor.AUTHOR SUMMARYParacoccidioides spp. are pathogenic fungi that cause paracoccidioidomycosis (PCM) in humans, the main deep mycosis of Latin America. Recently, by knocking down the paracoccin gene, our group showed that this lectin is necessary for the morphological transition from yeast to hyphae, and that this decrease results in low P. brasiliensis virulence. Here, after overexpress PCN, we revealed the importance of the yeast chitin hydrolysis to the host response. Infection of mice with ov-PCN yeasts causes severe lung disease compared to moderate disease caused by wt-PCN yeasts. The release of smaller chitin particles was as a result of an accelerated chitin hydrolysis provided by ov-PCN yeasts. Interestingly, these smallest chitin particles are able to modulate host response by increasing IL-10 in the meantime that decrease TNF-α secretion, thus hampering Th1 immune response that is crucial in the fight against this fungi. These findings represent a significant advance in the knowledge about the role of PCN chitinase in P. brasiliensis.