Teck Koon Tan

The degree of deacetylation of chitosan: advocating the first derivative UV-spectrophotometry method of determination

The degree of deacetylation (DD) is increasingly becoming an important property for chitosan, as it determines how the biopolymer can be applied. Therefore, a simple, rapid and reliable method of determining the DD for chitosan is essential. In this report, the DD of chitosan was determined by nuclear magnetic resonance (NMR), linear potentiometric titration (LPT), ninhydrin test and first derivative UV-spectrophotometry (1DUVS). The DD was calculated on a per mol basis instead of on a per mass basis. This is important as the molecular weights of N-acetyl-d-glucosamine and d-glucosamine are different. By converting the mass of N-acetyl-d-glucosamine and d-glucosamine into mols and calculating for the percentage of d-glucosamine present in the chitosan sample, a more accurate estimation of the DD can be obtained. Of the four methods, there is good correlation between 1DUVS and NMR. The concentration of chitosan solution for 1DUVS analysis was standardised as 0.1000 mg chitosan per ml of 0.0100 M acetic acid solution. The presence of d-glucosamine was corrected for by a reference curve for N-acetyl-d-glucosamine. 1DUVS is easy to perform, sensitive and the interference of other contaminants to the results is minimal compared with the other three methods. Therefore, we advocate 1DUVS to be used as the standard methods for routine determination of DD of chitosan.

The chitosan yield of zygomycetes at their optimum harvesting time

Fungi are a promising alternative source of chitosan. Fungi can be manipulated to give chitosan of more consistent and desired physico-chemical properties compared to chitosan obtained from crustacean sources. Chitosan was extracted from the mycelia of Rhizopus oryzae USDB 0602 at various phases of growth. The growth phase which produced the most extractable chitosan was determined to be the late exponential phase. In contrast to previous work on the screening of chitosan from fungal sources, mycelia of the fungi used in this study were harvested at their late exponential growth phase instead of at a fixed incubation time. The amount of extractable chitosan varied widely among the fungal strains. Gongronella butleri USDB 0201 was found to produce the highest amount of extractable chitosan per ml of substrate, followed by Cunninghamella echinulata and Gongronella butleri USDB 0428. However, in terms of yield of chitosan per unit mycelia mass, C. echinulata was the best strain among all fungi in the experiment. Therefore, besides G. butleri USDB 0201, C. echinulata can also be considered to be used in the commercial production of chitosan.

Concurrent production of chitin from shrimp shells and fungi

Crustacean shells constitute the traditional and current commercial source of chitin. Conversely, the control of fungal fermentation processes to produce quality chitin makes fungal mycelia an attractive alternative source. Therefore, the exploitation of both of these sources to produce chitin in a concurrent process should be advantageous and is reported here. Three proteolytic Aspergillus niger (strains 0576, 0307 and 0474) were selected from a screening for protease activity from among 34 zygomycete and deuteromycete strains. When fungi and shrimp shell powder were combined in a single reactor, the release of protease by the fungi facilitated the deproteinization of shrimp-shell powder and the release of hydrolyzed proteins. The hydrolyzed proteins in turn were utilized as a nitrogen source for fungal growth, leading to a lowering of the pH of the fermentation medium, thereby further enhancing the demineralization of the shrimp-shell powder. The shrimp-shell powders and fungal mycelia were separated after fermentation and extracted for chitin with 5% LiCl/DMAc solvent. Chitin isolates from the shells were found to have a protein content of less than 5%, while chitin isolates from the three fungal mycelia strains had protein content in the range of 10-15%. The relative molecular weights as estimated by GPC for all chitin samples were in the 10(5) dalton range. All samples displayed characteristic profiles for chitin in their FTIR and solid-state NMR spectra. All chitin samples evaluated with MTT and Neutral Red assays with three commercial cell lines did not display cytotoxic effects.

Identification and molecular phylogeny of Epulorhiza isolates from tropical orchids

24 isolates of Epulorhiza were obtained from the roots and protocorms of orchids in Singapore. Two groups were distinguished based on morphological and cultural characteristics. Group 1 comprised 20 isolates which were identified as E. repens, while those in group 2 were identified as E. calendulina-like Rhizoctonia. The ITS-5.8S rDNA sequence identity ranged from 88-100% among isolates of E. repens and six sub-groups were further delineated. The sequence identity was 98-100% among E. calendulina-like Rhizoctonia isolates. The sequence identity between E. repens isolates and E. calendulina-like Rhizoctonia isolates ranged from 18-44%. Apparently, isolates from both groups 1 and 2 were genetically distinct. Phylogenetic analysis showed that the distribution of the isolates correlated with the sites and the hosts from which the fungi were isolated. No matching sequences have been found in the GenBank database for the ITS region of E. repens and E. calendulina-like Rhizoctonia.

Production of fungal chitosan by solid state and submerged fermentation

The growth of the fungus Gongronella butleri USDB 0201 was compared in solid state fermentation (SSF) and submerged fermentation (SMF) using various nitrogen sources. The optimal production of biomass and chitosan by SMF was around 1.5–2.5 times higher than SSF. Urea is the best nitrogen source tested. SSF is to be preferred for the production of lower MW chitosan.

Evaluation of the allergenicity of tropical pollen and airborne spores in Singapore

Background: Sensitization to pollen and spores of the Southeast Asian tropical region is not well documented. This study evaluated the allergenicity of the tropical airspora in Singapore.

Variations in tropical airspora in Singapore

Nigrospora spherica was the major component in the airspora of three urban outdoor sites. Less frequent fungi include Alternaria and Cladosporium , and Curvularia which was represented by many species. Daily spore load showed a single peak at 10.00 h, dominated by N. sphaerica . This was replaced by Penicillium lapidosum as the dominant spore type at higher altitudes.

Properties of β-glucosidase purified from Aspergillus niger mutants USDB 0827 and USDB 0828

SummaryTwo extracellular β-glucosidases (EC 3.2.1.21) were isolated from Aspergillus niger USDB 0827 and A. niger USDB 0828, and their physical and kinetic properties studied. Both enzymes were very similar in terms of molecular size (230000 Da), pH optimum (pH 4.6), temperature optimum (65° C), stability at high temperatures and substrate preferences. They were capable of hydrolysing β-linked disaccharides, phenyl β-d-glucoside, p-nitrophenyl β-d-glucoside (PNPG), o-nitrophenyl β-d-glucoside, salicin and methyl β-d-glucoside but lacked activity towards α-linked disaccharides, a range of p-nitrophenyl monoglycosides and p-nitrophenyl diglycosides. Both β-glucosidases were better at hydrolysing cellobiose than cellotriose, cellotetraose or cellopentaose. For both enzymes, glucose showed competitive inhibition with PNPG as substrate but had no effect with cellobiose. However, the two β-glucosidases differed in inhibition by glucono-1,5-lactone and affinity for cellobiose. β-Glucosidase from A. niger USDB 0827 also gave lower specific activity, and was more susceptible to metal ions (Ag+, Fe2+ and Fe3+) inhibition than that of A. niger USDB 0828.

Kinetic properties of β-glucosidase from Aspergillus ornatus

SummaryKinetic properties of extracellular β-glucosidase from Aspergillus ornatus were determined. The pH and temperature optima for the enzyme were found to be 4.6 and 60°C, respectively. Under these conditions, the enzyme exhibited a Km (p-nitrophenyl-β-glucoside) value of 0.76±0.11 mM. The activation energy for the enzyme was 11.8 kcal/mol. Several divalent metal ions inhibited β-glucosidase activity, some of which showed inhibition of enzyme activity only at higher concentrations. Ag2+ was the most potent inhibitor. A metal chelating agent, EDTA, also inhibited β-glucosidase activity. Except for trehalose, glucose, glucono-δ-lactone, cellobiose, gentiobiose, laminaribiose, maltose and isomaltose inhibited β-glucosidase activity. Glucose was found to be a competitive inhibitor, whereas glucono-δ-lactone and other β-linked disaccharides were noncompetitive (mixed) inhibitors of the enzyme.

Lignicolous fungi of tropical mangrove wood

Thirty species of lignicolous marine fungi were observed on wood collected from Pontian Besar mangrove, Malaysia. The most frequent fungi consisted of Halosarpheia marina, Lulworthia sp., Lignincola laevis and Halosarpheia retorquens .