Production and partial characterization of biosurfactant produced by Streptomyces sp. R1

The present study focused on developing a wild-type actinomycete isolate as a model for a non-pathogenic filamentous producer of biosurfactants. A total of 33 actinomycetes isolates were screened and their extracellular biosurfactants production was evaluated using olive oil as the main substrate. Out of 33 isolates, 32 showed positive results in the oil spreading technique (OST). All isolates showed good emulsification activity (E₂₄) ranging from 84.1 to 95.8%. Based on OST and E₂₄ values, isolate R1 was selected for further investigation in biosurfactant production in an agitated submerged fermentation. Phenotypic and genotypic analyses tentatively identified isolate R1 as a member of the Streptomyces genus. A submerged cultivation of Streptomyces sp. R1 was carried out in a 3-L stirred-tank bioreactor. The influence of impeller tip speed on volumetric oxygen transfer coefficient (k _L a), growth, cell morphology and biosurfactant production was observed. It was found that the maximum biosurfactant production, indicated by the lowest surface tension measurement (40.5 ± 0.05 dynes/cm) was obtained at highest k _L a value (50.94 h⁻¹) regardless of agitation speed. The partially purified biosurfactant was obtained at a concentration of 7.19 g L⁻¹, characterized as a lipopeptide biosurfactant and was found to be stable over a wide range of temperature (20–121 °C), pH (2–12) and salinity [5–20% (w/v) of NaCl].

     

Esterification of Levulinic Acid Using ZrO<Subscript>2</Subscript>-Supported Phosphotungstic Acid Catalyst for Ethyl Levulinate Production

Ethyl levulinate (EL) is a versatile bio-based chemical with various applications such as fragrance and flavoring agents and also fuel blending component. EL can be produced through catalytic esterification of levulinic acid (LA) with ethanol. Herein, a series of zirconia (ZrO₂)-supported phosphotungstic acid (HPW) (HPW/Zr) catalyst, 15-HPW/Zr, 20-HPW/Zr, and 25-HPW/Zr, were prepared, characterized, and tested for EL production. The physicochemical properties of catalysts were characterized using Fourier-transformed infrared (FTIR) spectroscopy, x-ray diffraction (XRD), field emission scanning electron microscope (FESEM), N₂ physisorption, and ammonia temperature-programmed desorption (NH₃-TPD). The effect of reaction parameters: reaction time, temperature, catalyst loading, and molar ratio of LA to ethanol was inspected on LA conversion and EL yield. The catalyst with high surface area and high acidity seemed suitable for EL production. Among the catalysts tested, 20-HPW/Zr exhibited the highest EL yield of 97.3% at the following conditions: 150 °C, 3 h, 1.0 g of 20-HPW/Zr and 1:17 M ratio of LA to ethanol. The 20-HPW/Zr could be reused for at least four times with insignificant decrease in the EL yield. This study demonstrates the potential of ZrO₂-supported HPW for bio-based alkyl levulinate production at mild process conditions.     

Optimization of Biomass Conversion to Levulinic Acid in Acidic Ionic Liquid and Upgrading of Levulinic Acid to Ethyl Levulinate

Levulinic acid (LA) is a versatile platform chemical that can be derived from biomass as an alternative to fossil fuel resources. Herein, the optimization of LA production from glucose and oil palm fronds (OPF) catalyzed by an acidic ionic liquid; 1-sulfonic acid-3-methyl imidazolium tetrachloroferrate ([SMIM][FeCl₄]) have been investigated. Response surface methodology based on Box-Behnken design was employed to optimize the LA yield and to examine the effect and interaction of reaction parameters on the LA production. The reaction parameters include reaction temperature, reaction time, feedstock loading, and catalyst loading. From the optimization study, the predicted mathematical models for LA production from glucose and OPF covered more than 90 % of the variability in the experimental data. At optimum conditions, 69.2 % of LA yield was obtained from glucose, while 24.8 % of LA yield was attained from OPF and registered 77.3 % of process efficiency. The recycled [SMIM][FeCl₄] gave sufficient performance for five successive cycles. Furthermore, the optimum LA produced from glucose and OPF can be directly converted to ethyl levulinate through esterification over the [SMIM][FeCl₄] catalyst. This study highlights the potential of [SMIM][FeCl₄] for biorefinery processing of renewable feedstocks at mild process conditions.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity

Low reaction yields and the high cost of obtaining a single type of pure CD make γ-CD costly. Using rational design and with the aid of 3D modeling structures, recombinant CGTase from Bacillus sp. G1 was molecularly engineered with the aim of producing a higher percentage of γ-CD. A single mutation at subsite −3, denoted H43T, was found to increase γ-CD production from 10% to approximately 39% using tapioca starch. This novel increment was probably the result of reduced steric hindrance to the formation of γ-CD because of the shortened side chain together with the shortened loop at positions 86–89, at substrate-binding subsite −3. A mutation (Tyr188 → Trp) and a deletion at loop 139–144 showed little effect on product specificity; however, mutagenesis at these sites affected cyclization, coupling and hydrolysis activities as well as the kinetic properties of the mutant CGTase. Based on rational design, three further mutations of the mutant H43T (denoted H43T/Δ(139–144)/S134T/A137V/L138D/V139I, H43T/S85G and H43T/Y87F) were constructed and produced γ-CD with yields of 20%, 20% and 39%, respectively. The mutant H43T/Δ(139–144)/S134T/A137V/L138D/V139I had very low cyclization and coupling activities, however their hydrolysis activity was retained. Double mutation (H43T/S85G) caused the enzyme to exhibit higher starch hydrolysis activity, approximately 26 times higher than the native CGTase G1. Although the mutants H43T and H43T/Y87F could produce the same percentage (39%) of γ-CD, the latter was more efficient as the total amount of CD produced was higher based on the V_max and k_cat values.     

Catalysis by Glomerella cingulata cutinase requires conformational cycling between the active and inactive states of its catalytic triad

Cutinase belongs to a group of enzymes that catalyze the hydrolysis of esters and triglycerides. Structural studies on the enzyme from Fusarium solani have revealed the presence of a classic catalytic triad that has been implicated in the enzyme's mechanism. We have solved the crystal structure of Glomerella cingulata cutinase in the absence and in the presence of the inhibitors E600 (diethyl p-nitrophenyl phosphate) and PETFP (3-phenethylthio-1,1,1-trifluoropropan-2-one) to resolutions between 2.6 and 1.9 Å. Analysis of these structures reveals that the catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 Å away from its expected position. Solution-state NMR experiments are consistent with the disrupted configuration of the triad observed crystallographically. H204N, a site-directed mutant, was shown to be catalytically inactive, confirming the importance of this residue in the enzyme mechanism. These findings suggest that, during its catalytic cycle, cutinase undergoes a significant conformational rearrangement converting the loop bearing the histidine from an inactive conformation, in which the histidine of the triad is solvent exposed, to an active conformation, in which the triad assumes a classic configuration.     

Tissue allograft coding and traceability in USM Tissue Bank,Malaysia

In Malaysia, tissue banking activities began in Universiti Sains Malaysia (USM) Tissue Bank in early 1990s. Since then a few other bone banks have been set up in other government hospitals and institutions. However, these banks are not governed by the national authority. In addition there is no requirement set by the national regulatory authority on coding and traceability for donated human tissues for transplantation. Hence, USM Tissue Bank has taken the initiatives to adopt a system that enables the traceability of tissues between the donor, the processed tissue and the recipient based on other international standards for tissue banks. The traceability trail has been effective and the bank is certified compliance to the international standard ISO 9001:2008.     

Uromycladium tepperianum, the gall rust fungus from Falcataria moluccana in Malaysia and Indonesia

Batai (Falcataria moluccana) is a valuable tree species for forest plantations in Malaysia and Indonesia. Since 1993, a gall rust disease has caused severe damage to all growth stages, from seedlings in the nursery to mature trees in the field. To identify the fungus causing gall rust disease on F. moluccana in Malaysia and Indonesia, study of the mode of infection and changes in the anatomy of infected cells were carried out in the anatomy laboratory. The disease in Malaysia and Indonesia is caused by Uromycladium tepperianum. The fungus produces three longitudinally ridged teliospores on each head, with spores measuring 13–20 μm wide and 17–28 μm long. The fungus is microcyclic, completing its entire life cycle on F. moluccana. This study confirmed that the teliospores themselves cannot infect the host. Under favorable conditions, about 10 h after inoculation, teliospores germinate to produce basidiospores that form penetration pegs about 6 h later, and it is this peg which penetrates the host cells directly through the epidermis. Pycnia, recognized as small brown pustules, break through the epidermis about 7 days after inoculation.