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Fulltext A comparative study of extraction techniques for maximum recovery of glutamate decarboxylase (GAD) from Aspergillus oryzae NSK

Lee Ying Yeng A, Kadir MS, Ghazali HM, Raja Abd Rahman RN, Saari N

BMC Res Notes, 2013 Dec 10;6:526.
PMID: 24321181 DOI: 10.1186/1756-0500-6-526

BACKGROUND: γ-Amino butyric acid (GABA) is a major inhibitory neurotransmitter of the mammalian central nervous system that plays a vital role in regulating vital neurological functions. The enzyme responsible for producing GABA is glutamate decarboxylase (GAD), an intracellular enzyme that both food and pharmaceutical industries are currently using as the major catalyst in trial biotransformation process of GABA. We have successfully isolated a novel strain of Aspergillus oryzae NSK that possesses a relatively high GABA biosynthesizing capability compared to other reported GABA-producing fungal strains, indicating the presence of an active GAD. This finding has prompted us to explore an effective method to recover maximum amount of GAD for further studies on the GAD's biochemical and kinetic properties. The extraction techniques examined were enzymatic lysis, chemical permeabilization, and mechanical disruption. Under the GAD activity assay used, one unit of GAD activity is expressed as 1 μmol of GABA produced per min per ml enzyme extract (U/ml) while the specific activity was expressed as U/mg protein.
RESULTS: Mechanical disruption by sonication, which yielded 1.99 U/mg of GAD, was by far the most effective cell disintegration method compared with the other extraction procedures examined. In contrast, the second most effective method, freeze grinding followed by 10% v/v toluene permeabilization at 25°C for 120 min, yielded only 1.17 U/mg of GAD, which is 170% lower than the sonication method. Optimized enzymatic lysis with 3 mg/ml Yatalase® at 60°C for 30 min was the least effective. It yielded only 0.70 U/mg of GAD. Extraction using sonication was further optimized using a one-variable-at-a-time approach (OVAT). Results obtained show that the yield of GAD increased 176% from 1.99 U/mg to 3.50 U/mg.
CONCLUSION: Of the techniques used to extract GAD from A. oryzae NSK, sonication was found to be the best. Under optimized conditions, about 176% of GAD was recovered compared to recovery under non optimized conditions. The high production level of GAD in this strain offers an opportunity to conduct further studies on GABA production at a larger scale.

Matched MeSH terms: Aspergillus oryzae/enzymology*
Evaluation of commercial soy sauce koji strains of Aspergillus oryzae for γ-aminobutyric acid (GABA) production

Ab Kadir S, Wan-Mohtar WA, Mohammad R, Abdul Halim Lim S, Sabo Mohammed A, Saari N

J Ind Microbiol Biotechnol, 2016 Oct;43(10):1387-95.
PMID: 27541157 DOI: 10.1007/s10295-016-1828-5

In this study, four selected commercial strains of Aspergillus oryzae were collected from soy sauce koji. These A. oryzae strains designated as NSK, NSZ, NSJ and NST shared similar morphological characteristics with the reference strain (A. oryzae FRR 1675) which confirmed them as A. oryzae species. They were further evaluated for their ability to produce γ-aminobutyric acid (GABA) by cultivating the spore suspension in a broth medium containing 0.4 % (w/v) of glutamic acid as a substrate for GABA production. The results showed that these strains were capable of producing GABA; however, the concentrations differed significantly (P

Matched MeSH terms: Aspergillus oryzae/classification; Aspergillus oryzae/metabolism*
Fulltext Ulcerative keratomycosis--case reports on three different species of fungi

Sukumaran K

Med J Malaysia, 1991 Dec;46(4):388-91.
PMID: 1840452

Three clinical cases of fungal corneal ulcers are described to highlight the course, ocular morbidity and principles of treatment. A brief discussion of the diagnosis and management of ulcerative keratomycosis is presented.

Matched MeSH terms: Aspergillus oryzae*
Production of 3-Oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic Acid in the Fungus Aspergillus oryzae: A Step Towards Heterologous Production of Pyrethrins in Fungi

Mohamed ME, Pahirulzaman KA, Lazarus CM

Mol Biotechnol, 2016 Mar;58(3):172-8.
PMID: 26718544 DOI: 10.1007/s12033-015-9911-0

Pyrethrins are natural insecticides, which accumulate to high concentrations in pyrethrum (Chrysanthemum cinerariaefolium) flowers. Synthetic pyrethroids are more stable, more efficacious and cheaper, but contemporary requirements for safe and environmentally friendly pesticides encourage a return to the use of natural pyrethrins, and this would be favoured by development of an efficient route to their production by microbial fermentation. The biosynthesis of pyrethrins involves ester linkage between an acid moiety (chrysanthemoyl or pyrethroyl, synthesised via the mevalonic acid pathway from glucose), and an alcohol (pyrethrolone). Pyrethrolone is generated from 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid, which originates from α-linolenic acid via the jasmonic acid biosynthetic cascade. The first four genes in this cascade, encoding lipoxygenase 2, allene-oxide synthase, allene-oxide cyclase 2 and 12-oxophytodienoic acid reductase 3, were amplified from an Arabidopsis thaliana cDNA library, cloned in a purpose-built fungal multigene expression vector and expressed in Aspergillus oryzae. HPLC-MS analysis of the transgenic fungus homogenate gave good evidence for the presence of 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid.

Matched MeSH terms: Aspergillus oryzae/genetics*; Aspergillus oryzae/metabolism
Fulltext Evaluation of a Malaysian soy sauce koji strain Aspergillus oryzae NSK for γ-aminobutyric acid (GABA) production using different native sugars

Hajar-Azhari S, Wan-Mohtar WAAQI, Ab Kadir S, Rahim MHA, Saari N

Food Sci Biotechnol, 2018 Apr;27(2):479-488.
PMID: 30263772 DOI: 10.1007/s10068-017-0289-6

In this study, a selected γ-aminobutyric acid (GABA)-rich Malaysian strain Aspergillus oryzae NSK was collected from soy sauce koji. The strain was used to explore the effect of using renewable native sugar syrup, sugarcane, nipa, and molasses as fermentable substrates for developing a novel functional GABA soy sauce. We evaluated the strain using the chosen native sugars for 7 days using shake flask fermentation at 30 °C. The results showed optimum GABA concentration was achieved using cane molasses as the fermentable substrate (354.08 mg/L), followed by sugarcane syrup (320.7 mg/L) and nipa syrup (232.07 mg/L). Cane molasses was subsequently utilized as a substrate to determine the most suitable concentration for A. oryzae NSK to enhance GABA production and was determined as 50% g/L of glucose standard cane molasses. Our findings indicate that cane molasses can be used as a GABA-rich ingredient to develop a new starter culture for A. oryzae NSK soy sauce production.

Matched MeSH terms: Aspergillus oryzae
Fulltext Vital parameters for high gamma-aminobutyric acid (GABA) production by an industrial soy sauce koji Aspergillus oryzae NSK in submerged-liquid fermentation

Wan-Mohtar WAAQI, Ab Kadir S, Halim-Lim SA, Ilham Z, Hajar-Azhari S, Saari N

Food Sci Biotechnol, 2019 Dec;28(6):1747-1757.
PMID: 31807347 DOI: 10.1007/s10068-019-00602-y

In submerged-liquid fermentation, seven key parameters were assessed using one-factor-at-a-time to obtain the highest GABA yield using an industrial soy sauce koji Aspergillus oryzae strain NSK (AOSNSK). AOSNSK generated maximum GABA at 30 °C (194 mg/L) and initial pH 5 (231 mg/L), thus was able to utilize sucrose (327 mg/L of GABA) for carbon source. Sucrose at 100 g/L, improved GABA production at 646 mg/L. Single nitrogen sources failed to improve GABA production, however a combination of yeast extract (YE) and glutamic acid (GA) improved GABA at 646.78 mg/L. Carbon-to-nitrogen ratio (C8:N3) produced the highest cell (24.01 g/L) and GABA at a minimal time of 216 h. The key parameters of 30 °C, initial pH 5, 100 g/L of sucrose, combination YE and GA, and C8:N3 generated the highest GABA (3278.31 mg/L) in a koji fermentation. AOSNSK promisingly showed for the development of a new GABA-rich soy sauce.

Matched MeSH terms: Aspergillus oryzae
Fulltext Development of a pyrG mutant of Aspergillus oryzae strain S1 as a host for the production of heterologous proteins

Ling SO, Storms R, Zheng Y, Rodzi MR, Mahadi NM, Illias RM, et al.

ScientificWorldJournal, 2013;2013:634317.
PMID: 24381522 DOI: 10.1155/2013/634317

The ease with which auxotrophic strains and genes that complement them can be manipulated, as well as the stability of auxotrophic selection systems, are amongst the advantages of using auxotrophic markers to produce heterologous proteins. Most auxotrophic markers in Aspergillus oryzae originate from chemical or physical mutagenesis that may yield undesirable mutations along with the mutation of interest. An auxotrophic A. oryzae strain S1 was generated by deleting the orotidine-5'-monophosphate decarboxylase gene (pyrG) by targeted gene replacement. The uridine requirement of the resulting strain GR6 pyrGΔ0 was complemented by plasmids carrying a pyrG gene from either Aspergillus nidulans or A. oryzae. β -Galactosidase expression by strain GR6 pyrGΔ0 transformed with an A. niger plasmid encoding a heterologous β -galactosidase was at least 150 times more than that obtained with the untransformed strain. Targeted gene replacement is thus an efficient way of developing auxotrophic mutants in A. oryzae and the auxotrophic strain GR6 pyrGΔ0 facilitated the production of a heterologous protein in this fungus.

Matched MeSH terms: Aspergillus oryzae/physiology*
Fulltext Functional expression of a novel α-amylase from Antarctic psychrotolerant fungus for baking industry and its magnetic immobilization

He L, Mao Y, Zhang L, Wang H, Alias SA, Gao B, et al.

BMC Biotechnol, 2017 02 28;17(1):22.
PMID: 28245836 DOI: 10.1186/s12896-017-0343-8

BACKGROUND: α-Amylase plays a pivotal role in a broad range of industrial processes. To meet increasing demands of biocatalytic tasks, considerable efforts have been made to isolate enzymes produced by extremophiles. However, the relevant data of α-amylases from cold-adapted fungi are still insufficient. In addition, bread quality presents a particular interest due to its high consummation. Thus developing amylases to improve textural properties could combine health benefits with good sensory properties. Furthermore, iron oxide nanoparticles provide an economical and convenient method for separation of biomacromolecules. In order to maximize the catalytic efficiency of α-amylase and support further applications, a comprehensive characterization of magnetic immobilization of α-amylase is crucial and needed.
RESULTS: A novel α-amylase (AmyA1) containing an open reading frame of 1482 bp was cloned from Antarctic psychrotolerant fungus G. pannorum and then expressed in the newly constructed Aspergillus oryzae system. The purified recombinant AmyA1 was approximate 52 kDa. AmyA1 was optimally active at pH 5.0 and 40 °C, and retained over 20% of maximal activity at 0-20 °C. The K m and V max values toward soluble starch were 2.51 mg/mL and 8.24 × 10-2 mg/(mL min) respectively, with specific activity of 12.8 × 103 U/mg. AmyA1 presented broad substrate specificity, and the main hydrolysis products were glucose, maltose, and maltotetraose. The influence of AmyA1 on the quality of bread was further investigated. The application study shows a 26% increase in specific volume, 14.5% increase in cohesiveness and 14.1% decrease in gumminess in comparison with the control. AmyA1 was immobilized on magnetic nanoparticles and characterized. The immobilized enzyme showed improved thermostability and enhanced pH tolerance under neutral conditions. Also, magnetically immobilized AmyA1 can be easily recovered and reused for maximum utilization.
CONCLUSIONS: A novel α-amylase (AmyA1) from Antarctic psychrotolerant fungus was cloned, heterologous expression in Aspergillus oryzae, and characterized. The detailed report of the enzymatic properties of AmyA1 gives new insights into fungal cold-adapted amylase. Application study showed potential value of AmyA1 in the food and starch fields. In addition, AmyA1 was immobilized on magnetic nanoparticles and characterized. The improved stability and longer service life of AmyA1 could potentially benefit industrial applications.

Matched MeSH terms: Aspergillus oryzae/genetics; Aspergillus oryzae/metabolism
Fatal aspergillosis in imported parrots

Kaplan W, Arnstein P, Ajello L, Chandler F, Watts J, Hicklin M

Mycopathologia, 1975 Jul 30;56(1):25-9.
PMID: 1097931

Spontaneous fatal aspergillosis occurred in several species of parrots imported from Latin America, Australia, Malaya and Ghana for studies on the control of psittacosis. Over a period of 4 years, 655 parrots were imported for use in these studies. All birds that died during these investigations were necropsied, and the internal organs of 45 were found to have macroscopic lesions suggestive of aspergillosis. Of these 45 suspected cases, 32 were confirmed as aspergillosis by both histopathology and culture, and three others by histopathology alone. There was no evidence that the remaining 10 had this disease. Of the 32 culturally confirmed cases, 13 were found to be caused by Aspergillus fumigatus, 16 by A. oryzae, and three by both fungi. In this series, three sets of circumstances appear to have been associated with the development of fatal aspergillosis. Their capture and transport to the United States, the administration of chlortetracycline used in the control of psittacosis, and the administration of cortisone acetate in an attempt to activate existent latent psittacosis infections. The possible causal relationship of these factors are discussed.

Matched MeSH terms: Aspergillus oryzae/isolation & purification