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3. Enhancing Kojic Acid Production in Aspergillus oryzae: Leveraging Crude Cellulase from Achatina fulica for Strain Improvement via Protoplasting and UV Mutagenesis.

Author

Ocha Putri Mulia, Larosa, Febri L. S., Tri Hastuti Septiarini, Ratika Rahmasari, and Suryadi, Herman

Subjects
KOJI, CELLULASE, MUTAGENESIS, AMMONIUM sulfate, ACIDS, ENZYMES
Abstract

This study aims to prove the ability of crude cellulase enzymes from snails for protoplasting Aspergillus oryzae cells and its application for strain improvement with UV mutagenesis. Snail enzyme was obtained from Achatina fulica by dissolving its digestion track and fractionating it with ammonium sulfate. The activity of fractions was measured Spectrophotometrically and used for cell protoplasting for 2 hours, then irradiated with UV for 10, 15, and 20 minutes, respectively, with 5 cm in the distance. Screening of mutants is carried out with 1% FeCl3, and the potential mutant strain was tested for kojic acid production in an aerobic state and determined by Spectrophotometry at 268 nm. The cellulase activity in crude snail enzyme was 11.5807 U/ml and increased to 16.3984 U/ml after fractionation. The best protoplast formation was obtained with a 60% fraction, which showed transparent performance under the microscope. The UV mutagenesis of protoplasts showed that the highest number of potential mutants was obtained from UV treatment for 15 minutes (41.67%). The potential mutants look dark brown (DBC), such as strain 10H3, and produced higher kojic acid concentration than the parent strain. In conclusion, UV mutagenesis of Aspergillus oryzae through protoplasting by crude cellulase of snail enzyme was effective and improved kojic acid concentration. [ABSTRACT FROM AUTHOR]

Published
2024
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11. Development and validation of a UPLC-MS/MS method with volumetric absorptive microsampling to quantitate cyclophosphamide and 4-hydroxycyclophosphamide

Author

Harahap, Yahdiana, Steven, Steven, and Suryadi, Herman

Subjects
Pharmacology, Pharmacology (medical)
Abstract

Cyclophosphamide (CP) is an anti-cancer alkylating prodrug, metabolized by CYP450 into its active metabolite 4-hydroxycyclophosphamide (4-OHCP). Its therapeutic effectiveness is determined by the 4-OHCP concentration. Several analytical methods in plasma and dried blood spots have been developed to analyze cyclophosphamide and 4-OHCP; however, there are many disadvantages. The objective of this study was to develop and validate the ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method by volumetric absorptive microsampling (VAMS) and 4-hydroxycyclophosphamide-d4 (4-OHCP-d4) as an internal standard. VAMS requires small sample volumes, and it is not affected by the hematocrit values; therefore, it is an efficient sampling method. The samples were derivatized with 5 μL semicarbazide hydrochloride (SCZ) and 25 μL of the resulting 4-OHCP-SCZ; 4-OHCP-d4-SCZ derivatives were absorbed by VAMS and extracted by protein precipitation. The optimum conditions were obtained using the Waters Acquity® UPLC BEH C18 (2.1 × 100 mm; 1.7 μm) column; flow rate 0.15 ml/min; mobile phase 0.01% formic acid and methanol; gradient elution mode for 6 min by positive electrospray ionization; and multiple reaction monitoring of m/z 260.7 > 140.0 for CP, 333.7 > 221.0 for 4-OHCP-SCZ, and 337.7 > 225.1 for 4-OHCP-d4-SCZ. The method met the validation requirements set by the FDA. The cyclophosphamide LLOQ value was 5 ng/mL, and the calibration curve range was 5—60,000 ng/ml. Furthermore, the 4-OHCP LLOQ value was 2.5 ng/ml, and the calibration curve range was 2.5—1,000 ng/ml.

Published
2022

34. Methods of Random Mutagenesis of Aspergillus Strain for IncreasingKojic Acid Production

Author

Suryadi, Herman, Irianti, Marina Ika, and Septiarini, Tri Hastuti

Abstract

Abstract: Kojic acid is an organic acid that is commonly used in the pharmaceutical and cosmeticindustries. This acid compound is a secondary metabolite produced by various microorganisms, oneof which is Aspergillus oryzae. Typically, improving the strain can enhance kojic acid production. Amutation is one of the tools to perform strain improvement because the change in kojic acidproducinggenes effectively increases kojic acid yield. A random mutagenesis is a classic approachfor inducing and producing mutants with random mutations. The mutagenesis can be generated bythe individual physical and chemical mutagen, combined physical and chemical mutagens, or initiateby protoplast preparation. Aspergillus strains that are exposed to physical mutagens (e.g., UV) orchemical mutagens (e.g., N-methyl-N-nitro-N-nitrosoguanidine (NTG)) showed their abilities inincreasing kojic acid production. Several new mutation methods, such as Ion Beam Implantation andAtmospheric and room temperature plasma (ARTP), also showed good responses in enhancing theproduction of biological products such as kojic acid. This review compared different random mutagenesismethods of Aspergillus strain with various mutagen types to provide better insight for researchersin choosing the most suitable method to increase kojic acid production.

Published
2022
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47. Development and Validation of Doxorubicin Hydrochloride and Doxorubicinol in Plasma Using Liquid Chromatography-Tandem Mass Spectrometry.

Author

HARAHAP, YAHDIANA, SURYADI, HERMAN, and WINARTI, AGATHA C.

Subjects
*LIQUID chromatography-mass spectrometry, *DOXORUBICIN, *CONGESTIVE heart failure, *GRADIENT elution (Chromatography)
Abstract

BACKGROUND: Doxorubicin is an anthracycline antibiotic glicoside which has antineoplastic activity and is the most administrated chemotherapy agent to medicate solid tumour in adults including lungs, ovarium, and breast cancer1,2,3. Long term use of this chemotherapy agent is limited because the development of progressive dose-dependent cardiomyopathy which develops irreversibly towards congestive heart failure. The cardiotoxic impact of doxorubicin is highly determined by accumulation of its main metabolite, doxorubicinol4. The aim of this study was to obtain a validated analysis method of doxorubicin and doxorubicinol simultaneously in plasma with hexamethylphosphoramide as the internal standard utilizing liquid chromatography-tandem mass spectrometry. METHODS: Sample preparation was performed by protein precipitation using methanol. The separation was performed using UPLC C-18 BEH (2.1 x 100 mm), 1.7 µm column, 45°C temperature column. The mobile phase consisted of 0.1% acetate acid (eluent A) and acetonitrile (eluent B) at 0.15 ml/min with gradient elution. The detection of the mass was performed on Waters Xevo TQD using ESI positive (+) type MRM for doxorubicin, doxorubicinol, and hexamethylphosphoramide with m/z values: 544.22>361.05; 546.22>363.05; and 180.03>135.16, respectively RESULTS: This method is linear in the range concentration of 1-100 ng/mL for doxorubicin with r value = 0.9963; 0.5-50 ng/mL for doxorubicinol with r value = 0.9977. %Diff and %CV of the assay were less than 20% for LLOQ and less than 15% for other concentration. LLOQ for doxorubicin and doxorubicinol 1.0 ng/mL and 0.5 ng/mL respectively. CONCLUSIONS: This method has successfully fulfilled validation requirement refers to EMEA Guidelines 20115 and FDA guidelines 20186. [ABSTRACT FROM AUTHOR]

Published
2020

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