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Production of Chitosan from Oyster Mushroom for α-amylase Immobilization

Md Tanvir Kabir
Department of Botany, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Md Shahinur Kabir
Department of Botany, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Md Kamruzzaman Pramanik
Institute of Food and Radiation Biology, Bangladesh Atomic Energy Research Establishment, Savar, Dhaka, Bangladesh

Md Zahidul Islam
Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh

Effat Jahan Tamanna
Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh

Abstract/ Executive Summary:

Experiments were carried out to produce chitosan from a locally available mushroom and to use the produced chitosan as a matrix to immobilize α-amylase. On the basis of morphological characteristics and the sequence of the internal transcribed spacer region of the nuclear rDNA, the fungal sample was identified as Pleurotus ostreatus. The average crude chitin content in the dried fruit body of P. ostreatus was 24.11%. The average yield of chitosan from P. ostreatus was 163.3 mg/g dry weight and the degree of deacetylation of the produced chitosan was 73.42%. This chitosan was used as a matrix to immobilize α-amylase. The diameter of the α-amylase immobilized beads ranged from 1839 - 2273 μm. The amount of reducing sugar produced from starch by using free α-amylase and chitosan-immobilized α-amylase was 1.710 and 1.508 mg/ml, respectively. Immobilized enzyme produced only 11.81% less reducing sugar than that of the soluble enzyme in the first cycle. However, immobilized α-amylase was easily recovered from the product and reused for two more cycles which was not possible with the same soluble free enzyme. Considering the total production of reducing sugar in three cycles, chitosan-immobilized α-amylase was found to be more productive and cost-effective than conventional soluble enzymatic reaction.

Keywords: α-amylase, Chitosan, Enzyme, Immobilization, Mushroom

Location: The study was conducted in the laboratory of Microbiology and Industrial Irradiation Division, Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Savar, Dhaka and in the Department of Botany, Jahangirnagar University (JU), Department of Biotechnology and Genetic Engineering, JU, Bangladesh.

Start Date:

End Date:

Program Area: Postharvest and Agro-processing

Commodity: Mushroom

Objectives:

The present study was done to produce chitosan from a locally available oyster mushroom and to use it as a matrix for immobilization of α-amylase.

Methodology:

For extraction of chitin and then production of chitosan, mushroom sample was collected from Savar, Dhaka, Bangladesh (Fig. 1). The enzyme α-amylase (1,4 α-D-glucan glucanohydrolase, EC 3.2.1.1) was purchased from Sigma-Aldrich. The study was conducted in the laboratory of Microbiology and Industrial Irradiation Division, Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Savar, Dhaka and in the Department of Botany, Jahangirnagar University (JU), Department of Biotechnology and Genetic Engineering, JU, Bangladesh The preliminary identification of the sample was done based on the shape, diameter, margin, texture and color of the pileus, gill attachment to stipe, gill spacing, stipe length, stipe diameter, stipe color and stipe texture. To confirm the identity of the fungal sample, molecular identification based on DNA sequence of the internal transcribed spacer (ITS) region of the nuclear rDNA was done. Nuclear DNA was extracted from the fruit body of the mushroom by using Maxwell DNA Kit (Promega, USA) according to the manufacturer’s instruction. The concentration of the purified DNA was measured by using Nano Drop Spectrophotometer (Model: ND2000, Thermo Scientific, USA). The internal transcribed spacer (ITS) region of the nuclear rDNA was amplified by polymerase chain reaction (PCR). The primer ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) and ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) was used as described by White et al. (1990). After PCR reaction, agarose gel electrophoresis was done to resolve the PCR product and determine its size. Electrophoresis was carried out at 100 V for 30 min. The gel was then viewed under a gel documentation system and photographed. The PCR product was purified and the DNA sequencing was done at 1st base Laboratory, Malaysia. After sequencing the resulted nucleotide sequences were edited and compared using Basic Local Alignment Search tool (BLAST) of National Center for Biotechnology Information (NCBI). The obtained DNA sequence of this study (Gen Bank accession No. MT672600) was compared with the ITS sequence of related species by ClustalW using MEGA 7.0 (Kumar et al. 2016) and a phylogenetic tree was constructed based on Tamura and Nei (1993). At first chitin was extracted from the mushroom and then converted to chitosan. For production of chitosan the methods described by Johney et al. (2017) and Yang et al. (2017) were followed with some modifications. The solubility of chitosan was tested at different concentrations of (0.2, 0.5 and 1%) acetic acid at 50°C on a magnetic stirrer. Acid-base titrametric method described by Domard and Rinaudo (1983) with some modifications was used to determine the degree of deacetylation (DD) of chitosan. For testing the potentiality of using the produced chitosan as a matrix to immobilize enzyme, a widely used enzyme α-amylase was used considering its industrial application, cost and availability. The beads were formed by dipping the α-amylase, chitosan and sodium alginate solution to 0.2 M CaCl2 solution using a syringe. The beads were kept into CaCl2 solution for 30 minutes. Then the beads were separated by using sieve and washed twice with distilled water and kept at 4°C for further use. Twenty (20) enzyme-immobilized beads were randomly selected for determination of their size. The diameter of the beads (μm) was measured under a microscope (Micros, Austria), integrated with the Olympus CellSens Entry software. The enzymatic activity of the free and immobilized α-amylase was measured by using starch as a substrate. In brief, 5 ml of 1% starch solution was added to the α-amylase immobilized chitosan beads. In another tube 5 ml of 1% starch solution was added to 1 ml distilled water instead of the enzyme and it served as a negative control. For positive control, 1 ml of 30 U α-amylase was added to a tube containing 5 ml of 1% starch solution. The tubes were incubated at 37°C for 30 minutes. Two ml solution from each sample was taken into fresh test tube. Freshly prepared 2 ml dinitrosalisylic acid (DNS) reagent and 1 ml Rochelle salt solution were added to the tubes. The optical density of the solution was measured at 575 nm by using a UV-VIS spectrophotometer. The reusability of the immobilized α-amylase was determined by repeated batch experiments. At the end of each cycle the immobilized enzyme was removed, washed with distilled water and the reaction medium was changed with fresh substrate solution. The assay was carried out for 3 cycles under standard assay conditions.

Source of Information: Bangladesh J. Bot. 49(3): 593-599, 2020 (September)

Article Link (Online Journal): DOI: https://doi.org/10.3329/bjb.v49i3.49989

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

it can be concluded that the locally available Pleurotus ostreatus is a good source of extracting chitin and then producing chitosan. Furthermore, the chitosan produced from the chitin of P. ostreatus can be used in immobilization of α-amylase. The immobilized enzyme was usable for 3 cycles instead of one time use of soluble free enzyme. The cumulative total production of reducing sugar from starch by using the immobilized α-amylase was 238% more than the reducing sugar produced by free α-amylase. Thus, immobilization of αamylase using chitosan may be useful in industrial sector. However, further research to optimize the conditions will be required before such endeavor.

Knowledge Management: Journal