Isolation, selection and identification of aspergillus oryzae producing high salt tolerant neutral protease

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE VU THI LAN ISOLATION, SELECTION AND IDENTIFICATION OF ASPERGILLUS ORYZAE PRODUCING HIGH SALT TOLERANT NEUTRAL PROTEASE Major: Food technology Code: 24.18.05.54 Supervisor: Dr Nguyen Hoang Anh AGRICULTURAL UNIVERSITY PRESS - 2017 DECLARATION I hereby declare that the thesis entitled “Isolation, selection and identification of Aspergillus oryzae producing high salt tolerant neutral protease” is the result of the research work carried out by me under the guidance of Dr Nguyen Hoang Anh in the Central Laboratory of Food Science and Technology, the faculty of Food Science and Technology, Vietnam National University of Agriculture I certify that the work presented in this thesis has not been submitted to any other universities Any help received in preparing this thesis and all sources used have been specifically acknowledged Hanoi, May 10th, 2017 Master candidate Vu Thi Lan i ACKNOWLEDGEMENT I would like to express my deep gratitude and appreciation to my supervisor, Dr Nguyen Hoang Anh, Vice Dean as well as Head of Central Laboratory of the faculty of Food Science and Technology whose encouragement and guidance supported me to this thesis His patience, motivation, enthusiasm, and immense knowledge helped me during the time of my research and thesis writing I am grateful to Research and Teaching Higher Education Academy-Committee on Development Cooperation (ARES-CDD) for generous financial support for the course work and research work I sincerely thank all the teachers in the Department of Food Safety and Quality management, Faculty of Food Science and Technology, who gave me many valuable suggestions and ideas for my thesis Finally, I would like to acknowledge my family and friends for their love and encouragement during the completion of the thesis Hanoi, May 10th, 2017 Master candidate Vu Thi Lan ii TABLE OF CONTENT Declaration i Acknowledgement ii Table Of Content iii List Of Abbreviations v List Of Tables vi List Of Figures vii PART I INTRODUCTION 1.1 Introduction 1.2 Objectives of study PART II LITERATURE REVIEW 2.1 Enzyme protease 2.1.1 2.1.2 2.1.3 2.1.4 2.2 2.2.1 2.2.2 2.2.3 Enzyme protease Classification of proteases Application of proteases in industries Sources of proteases Aspergillus group General characteristics of Aspergillus oryzae 10 Use of Aspergillus oryzae 13 Enzyme production of A oryzae 14 PART III MATERIAL AND METHOD 16 3.1 Material 16 3.1.1 3.1.2 3.1.3 Sample collection 16 Reference fungi 18 Fungal media and buffers 18 3.2 3.2.1 Methods 20 Isolation of Aspergillus oryzae from natural substrates 20 3.2.2 3.2.3 Primary identification of Aspergillus oryzae 21 Determination of protease activity by well diffusion and enzymatic assay 21 Effect of pH on activity and stability of protease 23 3.2.3 iii 3.2.4 3.2.5 Effect of NaCl concentrations on activity and stability of protease 23 Identification of Aspergillus oryzae by molecular biological method 23 PART IV RESULTS AND DISCUSSION 25 4.1 Isolation and primary identification of Aspergillus oryzae 25 4.1.1 Isolation of Aspergillus oryzae from the natural sources 25 4.1.2 4.2 Primary identification of the isolated fungal isolates 27 Determination of protease activity produced from isolated A oryzae 29 4.2.1 Determination of protease activity produced from the isolates……… 30 Growth rate of the fungi on the different media 32 4.2.2 4.3 Effect of Sodium chloride (NaCl) on protease activity and stability 33 4.3.1 4.3.2 4.4 4.4.1 4.4.2 4.5 4.5.1 4.5.2 Effect of NaCl on protease activity 33 Effect of salt on protease stability 35 Effect of pH on the protease activity and stability 36 Effect of pH on the protease activity 36 Effect of pH on the protease stability 37 Identification of the fungi by molecular biological method 38 DNA extraction and PCR 38 BLAST search 39 PART V CONCLUSION AND RECOMMENDATION 41 5.1 Conclusion 41 5.2 Recommendation 41 REFERENCES 42 iv LIST OF ABBREVIATIONS Acronym Abbreviations A.flavus Aspergillus flavus A oryzae Aspergillus oryzae A sojae Aspergillus sojae A nomius A parasiticus Aspergillus nomius Aspergillus parasiticus ITS Internal Transcribed Spacer PCR Polymerase Chain Reaction BLAST Basic Local Alignment Search Tool bp Base pair v LIST OF TABLES Table 2.1 Characteristics of types of proteases Table 3.1 Characteristics of the collected samples 16 Table 3.2 The enzymatic assay procedure of protease 22 Table 4.1 Natural sources of Aspergillus oryzae and isolation results 25 Table 4.2 Morphological characteristics of four isolates on PDA 28 Table 4.3 Diameter of clear zones of protease produced from isolates 30 Table 4.4 Diameter (mm) of colony on PDA and CYA 32 vi LIST OF FIGURES Figure 2.1 Crystal structure of protease from Aspergillus oryzae Figure 2.2 Aspergillus oryzae morphology 11 Figure 2.3 Conidial head of A oryzae 12 Figure 2.4 Conidial head of A flavus 12 Figure 3.1 The hyphae on the surface of soybeans (Hung Yen) and rices (Nam Dinh) 16 Figure 3.2 Aspergillus oryzae from Institute of Microbiology and Biotechnology 18 Figure 4.1 Morphological characterization of strain TB1 29 Figure 4.2 Aspergillus oryzae in 4-day PD broth culture 30 Figure 4.3 The clear distinct zones of proteases on the casein agar plates flooded with BCG reagent after day incubation 31 Figure 4.4 The growth rate of fungus TB1 on CYA and PDA after days and days 33 Figure 4.5 Effect of NaCl concentrations on protease activity of two isolates TB1 and G2 34 Figure 4.6 The NaCl tolerance of the protease from TB1and G2 at 16% NaCl 35 Figure 4.7 Effect of pH on protease activity from TB1 and G2 37 Figure 4.8 The pH stability of protease produced from A.oryzae TB1 and G2 38 vii THESIS ABSTRACT Master candidate: Vu Thi Lan Thesis title: Isolation, selection and identification of Aspergilus oryzae producing high salt tolerance neutral protease Major: Food technology Code: 24180554 Education organization: Vietnam National University of Agriculture (VNUA) This study was to isolate, select and identify Aspergillus oryzae producing high salt tolerant neutral protease Four isolates (TB1, TB2, G2 and M1) in 12 isolates were primarily assumed to be A oryzae by morphological characterization TB1 and G2 revealed the highest protease activity with 49.26 u/l and 29.10 u/l, respectively The protease was labile in the sodium chloride solution alternated from 0% to 20% The protease activity of TB1 and G2 behaved high salt tolerance in 16% NaCl and retained 49.2% and 34.8%, respectively, of initial activity after hours The optimum pH for activity of the extracellular protease of both isolates TB1 and G2 were shown to be 7.0 The protease was more stable in the neutral condition than in acid or alkaline environments After incubation at 37oC for 12 hours at pH 7.0, the enzyme activity left were detected only 37% for TB1 and 41% for G2 TB1 was determined to be Aspergillus oryzae by the molecular method Key words: Aspergillus oryzae (A.oryzae), protease, salt tolerance viii PART I INTRODUCTION 1.1 INTRODUCTION Proteases are multifunctional enzymes and represent a fundamental group of enzymes due to diversity of their physiological roles and biotechnological applications (Silva et al., 2011) These enzymes are extremely important in the pharmaceutical, medical, food, and biotechnology industries, accounting for nearly 60% of the whole enzyme market (Ramakrishna, Rajasekhar et al., 2010) It has been estimated that microbial proteases represent approximately 40% of the total worldwide enzyme sales (Rao et al., 1998) Proteases are ubiquitous but to get high salt tolerant neutral proteases is still receiving considerable attention Proteases can be classified into three types based on their optimum functional pH Neutral protease is more important for food industry because it can hydrolyze the proteins of the raw materials thoroughly and reduce the bitterness It is mainly used in the industry of food fermentation, brewing and feed additives etc In addition, some kinds of food are unique due to its high concentration of sodium chloride The higher sodium chloride content provided a lower degree of protein degradation The salt stable proteases are used in fermented food production, antifouling coating preparation and waste treatment, especially at marine habitat (Gao et al., 2016) The protease activity and stability decreased sharply when the materials is mixed with sodium chloride at high concentration, which is used for inhibiting spoilage bacteria, selectively retaining the slow growth of osmotolerant yeast and lactic acid bacteria as well as prolonging the preservation time Consequently, a protease which could tolerate high concentration of sodium chloride is important in order to improve food quality, to shorten the time for the maturation process and to improve the efficiency of raw material utilization (Wang et al., 2013) Since proteases are physiologically necessary for living organisms, being found in a wide diversity of sources such as plants, animals, but commercial proteases are produced exclusively from microorganisms Fungi of the genera Aspergillus, Penicillium and Rhizopus are especially useful for producing proteases, as several species of these genera are generally regarded as safe, of which, Aspergillus oryzae (A.oryzae) is mentioned (Chutmanop, Chuichulcherm et al 2008) This fungus is also a potential source of proteases due to their high 700bp~ Figure 4.9 Gel electrophoresis of the total DNA extracted from the fungal biomass Figure 4.10 Amplification profile of two fungi TB1 and G2, showing 700bp fragment Lane M, DNA ladder (100bp DNA ladder, New England Biolabs) 4.5.2 BLAST search The ITS sequence of the isolates TB1 and G2 was searched for the homologous sequences in the database using the BLAST program The BLAST searches evidenced that the isolate TB1 was most closely related to an Aspergillus oryzae originated from a wide range of sources throughout the worlds with the query coverage of 97-98% and the max nucleotide identities 99% (table 4.5) and unfortunately, G2 was Aspergillus flavus Few samples of the most closely isolates were isolated in Korea and China Table 4.5 Blast search using complete ITS sequence of the fungi No GenBank fungi Aspergillus oryzae ,strain AS-A9 Aspergillus oryzae, strain QRF399 Aspergillus oryzae, strain BS-A2 Aspergillus oryzae, strain GS-A3 Aspergillus oryzae, strain SJ-A4 Host, country Nuruk, Korea Galapogas finches, China Nuruk, Korea Nuruk, Korea Nuruk, Korea 39 Accession Query cover (%) Identity (%) HQ285528.1 97 99 KP278179.1 98 99 HQ285529.1 97 99 HQ285557.1 97 99 HQ285576.1 97 99 Based on analysis of the ITS sequence, we conclude that the fungus TB1 was Aspergillus oryzae, whereas G2 was Aspergillus flavus As mentioned in the Literature review part, Aspergillus oryzae has a world-wide distribution and is regarded to be safe in many industries (Chancharoonpong, Hsieh et al., 2012) 40 PART V CONCLUSION AND RECOMMENDATION 5.1 CONCLUSION o Four strains TB1, TB2, G2, M1 were assumed to be Aspergillus oryzae by morphological identification o TB1 and G2 revealed the larger clear zones and higher protease activity than TB2 and M1 o TB1 and G2 showed the decrease of protease activity from 0% to 20% of NaCl concentration The salt tolerance of protease from two strains TB1 and G2 was conducted by incubation with 16% NaCl for hours o pH 7.0 was the optimal condition for protease activity of both TB1 and G2 The pH stability was conducted during 12 hours with a decrease of residual activity less than 50% compared to the initial point t = o TB1 was identified to be Aspergilus oryzae by the ITS sequencing, whereas G2 was Aspergillus flavus 5.2 RECOMMENDATION Due to the limited experiment time, we have some recommendations for the further study as follows: o Determining some effects on protease such as temperature, metal ions o Identifying and characterizing the pH and salt effect on protease of the isolate TB2 which is being considered 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Journal of Food Processing2014 75 Doyle, Jeff J (1987) "A rapid DNA isolation procedure for small quantities of fresh leaf tissue" Phytochem bull 19: 11-15 76 Fayyad A (2008) "Isolation of Aspergillus oryzae and New Aroma Production", the Islamic University-Gaza 77 Fernandes, L Fernandes, Lia Zimmermann, Narjara Leite, Clarice Loguercio (2010) "Enzyme screening of dikaryotic cultures from lignocellulolitic Basidiomycetes (fungi) collected in southern Brazil doi: 10.5007/2178-4574.2008 v37p07." 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Journal of food science and technology51(6): 1185-1190 52 ANNEX The standard curve of protease activity Tyrosin solution (ml) 0.05 Tyrosine µM Abs (660nm) 0.055 0.08 0.1 0.111 0.173 0.2 0.221 0.359 0.4 0.442 0.7 0.5 0.553 0.86 The ITS of two isolates TB1 and G2 >TB1-615 Nu- Aspergillus oryzae AATGATCGTTCGTAGGGGTGACCTGCGGAAGGATCATTACCGAGTGTAGGGTTCCTAGC GAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTC ATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTG TCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGG ATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTG CAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGG GGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTC GTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCG AGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAAT CAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATAT CAATAAGCCGGAGGAAAATCATTA >G2-541 Nu-Aspergillus flavus CTAGCGAGCCCACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCC ATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAAC TCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACA ATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGA ATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCC GGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGT CGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATC CTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGC AAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGC ATATCAAAAA 53 ... entitled ? ?Isolation, selection and identification of Aspergillus oryzae producing high salt tolerant neutral protease? ?? is the result of the research work carried out by me under the guidance of Dr... biochemistry and molecular biology is extremely necessary In this context, the study "Isolation, selection and identification of Aspergillus oryzae producing high salt tolerant neutral protease" ... Effect of pH on activity and stability of protease 23 3.2.3 iii 3.2.4 3.2.5 Effect of NaCl concentrations on activity and stability of protease 23 Identification of Aspergillus oryzae