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. 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 do 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.
ii Table Of Content. iii List Of Abbreviations. v List Of Tables. vi List Of Figures.
Objectives of study. Classification of proteases. Application of proteases in industries. Sources of proteases.
General characteristics of Aspergillus oryzae. Use of Aspergillus oryzae. Enzyme production of A. MATERIAL AND METHOD.
Fungal media and buffers. Isolation of Aspergillus oryzae from natural substrates. Primary identification of Aspergillus oryzae. Determination of protease activity by well diffusion and enzymatic assay.
Effect of pH on activity and stability of protease. Effect of NaCl concentrations on activity and stability of protease. Identification of Aspergillus oryzae by molecular biological method. RESULTS AND DISCUSSION.
Isolation and primary identification of Aspergillus oryzae. Isolation of Aspergillus oryzae from the natural sources. Primary identification of the isolated fungal isolates. Determination of protease activity produced from isolated A.
Determination of protease activity produced from the isolates………. Growth rate of the fungi on the different media. Effect of Sodium chloride (NaCl) on protease activity and stability. Effect of NaCl on protease activity.
Effect of salt on protease stability. Effect of pH on the protease activity and stability. Effect of pH on the protease activity. Effect of pH on the protease stability.
Identification of the fungi by molecular biological method. DNA extraction and PCR. CONCLUSION AND RECOMMENDATION. 42 iv LIST OF ABBREVIATIONS Acronym Abbreviations A.flavus Aspergillus flavus A.
oryzae Aspergillus oryzae A. sojae Aspergillus sojae A. nomius Aspergillus nomius A. parasiticus 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.
Characteristics of types of proteases. Characteristics of the collected samples. The enzymatic assay procedure of protease. Natural sources of Aspergillus oryzae and isolation results.
Morphological characteristics of four isolates on PDA. Diameter of clear zones of protease produced from isolates. Diameter (mm) of colony on PDA and CYA. 32 vi LIST OF FIGURES Figure 2.
Crystal structure of protease from Aspergillus oryzae. Aspergillus oryzae morphology. Conidial head of A. Conidial head of A.
The hyphae on the surface of soybeans (Hung Yen) and rices (Nam Dinh). Aspergillus oryzae from Institute of Microbiology and Biotechnology. Morphological characterization of strain TB1. Aspergillus oryzae in 4-day PD broth culture.
The clear distinct zones of proteases on the casein agar plates flooded with BCG reagent after 3 day incubation. The growth rate of fungus TB1 on CYA and PDA after 2 days and 5 days. Effect of NaCl concentrations on protease activity of two isolates TB1 and G2. The NaCl tolerance of the protease from TB1and G2 at 16% NaCl.
Effect of pH on protease activity from TB1 and G2. 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. 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 49.8%, respectively, of initial activity after 9 hours.
The optimum pH for activity of the extracellular protease of both isolates TB1 and G2 were shown to be 7. 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. 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. 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. It has been estimated that microbial proteases represent approximately 40% of the total worldwide enzyme sales (Rao et al.
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. 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. 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. This fungus is also a potential source of proteases due to their high 1 proteolytic activity, broad biochemical diversity, their susceptibility to genetic manipulation, high productivity, and being extracellular and are easily recoverable from the fermentation medium (de Castro and Sato 2014).
Many studies have characterized the proteases from A.oryzae and disclosed their role in food processing technology. However, there have been only a few reports on the mechanism of the protease stability under high concentration of sodium chloride. In order to enhance the performance of the enzyme in shortening the production cycle and conversion rate of raw materials, studies on the protease properties under high concentration of sodium chloride are necessary. Besides morphological and physical chemical characteristics, identification of the accurate A.
oryzae by methods of 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" is conducted. OBJECTIVES OF STUDY General objective The aim of this study is to isolate, select and identify the Aspergillus oryzae producing high salt tolerant neutral protease from some Vietnam natural sources. Specific objectives - Isolate A.
oryzae from some Vietnam natural sources and primarily identify by morphological method; - Select strains producing protease by well diffusion and enzymatic assay; - Determine the high salt tolerance of protease activity and stability; - Determine the effect of pH on protease activity and stability; - Identify isolated strains using molecular biology method. Enzyme protease Protease or peptidase is one of member in hydrolysis enzyme group that is capable of cutting the peptide link of polypeptide molecules, proteins and some other similar substrates into free amino acids and low molecular peptides. Crystal structure of protease from Aspergillus oryzae (Kamitori, et al., 2003) The characters of this enzyme are common with respect to optimum pH, temperature and stability. The biochemical characterization showed that the enzyme was most active over the pH range 5.5 and was stable from pH 4.
The optimum temperature range for activity was 55–60°C, and the enzyme was stable at temperatures below 45°C (Vishwanatha, 2009). Majority of these enzymes show low thermostability and lose their activities and structure at high temperature (Rao et at. In the body, proteins in food are digested in the digestive tract by protein- degrading enzymes, first pepsin in gastric juice and then secretions in the pancreas and from mucosal cells, intestine. Amino acids are absorbed into the liver and then involved in the metabolism.
Protein hydrolysis plays an important role in the production of many foods. This process can be accomplished by the protease of the food itself or the microbial protease introduced into the food processing process. 3 Protease is one of the most important commercial enzymes, and is used in food processing, detergents, diary industry and leather making. Proteases occur widely in plants and animals, but commercial proteases are produced exclusively from microorganism.
Molds of the genera Aspergillus, Penicillium and Rhizopusare especially useful for producing proteases, as several species of these genera are generally regarded as safe (Chutmanop, Chuichulcherm et al. Classification of proteases As reported by Pushpam, proteases are classified into six types based on the functional groups in their active sites. They are aspartic, cysteine, glutamic, metallo, serine, and threonine proteases. They are also classified as exo- peptidases and endo-peptidases, based on the position of the peptide bond cleavage.
Proteases are also classified as acidic, neutral or alkaline proteases based on their pH optima. Exopeptidases: The exopeptidases act only near the end of polypeptide chains. Based on their site of action at the N or C terminus, they are classified as aminopeptidases and carboxypeptidases, respectively (Barrett, 1994). The former act at a free N terminus of the polypeptide chain and liberate a single amino acid residue, a dipeptide, or a tripeptide while the later act at C terminals of the polypeptide chain and liberate a single amino acid or a dipeptide.
Endopeptidases: The peculiar characteristics of endopeptidases are by their preferential action at the peptide bonds in the inner regions of the polypeptide chain away from the N and C termini.