Synthesis of a chitosan-zinc oxide dihybrid matrix for immobilization of lipase enzyme in industry

Abstract

Lipase enzyme from Cyclobacter species c18 was introduced into susceptible BL21 cells via plasmid pet 28 containing lipase enzyme gene and lipase enzyme was induced and expressed with 0.5 mM IPTG concentration. Then, through nickel-agarose purification column, protein was bound to nickel ion through polyhistidine sequence and the quality of enzyme purification and its molecular size were examined by SDS PAGE; and for the first time, lipase enzyme was immobilized on chitosan-zinc oxide substrate by physical adsorption and covalent bonding method. To perform the stabilization process, a chitosan-zinc oxide nanohybrid substrate was synthesized, and with the help of energy dispersive X-ray spectroscopy (EDX), cross-linking between the two nanoparticles was confirmed. The physical and covalent stabilization process of the lipase enzyme was performed on the synthetic substrate. The results of lipase enzyme immobilization showed that the enzyme maintained its catalytic efficiency at about 90 and 96 percent in physical and covalent immobilization, respectively. At the same time, the stability of the enzyme increased. For example, the half-life of the enzyme at a temperature of 35 degrees showed that the half-life increased by 7% in physical immobilization and by 15% in covalent immobilization. At the same time, the delta star also increased in immobilization, which indicates that we must spend more energy to denature the enzyme and cause the loss of enzyme activity. The storage stability of the enzyme in physical immobilization at 25 degrees Celsius after 35 days has increased by 200% compared to the solution conditions, and in covalent immobilization it has increased by 320%. The enzyme has more than 50% activity in physical and covalent immobilization up to the tenth cycle, and in the eighth cycle it maintains more than 80% activity, and this is an important point because it saves money in industrial use. Covalent and physical stabilization was able to resist well against increasing salt concentrations and had a protective effect. For example, KCl salt was able to be active in covalent stabilization by increasing the concentration up to 180%. The enzyme was also able to have a protective role against solvents, detergents and chelators.\n\nDesigning and production of stabilized recombinant lipase enzyme from Psychrobacter sp. C18 on Synthesized nanocomposite chitosan-zinc oxide with high stability for use in industrial applications

Description

Description of the invention Title of the invention Synthesis of chitosan-zinc oxide dihybrid matrix for lipase enzyme stabilization in industry Technical background of the relevant invention This invention relates to the synthesis of a chitosan-zinc oxide nanohybrid support matrix to which the lipase enzyme is physically and covalently attached, and which can provide greater stability, increase the half-life, and recycle the protein for use in industrial applications. Technical problem and stating the objectives of the invention The main weakness of enzymes is their instability, which reduces their half-life when exposed to temperature and pH fluctuations. In addition, enzyme purification and recovery are very expensive processes. Also, the short half-life of enzymes limits their use in industries. When enzymes are used in a soluble form in reactions, their recovery from the reaction mixture is very difficult. Their main problem is that they are sensitive and fragile to harsh environmental conditions, which leads to a reduction in the functional half-life of the enzyme. Enzymes are sensitive, unstable, and usable in aqueous environments, which are non-ideal for a catalyst and undesirable in many syntheses. In many cases, one way to avoid these problems is to immobilize the enzyme; therefore, since enzyme immobilization allows for intermittent processing in industrial scale applications, it is a good way to improve enzyme stability and increase its practical applications.Increasing enzyme stability can lead to a reduction in the amount of enzymes consumed, an increase in the life of enzyme reactors, an increase in the chance of reusing the enzyme, and also to obtaining a stronger signal in the biosensor. The stability of enzymes can be increased by various methods, including: enzyme stabilization, enzyme modification, protein engineering, and engineering the environment surrounding enzymes. Considering the diverse applications of lipase and its important role in biotechnological and industrial processes, including papermaking and oleochemical, it can be used as one of the most widely used enzymes. Considering the aforementioned points about the problems of the enzyme, its application has become relatively difficult; therefore, the stabilization technique can be used to improve lipase performance. Lipases are limited due to low efficiency, poor reproducibility, and inconsistent optimal performance in their native form. Protein engineering with recombinant DNA technology provides a suitable tool to overcome these shortcomings and produce strong enzyme catalysts with high efficiency. In fact, the purpose of selecting and producing lipase enzyme among a wide range of enzymes is due to its widespread use in industry, which is the third commercially important enzyme after amylase and proteases. Microbial lipases have many applications in industries due to their extensive enzymatic properties. The global market value of industrial enzymes reached $5 billion in 2016, with lipase and esterase enzymes accounting for approximately 10% of global industrial enzymes and ranking third after carbohydrases and proteases. For this reason, studies on lipase enzyme were conducted in this research. Lipases are important industrial biocatalysts that have extraordinary applications in the production of a wide range of products. Due to the widespread use of this enzyme and its limited availability for industrial use, this enzyme is sold at exorbitant prices, which is not economically viable because this enzyme alone has a short half-life and can only be used once. It also operates within a specific range of temperature and pH.In fact, in this research, we were able to mass-produce this enzyme cost-effectively as a team at Shahrekord University and overcome some limitations through the stabilization process, such as the short half-life of the enzyme and its single-use nature, etc. To overcome these problems, we used the stabilization process on chitosan-zinc oxide hybrid nanosurfaces, which are cheap, available, and safe, and can even be used in the food industry. A suitable support material (substrate) must be physically stable and resistant to mechanical deformation, inert to the target protein, biologically compatible, resistant to microbial invasion, commercially available, and affordable. The support materials selected in this project have all the characteristics. Chitin and chitosan are increasingly used as supports due to their abundance and availability. Today, the use of chitosan along with magnetic nanoparticles as a combined support has been proposed and has gained great importance for the stabilization of enzymes.The most common inorganic precursors used for support include silica, inorganic oxides such as zinc oxide and titanium, which can be combined with biopolymers such as chitosan, lignin and alginate, which are mainly used for adsorption or covalent immobilization of hydrolases, oxidoreductases and transferases. Organic-inorganic hybrids have great potential as support materials for enzymes because these hybrids have good mechanical stability and strength and very high affinity for biological molecules. The high stability and often chemical inertness are related to the characteristics of the inorganic precursor and the good ability to bind enzymes due to the organic components because synthetic polymers and biopolymers have many functional moieties in their structure that are able to interact with the chemical groups of biocatalysts. Therefore, due to their stability and performance, hybrids of this group can be used for many practical applications. Nanochitosan is an amino-containing polysaccharide that is abundant in nature.Chitin and chitosan are the second most abundant biopolymers after cellulose. As a natural resource, chitosan exhibits unique and interesting properties such as biocompatibility, biodegradability, and non-toxicity. For these reasons, chitosan polymer has important applications in the following areas: food packaging, agriculture, biomedicine, cosmetics, wastewater treatment, enzyme immobilization, and carrier for controlled drug delivery. ZnO is also an environmentally friendly solid ion semiconductor. Zinc oxide is a versatile material with its unique physical and chemical properties, such as high chemical stability, high electrochemical coupling coefficient, increased photostability, and so on. Zinc oxide is used in various industries due to its safety, including medical, pharmaceutical, plastic, paint, etc. For example, among all the prepared particles, zinc oxide nanoparticles with the smallest size (~ 15 nm) prepared by refluxing zinc acetate dihydrate in diethylene glycol for 3 hours show significant antibacterial and antibiofilm activity, which may serve as a potential alternative in biomedical applications.Therefore, a dihybrid nanosubstrate consisting of these two materials was synthesized to stabilize the lipase enzyme, and the immobilization of the lipase enzyme occurred for the first time on a synthetic chitosan-zinc oxide substrate. Through the immobilization process on a chitosan-zinc oxide substrate, enzymes are less sensitive to their environment due to their stability. Processes that use insoluble enzymes suspended in hydrophobic aqueous media require immobilization to optimize enzyme dispersion to improve substrate accessibility and prevent aggregation of hydrophilic protein particles. In addition, immobilization on a chitosan-zinc oxide substrate allows for recycling. Enzyme immobilization on an insoluble solid should be simple and cost-effective. Enzyme immobilization on a chitosan-zinc oxide substrate facilitates enzyme utilization; however, it should be studied separately for each case. The immobilization of an enzyme requires the interaction of two species: the enzyme and the carrier, and therefore the surface properties of both are important. In the case of enzymes, polar groups (such as amino groups on lysine or acidic groups on glutamic acid), nonpolar surface regions, or sugar moieties can affect the surface properties. The carrier can be tailored to match any of these enzyme surface properties.For any carrier, it is essential to have a high surface area, which can be achieved by small particle size, although this makes separation difficult, or by highly porous materials with pores of sufficiently large dimensions that do not restrict the penetration of substrates. In addition, the materials must be chemically and mechanically stable. In the method of encapsulating the enzyme within the carrier, the pores must be of an appropriate size to allow unhindered penetration of the substrate and to ensure that the enzyme is trapped inside. The binding of the enzyme to the carrier can be enhanced by covalent bonding. Reactivation of the functional groups can be achieved by choosing the appropriate organic monomer on the polymeric carrier matrix. In the case of silicates, surface derivatives can be obtained by modifying the surface hydroxy groups with a suitable reagent.In extreme cases of binding, no carrier is used and the enzyme molecules are bound to each other and form the carrier itself. In the process of immobilizing the enzyme on a chitosan-zinc oxide substrate, the enzyme molecules are bound to a high-surface-area structure by physical adsorption or covalent bonds or are encapsulated in gels or microcapsule structures. In particular, the multi-point binding between the enzyme and the material or surface used for immobilization prevents the unfolding of the complex structure of the protein molecule and, as a result, increases stability. Cross-linked enzyme crystals and cross-linked enzyme aggregates are based on multi-point linkages of enzyme molecules. A stable enzyme is an enzyme bound to an inert, insoluble material. This can provide greater resistance to changes in conditions such as changes in pH or temperature. It also allows the enzymes to remain in a fixed location during the reaction and then they can be easily separated from the products and may be reused.Enzymes immobilized on the chitosan-zinc oxide surface are dispersed and no longer aggregate, preventing inactivation phenomena, while free enzyme may aggregate, which greatly reduces enzyme activity. Also, enzyme immobilization may result in the preservation of enzyme properties under extreme conditions where the enzyme tends to distort and consequently its activity decreases. Furthermore, immobilization of enzymes on a porous support may in many cases have a positive effect on the observed enzyme behavior that is not actually related to structural changes. The immobilized enzyme is bound to the chitosan-zinc oxide substrate and cannot move freely in solution. The most prominent advantage of enzyme immobilization technology is the increased thermal stability of the enzyme, which allows for the reuse of a specific enzyme multiple times as well as easy separation of the enzyme from the reaction mixture, which occurred in the physical immobilization of the enzyme, and the thermal stability for the lipase enzyme was increased and the enzyme was recycled up to 14 times. When the enzyme is attached to the support material by ionic interactions, hydrophobic forces, or disulfide bonds, the resulting immobilization is reversible; since the enzyme and the support material can be separated under certain conditions. When the enzyme and the support material are linked by covalent bonds, entrapment, entrapment, crosslinking, or network formation, the immobilization is irreversible. This means that the structure of the immobilized enzyme can be modified by binding to a support material.One of the most practical examples of covalent attachment is the cross-linking of enzymes to solid supports using glutaraldehyde, which often results in reduced enzyme activity. Structural changes in proteins during immobilization and side reactions involving amino acid residues exposed to the solvent lead to loss of enzyme function. A description of the state of the prior art and the history of developments related to the claimed invention. In various papers, hydrophobic sol-gel, polyvinyl chloride, chitosan, chitin, agarose, sepharose and trisacryl, mesoporous silica, microporous polymer matrix have been used for lipase immobilization, which shows that immobilized enzymes show higher activity than free enzymes. According to Roger, the advantages of using immobilized enzymes include easier handling, easy separation, more efficient recovery and reuse, continuous operation capability of the fixed substrate, and often increased stability; therefore, due to their larger specific surface area, lower diffusion limitation, nanomaterials (NMs) have been used as new and promising supports for lipase immobilization. Nanoparticles of inorganic and organic origin have attracted much attention as they significantly improve the immobilization efficiency and the efficiency of biocatalytic systems. These nanoparticles provide a large surface area for enzyme binding, which leads to a higher enzyme loading on the matrix surface and an increase in immobilization efficiency; therefore, the greatest advantage of nanoparticles over other inorganic materials is their ability to minimize diffusion limitations. Enzyme molecules are attached to the surface of non-porous particles and their active sites are in extensive contact with the substrates, which means that nanoparticle-based biocatalytic systems usually maintain high catalytic activity. Various inorganic nanomaterials, such as nano-gold and graphene, can be used as matrices for enzyme immobilization. However, inorganic oxide nanoparticles are most often used.In one study, lipase from Rhizomucor miehei was covalently immobilized on silica nanoparticles modified by octyltriethoxysilane and glycidoxypropyltrimethoxysilane, and the immobilized lipase was a highly thermally stable biocatalyst, with this biocatalytic system retaining more than 90% catalytic activity. The optimum temperature of Yarrowia lipolytica lipase immobilized on macroporous resin was 40°C, which was 5°C higher than free lipase, and the activity of immobilized Yarrowia lipolytica lipase at alkaline pH (more than 50% at pH=12) was much higher than free lipase, and free lipase lost the most activity (35.3%) and immobilized lipases retained more than 46.4% of their initial activity after 3 h of heat treatment at 70°C. At alkaline pH, immobilized lipases were more stable than free lipase, and immobilized lipases retained 80% of their activity after 5 cycles. In one study, Fe3O4 nanoparticles were used to immobilize lipase from Mucor javanicus. They covalently immobilized the lipase onto the surface of activated magnetite nanoparticles, which were then crosslinked to form enzyme aggregates. The immobilized lipases lost only 10% of their activity after 10 cycles of 1,3-diacylglycerol production at 55°C. In patent number B1 0140542 in 1984 in Europe, lipase was immobilized on a Rhizopus, Geotrichum or Aspergillus carrier. Immobilized lipase preparations can be used for the esterification of fats. Immobilized lipase preparations are known for the transesterification of fats. Immobilized lipase preparations can be prepared by simply mixing an aqueous solution of lipase and an ion exchange resin containing a specific combination of a specific class of ion exchange resins and a specific proportion of water in the final immobilized lipase preparation, allowing continuous interesterification to be carried out, which is a cost-effective method. In patent number 5,021,345 in 1984, immobilized lipase is used to solubilize the racemate of a racemic alcohol ester such as glycidyl butyrate racemate. Fortunately, immobilization patents in the field of lipase represent other goals and have not been implemented on a dihybrid chitosan-zinc oxide nano-substrate to increase stability, enzyme life cycle, increase half-life and resistance to organic solvents, and its use in industry is economically viable. Patent No. 8,951,761, published in 2015, relates to immobilized enzymes and methods of use thereof. In fact, in one aspect, it discusses immobilized enzymes that include transesterification enzymes, esterification enzymes immobilized in a food matrix. Depending on the choice of enzyme and matrix, the enzyme can be immobilized on the matrix through covalent or non-covalent bonds (e.g., electrostatic, ionic, hydrogen bonds, adsorption, entrapment, encapsulation, etc.). As immobilization conditions Without wishing to be limited by theory, it is believed that immobilization of the enzyme on the matrix makes the enzyme significantly more stable due to the stabilization of the enzyme structure. If possible, the enzyme should not be washed out of the matrix, which increases the efficiency of the esterification, transesterification, or interesterification / in-esterification process as 60 and also increases the life of the immobilized enzyme for future use. It is also desirable to have enzymes attached to the surface of the matrix in such a way that the enzymes expose themselves. In the patent No. 8715982 in the United States published in 2014, it is related to the subject of immobilization of enzymes on a solid porous support and enzymes immobilized on solid porous supports. In particular, the present invention uses a fluid containing an enzyme under high pressure (e.g. 25 to 50 MPa) to immobilize an enzyme (e.g. CALB) on a porous support (e.g. silica). The process may include recycling the fluid through the porous support under high pressure. The biological species may be a protein, a protein fragment, a saccharide, a DNA fragment, a peptide, or a combination of two or more of these. The biological species may be an enzyme. The fluid containing the biological species may be a liquid and may be an aqueous liquid and may be an aqueous solution, etc. The results showed that the new pressure-based enzyme immobilization method greatly increased the loading of the enzyme onto a hydrophobic mesoporous silica support. In the patent number US 20100209968A1 published in 2008, it is described that the immobilized enzymes can be used for various chemical transformations, separations and purifications and used in sensors and diagnostics. Since the activity of native enzymes is reduced by biological, chemical or physical effects during storage or applications, there is a need to immobilize enzymes due to high production costs. Through immobilization, enzymes can be reused. After use, the enzymes are easily removed from the reaction mixture. In this way, they can be used under different processing conditions. Desirably, the substrate and reaction specificity and the enzyme reaction should not be lost as a result of immobilization. Immobilized enzymes are used in particular in commercially important biotechnological processes. In the food industry, the use of glucose isomerase for the conversion of glucose to fructose is important. The use of lipase for transesterification of edible oils is another standard process. Comparison of different lipase parameters in different research studies in research Recycling Ability Activity Retention (%) Immobilization mechanism support - 67 Covalent Coupling Gold-Silica 91% 5 cycles 66.7 Cross-Linking Chitosan 72% 15 cycles 118 Covalent Coupling SiO2-NSD-3 >50% 10 cycles 58.2 Covalent Coupling Amino-Silane modified superparamagnetic Fe3O4 89% 4 cycles 70 Covalent Coupling Fe3O4 treated with (3-aminopropyl) triethoxysilane >50% 6 cycles 155 (specific activity) Adsorption Hydrophobic nano-sized magnetite NPs 70% 21 cycles 73.9 (specific activity) Covalent Coupling Polydopamine modified iron oxide 88%, 5 cycles 55.6 Adsorption Fe3O4-Chitosan 56.4% 7 cycles 180 Covalent Coupling Lauric acid-stabilized magnetic NPs 70% 5 cycles 70.4 Electrostatic adsorption and covalent binding Polymer modified-Fe3O4 NPs 10 cycles 57 Covalent binding MWCNTs 74%, 5 cycles 108 Cross-Linking Chitosan-beads 69%, 10 cycles 72.8% Covalent Coupling magnetic chitosan beads 7 times, 83% 51.4 adsorption porous chitosan beads Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention Enzyme design, synthesis, and purification: First, we synthesize the designed primer, then introduce it into the bacteria through transformation, and then perform a PCR colony test to ensure that the bacteria have been transformed. We used the heat shock technique to transform the vector into susceptible Escherichia coli bacteria: 1. Add 5 μL of the plasmid of interest to 100 μL of susceptible cells, mix gently with a sampler, and allow to stand on ice for 30 minutes (test). In the positive control vial, add 5 μL of the other plasmid to 100 μL of susceptible cells, and place on ice for 30 minutes, gently tapping the vials every five minutes. 2. Place the vial in a 42°C water bath for 90 seconds, then immediately transfer the sample to ice and wait 5 minutes. 3. Add 900 microliters of liquid LB and place it in a shaking incubator at 37°C with a speed of 200 rpm for one hour to shake. 4. Then centrifuge the vial for 10 minutes in a refrigerated centrifuge at 200 rpm. 5. Discard 90% of the supernatant from each vial and inoculate the remaining 10% of each vial (100 μL) onto solid LB plates containing labeled kanamycin. 6. Place the plates in a 37°C incubator for 12 to 16 hours to grow. 7. We prepare a stock from the single clones obtained from the test plate and store it in a -70 freezer. Three sterile 0.2 microvials were taken and 10 μl of water for injection was added to each. Then, a single clone of the transformed bacteria was inoculated into each of the microvials and pipetted to dissolve the bacteria in water. Then, 1 μl of the 10 μl in each of the microvials was taken and each was cultured in its own section on a solid LB plate containing kanamycin (plate containing the replica culture). After drying, the plate containing the replica culture was placed in an incubator at 37°C for 16 hours and after the single colonies, a stock was prepared. Then, the remaining 9 μl was placed in the microvials for 10 minutes at 96°C (in PCR) to boil. Then, the microvials were placed on ice for 10 minutes and then the microvials were microfuged for 5 minutes. After microfuge, the supernatant containing the plasmid is the sample of interest. Then, the PCR equipment was prepared so that each microvial contained 10 μl of Master-Mix, 2 μl of sample, and 0.5 μl of T7-Forward primer and 0.5 μL of Reverse T7 primer and 7 μL of water for injection were poured to make the final volume of each microvial 20 μL. Then the microvials were placed in the PCR machine and the machine was turned on according to the instructions. Then the electrophoresis gel was prepared and after the gel was closed, it was transferred into the electrophoresis tank and TBE buffer was poured on it to be applied to the wells. Then after the machine was finished, the microvials were removed from the machine and about 1 μL of Lader-DNA (1Kb) was run into the first well with a crystal sampler. Then 3 μL of each sample was mixed separately with 1 μL of Lodig buffer on adhesive tape and pipetted and each was run into the second, third and fourth wells, respectively. Then the red wire, which is +, was connected to the bottom of the electrophoresis tank and the black wire, which is -, was connected to the top of the electrophoresis tank, and the electrophoresis device was turned on and the voltage was set to 100 volts.After about 30 minutes and when the run material in the well reached the lower red line, the electrophoresis machine was turned off and the gel was removed from the electrophoresis tank and transferred to the gel duct machine. The gel was photographed and the site of band formation was examined for bacterial transformation (38). Plate containing replica culture = For replica culture, the plate was divided into three equal parts and numbered, and based on the numbering on the microvials, each was cultured in its own part. Purification column preparation protocol: Inside a 10 ml syringe, layers of wool and glass were placed, then 70% alcohol was poured on it and the piston was pressed to compress it. Wool and glass were placed again, then 70% alcohol was poured on it and the piston was pressed to compress it, and this process was repeated until the diameter of the wool and glass was the size of a boiling tablet. Then it was waited for to dry. After drying, the wool and glass should be washed several times with deionized water and then lysis buffer was added and washing was performed. Then a 3 ml nickel resin column was poured on the wool and glass (the darker part is called the bed volume) and 5 times the bed volume of lysis buffer was poured into the syringe and washing was performed 3 times, that is, 15 cc of lysis buffer was poured into the column to wash it, and this process was repeated three times. The polyhistidine sequence allows purification of wild-type recombinant proteins. The polyhistidine sequence can bind tightly to metal and divalent ions such as nickel and cobalt; therefore, when the protein passes through a chromatography column containing nickel ions, the protein is separated from the rest of the material, causing the histidine sequence to bind to the column. At low imidazole concentrations, the 6-13 histidine sequences bind tightly to the nickel column, while nonspecific proteins are eluted without binding to the column along with the buffers. First, a preculture was prepared from the transformed bacterial stock. To prepare the preculture, 5 μl of the transformed bacterial stock was added to 5 ml of liquid LB containing kanamycin and it was placed in a shaking incubator at 150 RPM and 37°C for 10-12 hours. After 10-12 hours, 2 cc of the shaken solution was inoculated into 200 cc of liquid LB containing kanamycin and it was placed in a shaking incubator at 150 RPM and 37°C until the OD of the medium reached 0.6-0.7. Then, after the OD of the medium reached 0.6-0.7, IPTG was added to the culture medium in the Falcon at a final concentration of 0.5 mM (about 200 μl of IPTG was added for every 40 ml). After adding IPTG, the falcon containing the culture medium was placed in a shaking incubator for 8 hours at 180 RPM and 20°C to perform the induction and expression process. After 8 hours, the contents inside the falcon were centrifuged for 20 minutes at 1000 RPM and 4°C. Then, Lysis, Washing, and Elution buffers were added with pH 7.4 was prepared, which was prepared in a volume of 60 cc, by mixing 1 g of NaCl with a concentration gradient of 300 mM with 0.4 g of Tris-Hcl with a concentration gradient of 50 mM, 0.04 g of imidazole with a concentration gradient of 10 mM, and 428 μl of 2-mercaptoethanol with a concentration gradient of 100 mM and 60 cc of distilled water to prepare the lysis buffer in a volume of 60 cc. To prepare the washing buffer in a volume of 20 cc, 0.35 g of NaCl with a concentration gradient of 300 mM was mixed with 0.13 g of Tris-Hcl with a concentration gradient of 50 mM, and 0.05 g of imidazole with a concentration gradient of 40 mM and 20 cc of distilled water. To prepare the Elution buffer, 0.87 g NaCl with a 300 mM concentration gradient was mixed with 0.34 g Tris-Hcl with a 50 mM concentration gradient, 0.85 g imidazole with a 250 mM concentration gradient, 72 μl 2-mercaptoethanol with a 50 mM concentration gradient, and 50 cc of distilled water in a volume of 50 cc. Then, the nickel-sepharose resin column was washed several times with Laysis buffer until equilibrium was reached.Then, after centrifugation, the supernatant was discarded and about 10 ml of lysis buffer was added to the sediments in the falcon, and the falcon containing the sediment and 10 ml of lysis buffer was sonicated to rupture the bacterial wall (the sonicate process is 20 cycles of 30 seconds and a power of 80, with a 1-minute rest between each cycle of the device). After the sonication process, the crushed suspension of bacteria was centrifuged for 15 minutes at 12,000 RPM and 4°C to produce sediment and supernatant. Then, the supernatant was transferred to a new sterile falcon and centrifuged again for 15 minutes at 12,000 RPM and 4°C. After centrifugation, the supernatant (enzyme extract) was poured into a column that had previously reached equilibrium with the lysis buffer. Then the column cap was closed and ice was placed around the column and waited for 20 minutes. After 20 minutes, the cap was removed and the column valve was opened to allow the lysate to be removed dropwise, and finally about 1 ml of lysate buffer (the size of the diameter of a boiling tablet) remained on the column to prevent the column from drying out.The column valve was closed again and then 5 times the bed volume (i.e. about 15 cc) of washing buffer was poured into the column and waited for one minute. After 1 minute, the column valve was opened so that the washing buffer was removed dropwise, and finally about 1 ml of washing buffer remained on the column. The column valve was closed again and 10 ml of elution buffer was poured into the column and waited for one minute. 20 sterile vials were taken and 50 microliters of 60% glycerol were poured into each of them. After one minute, 20 vials containing 60% glycerol were placed under the column and 500 microliter volumes of elution buffer were collected in the vials and pipetting was performed quickly so that the enzyme did not precipitate and the vials were placed in a -20 freezer. After collecting the different elution fractions, the column was washed with 15 cc of lysis buffer without 2-mercaptoethanol. Then, to read the absorbance of different elution fractions at a wavelength of 280 nm, approximately 50 microliters of different elution fractions were poured into the cuvette with 1950 microliters of elution buffer, and their absorbance was read at a wavelength of 280 nm. After purifying the enzyme, we use the SDS Page test to ensure that the purification has been done correctly. The band in the specified area indicates good purification of the enzyme. After that, we prepare the substrate to immobilize the enzyme on the hybrid substrate. For this task, we need: Dissolve 12 mg of nanochitosan in 25 ml of 1% acetic acid (v / v), then add 350 mg of zinc oxide to the nanochitosan and place on a stirrer for 30 minutes to mix together. The next step is to add 25 ml of 1M sodium hydroxide to the previous mixture and then centrifuge and wash the resulting precipitate with distilled water, then centrifuge and place in an oven at 100 degrees for 3 hours to dry. Then, to bind the enzyme through electrostatic bonding, we need to modify the substrate and make the surface charge of the substrate more positive so that the enzyme binds to the substrate well. For this Dissolve 200 mg of the cross-linked substrate in 4 ml of dimethylformamide (DMF) and place it on a heater that has already reached a temperature of 80 ° C. Place it on the stirrer for three minutes and put the lid on the container to mix them together. Then add 700 μl of thionyl chloride to the sample and leave it at 80 ° C for four hours to interact with it. In the next step, add ammonia gradually until it reaches pH = 7. Then centrifuge the sample at 11,000 rpm for 5 minutes and place it in an oven at 25 ° C to dry. Finally, weigh the samples from the previous step in a vial and add 11 ml of ethylene diamine per 0.2 g of chlorinated substrate. Then place it in a water and oil bath for three hours. Finally, the solution was centrifuged, washed twice with water, centrifuged, and placed in a freeze-dryer to dry.The modified substrate is prepared for binding the enzyme to the substrate, so we need to read the Bradford absorbance of the purified enzyme from the column through a spectrophotometer at a wavelength of 595 nm and put the resulting number into the Bradford formula to obtain the amount of enzyme in milligrams. In the next step, we measure the activity of the purified enzyme by mixing 100 microliters of substrate, 800 microliters of assay buffer, and 100 microliters of enzyme and reading the absorbance of the mixed sample at a wavelength of 410 to find out the level of enzyme activity. Now, in order to stabilize the purified enzyme on the substrate, we need to examine different substrate ratios with ratios of one to one, one to three, one to ten, one to twenty, and five to find out in which of the ratios the enzyme has the highest percentage of stability. For example, in a one-to-one ratio, if we weigh 0.5 mg of substrate, we need 0.5 mg of enzyme along with a pH=8 buffer to stabilize the enzyme on the substrate.To stabilize the enzyme on the substrate, pour the mixture of enzyme, substrate and buffer into a vial and place it in a shaking incubator with a shaker at 150 rpm at 4 ° C to stabilize the enzyme on the substrate. For each ratio, the sample was examined every hour in terms of enzyme activity in the supernatant and enzyme activity in the sediment and Bradford enzyme to see at which hour the enzyme had the best activity. Based on the Bradford absorption of the enzyme and placing it in the Bradford formula, the percentage of stabilization can be obtained. In other words, to stabilize the enzyme on the hybrid nano substrate of chitosan and zinc oxide, about 2 mg of the amined nano hybrid substrate was dissolved in 3500 μl of optimal buffer with pH = 8 and 500 μl of purified lipase enzyme was added to it (0.5 to 0.5 ratio of substrate and enzyme). Then, the contents of the Falcon were placed in a shaking incubator at 100 RPM and 4°C for 1 hour to stabilize.Then, after 1 hour, about 100 microliters of the Falcon contents were removed and centrifuged for 10 minutes at 12,000 RPM, and the Bradford test was performed with the supernatant to determine the free enzyme activity (for the Bradford test, the blank contains only 1,000 microliters of Bradford solution, and the sample vial contains 100 microliters of enzyme incubated at 4°C and 900 microliters of Bradford solution). Then, the obtained absorbance was placed in the formula of the standard Bradford chart, and the amount of free enzyme and the percentage of surface binding were calculated. How to covalently immobilize an enzyme on a glutaraldehyde-containing substrate To determine the appropriate percentage of glutaraldehyde, first prepare a stock with a concentration of ten percent glutaraldehyde and then convert it to concentrations of 5%, 5.2%, and 1%. Then, add different concentrations of glutaraldehyde to the substrate and immobilize the enzyme on the substrate to see which concentration level the enzyme has the best activity. The appropriate percentage of glutaraldehyde for enzyme activity is 2.5%. Then, to perform covalent immobilization, add 10 ml of 5.2% glutaraldehyde (v / v) to 330 mg of chitosan and zinc oxide amine substrate and place it on a stirrer for two hours at room temperature to shake. Then, centrifuge the solution and then wash it with water to remove excess glutaraldehyde from the solution. Then, place it in an oven at 25°C to dry. Then, at a ratio of one to five, the enzyme is placed in a shaking incubator for one hour to shake and stabilize the enzyme on the surface. Then, by reading the Bradford absorbance of the solution, the degree of stabilization on the surface can be determined.After immobilizing the enzyme by two methods of surface adsorption and covalent adsorption, we must examine the enzyme in terms of temperature profile, pH profile, temperature stability, reuseability test, investigation of the effect of salts, detergents, and organic solvents on the activity of the surface and covalently immobilized enzyme, storage stability, and enzyme kinetics. To check the results of these tests, we proceed as follows: Investigating the pH profile of the enzyme in physical and covalent immobilization: To determine the pH profile, it is necessary to immobilize the enzyme on the substrate in a ratio of one to one for physical immobilization and one to five for covalent immobilization. Then, pH values ​​of 2 to 10 were prepared using buffers, and after adding the substrate and buffer, the enzyme solution containing the substrate was added and allowed to incubate for 15 minutes. Then, to stop the reaction, the enzyme was boiled for five minutes, and then centrifuged for ten minutes at 10,000 rpm, and finally, the absorbance of the solution was read at 410 nm. Study of enzyme temperature profile in physical and covalent immobilization: We measure the temperature profile at that pH, according to the pH at which the enzyme has optimal activity. To determine the temperature profile for physical immobilization, we immobilize the enzyme on the substrate in a ratio of one to one, and for covalent immobilization, we immobilize the enzyme on the substrate in a ratio of one to five. We should note that the buffer in which the enzyme is located and is immobilized on the substrate is the same pH at which the enzyme has optimal activity. Then, we pour the substrate, the assay buffer, and the enzyme immobilized on the substrate into the vials and prepare different temperatures of 4, 10, 20, 25, 30, 35, 40, 50 for the protein to incubate. Then, we place the vials at different temperatures for 15 minutes and allow them to incubate. After fifteen minutes of incubation, to stop the reaction, the contents of the vials need to be boiled for five minutes, then centrifuged for ten minutes at 10,000 rpm, and finally the absorbance of the solution is read at 410 nm. Study of enzyme thermal stability in physical and covalent immobilization: First, the enzyme is immobilized in physical immobilization with a ratio of one to one on a chitosan-zinc oxide substrate for one hour in a shaking incubator. For covalent immobilization, the enzyme is immobilized on the substrate with a ratio of one to five. In accordance with the maximum enzyme activity at the optimal temperature, we consider the optimal temperature and three temperatures lower than it and measure the enzyme activity at those temperatures. Therefore, it is necessary to provide different temperature conditions in advance and label the vials. Before starting the experiment, the immobilized enzyme, which comes with the substrate, is poured into the vials. We immediately place the zero-point and blank vials on ice, then allow ten minutes to pass, and place the vials in the pre-set temperature incubators for the first ten minutes, then allow them to incubate for ten minutes, then remove them and place them on ice for 30 minutes, then add 100 microliters of substrate and 800 microliters of assay buffer to the enzyme, and allow them to incubate for ten minutes at room temperature.Finally, it was placed in boiling water for ten minutes to inactivate the enzyme. Finally, it was centrifuged for five minutes at 10,000 rpm and then the sample absorption was read at a wavelength of 410 nm. After twenty minutes from the zero moment, the 20th minute vials were placed in the incubator and allowed to incubate for ten minutes, and the same steps were performed for the 20th to 50th minute vials. It should be noted that a separate blank is required for each temperature. To investigate the effect of salts, we use concentrations of 5, 10, 20 mM, and for detergents, we use 1% w / v. Only before performing this experiment, we must immobilize the enzyme on the substrate, either for physical immobilization or covalent immobilization. Then, we prepare 20 mM stocks for the salts of copper chloride, copper bisulfate, mercuric chloride, aluminum chloride, ammonium chloride, magnesium chloride, sodium chloride, zinc chloride, and barium chloride. Then, we label the vials and for each salt with each concentration, we need a blank and a sample. To measure the effect of salts, the substrate, assay buffer, salt, and immobilized enzyme are poured into each vial and compared to the blank that does not contain enzyme. Then, we allowed them to incubate for 15 minutes at the optimum temperature of the enzyme, then boiled for ten minutes to stop the reaction, and finally centrifuged at 12,000 rpm and the sample absorbance was read at a wavelength of 410 nm. For concentrations of ten mM and five mM, the same steps are repeated, only the salt concentration changes and is halved. To investigate the effect of detergents, we used SDS, Triton, and EDTA at 1% w / v, by weighing 0.01 g of SDS, EDTA, dissolving them in 900 μl of assay buffer, adding 100 μl of enzyme immobilized on the substrate, and finally adding 100 μl of substrate and allowing it to incubate for fifteen minutes at the temperature that has the optimal activity. After ten minutes, the contents of the vial are boiled and centrifuged, and the sample absorbance is read by spectrophotometry at a wavelength of 410 nm. To investigate the effect of organic solvents on the enzyme, we used organic solvents such as ethanol, methanol, and di-propanol. In this way, the enzyme immobilized on the substrate, assay buffer, organic solvent, and substrate were poured into the vials in this order and allowed to incubate for 15 minutes at the temperature at which the enzyme has optimal activity. Then, the contents of the vial were boiled and centrifuged for ten minutes, and the sample absorbance was read by spectrophotometry at a wavelength of 410 nm. It should be noted that to investigate the effect of ions, organic solvents, and detergents on the enzyme, the enzyme activity should be examined in the absence of the aforementioned items so that their effects can be determined through their ratio. Kinetic study of physical and covalent immobilization of lipase enzyme To calculate kinetics, it is better to use the UV4100phrmacia spectrophotometer. First, the temperature of the device must be set to 30 degrees. Then, the wavelength must be set to 410 nm, then we pour 50, 100, 150, 200, 250 mM of the substance into the sample cell, depending on the concentration. In this way, to check the concentration of 50 mM substrate, 50 microliters of substrate, 930 microliters of assay buffer, and 20 microliters of enzyme immobilized on the substrate are poured into the sample cell, respectively. Then, we place the cell inside the device, run the program settings, and give the device one minute to check the sample. Before placing the sample in the device, we need a blank cell. For concentrations of 100 to 250 mM substrate, we performed the same steps as mentioned earlier. Recycling of physical and covalent immobilization of lipase enzyme In this test, we find out how many times we can use an enzyme. In this method, the activity of the enzyme immobilized on the substrate is measured using 100 microliters of substrate, 800 microliters of assay buffer, and 100 microliters of enzyme at a wavelength of 410 nm, then centrifuged, measured again with the substrate and assay buffer, and centrifuged again to find out how many times it can be reused. Storage stability In this test, we examine the long-term stability of the enzyme on the substrate. In fact, we keep about two cc of the enzyme immobilized on the substrate in a vial at room temperature and in the refrigerator and measure its activity every six days. To check the enzyme activity, we need a substrate and an assay buffer, and we read its absorbance at a wavelength of 410 nm and check the activity every six days for up to 35 days. Explanation of shapes, maps and diagrams Figure 1: After designing the primer and the transformation process and growing the bacteria on a plate containing kanamycin, colonies containing the recombinant plasmid were randomly selected and PCR was performed with forward and reverse primers and transferred onto an electrophoresis gel. The band observed in Figure 1 indicates that the colonies containing the recombinant plasmid were correctly transformed. Figure 2: Purification of lipase enzyme with a nickel-agarose affinity column. Its presence was confirmed on a 12% polyacrylamide gel by SDS-PAGE electrophoresis. A protein sample extracted from the culture 8 hours after induction was run on an SDS-PAGE gel to examine the expression of the target protein. The band observed in Figure 2 shows that the extracted protein sample has the highest expression after 8 hours of induction and expression. Figure 3: Examination of spectra, functional groups, and types of bonds such as covalent, ionic, etc. in zinc oxide, chitosan, and synthetic substrate chitosan-zinc oxide, and modified substrate (amine-containing) through FTIR testing showed that chitosan and zinc oxide nanoparticles were connected to each other and a nanohybrid substrate was created, and through the NH2 functional group at the peak of 3300 to 3400, we found that they were well amined through ethyleneamine, because the peak of the graph in this region is sharper. Figure 4: Examination of the morphology of surfaces with a scanning electron microscope (SEM), which are chitosan-zinc oxide hybrid nano-substrates before and after sintering, and before and after amination, showed that the morphological changes indicate the immobilization of the enzyme on the substrate. Figure 5: FTIR bond analysis before and after physical immobilization of the enzyme with 0.5 mg of enzyme using an Irprestig FT-IR instrument. Which indicates that the enzyme has physically bound to the substrate. Figure 6: FTIR analysis of the bonds before and after covalent immobilization with 0.5 mg of enzyme using an Irprestige FT-IR instrument, indicating that the enzyme has covalently bound to the substrate. Figure 7: Study of the effect of storage stability on lipase enzyme activity in solution, covalent immobilization, and physical immobilization over a specific period, which indicates to what extent the enzyme can remain stable and function on surfaces. The results of storage stability showed that in the covalent immobilization state, the enzyme stability is greater than in the physical immobilization state, because after 35 days it was able to maintain 41% of its activity, and in the physical immobilization state it is greater than in the free state. Figure 8: Investigation and determination of the pH of the enzyme in the free state, covalent and physical immobilization. First, the enzyme was immobilized physically and covalently on a chitosan-zinc oxide hybrid nano-substrate. Then, the substrate and buffer with different pHs and the enzyme were mixed together and incubated for 15 minutes at 30°C. Then, they were boiled for 5 minutes to stop the reaction. After centrifugation, their absorption was read at a wavelength of 410 nm. The pH profile examination showed that the optimum pH of the enzyme solution is 8; but its pH range is not very wide; but in physical and covalent immobilization, this pH range has become wider, which is one of the advantages of good immobilization. Also, after immobilization, the pH has gone towards neutralization, which is very widely used in industries. Figure 9: Diagram of the effect of different temperatures on enzyme activity in solution, physical and covalent immobilization. To determine the optimal temperature of the enzyme in the immobilization state, it was immobilized on a chitosan-zinc oxide hybrid nano-substrate. Then, the substrate, assay buffer, and immobilized enzyme were mixed together and incubated for 15 minutes at different temperatures. Then, they were boiled for 5 minutes to stop the reaction. After centrifugation, their absorbance was read at a wavelength of 410 nm. The results of the enzyme temperature profile showed that in covalent and physical immobilization, in addition to increasing enzyme activity, the temperature range also increased, which was a good advantage of immobilization compared to free enzyme in this study. Figure 10: To investigate the reusability of the enzyme in covalent and physical immobilization of the enzyme. First, the enzyme was covalently immobilized on a chitosan-zinc oxide hybrid nano-substrate, then substrate and assay buffer were added to the precipitate resulting from enzyme immobilization, and its absorbance was read at a wavelength of 410 nm. Then, after centrifugation and discarding the supernatant, substrate and assay buffer were added to the remaining precipitate for several cycles, and its absorbance was read at a wavelength of 410 nm. The graph shows that the reusability of the enzyme in covalent and physical immobilization continues for up to 14 cycles, and in physical and covalent immobilization, they have maintained approximately 80% of their activity until the eighth cycle. Figure 11, 12, 13: By examining the effect of salts on enzyme activity, we found that in covalent and physical immobilization, the substrate protects the enzyme from the effects of high salt concentrations, and the effect of salts on the free enzyme state is different between physical and covalent immobilization; therefore, this test helps to remove waste from the environment (such as salts) and allows us to choose the type of immobilization appropriate to the environment in which we are conducting the research, because we know what concentration of salts the enzyme has had and what activity it has. First, the enzyme was immobilized superficially and covalently on the chitosan-zinc oxide hybrid nanosubstrate, then 5, 10, and 20 mM concentrations of salts were applied to the surface immobilized enzyme and incubated for 15 minutes at 40°C. Then, they were boiled for 5 minutes to stop the reaction, and after centrifugation, their absorption was read at a wavelength of 410 nm. Figure 14 and 15: Effect of detergents and organic solvents on lipase enzyme activity in solution, surface immobilization, and covalent immobilization. To investigate the effect of detergents on surface immobilization enzyme activity, first the enzyme was immobilized superficially on a chitosan-zinc oxide hybrid nano-substrate. Then, a concentration of 1% w / v of detergents and organic solvents was applied to the surface and covalent immobilization enzyme and incubated for 15 minutes at 40°C. Then, they were boiled for 5 minutes to stop the reaction. After centrifugation, their absorption was read at a wavelength of 410 nm. The results of the effect of organic solvents (ethanol, methanol, di-propanol) on soluble lipase enzyme activity, physical and covalent immobilization of lipase enzyme showed that the substrate was able to protect the enzyme well during immobilization and the enzyme works well against organic solvents.The study of detergents and chelators on the physical and covalent immobilization activity of the enzyme showed that physical immobilization of the substrate in the presence of detergents and chelators can protect the protein well, and in covalent immobilization, the substrate was still able to protect the enzyme in the presence of SDS and Triton; however, in the presence of EDETA, it was able to be active to a lesser extent compared to physical immobilization of the enzyme. Figure 16: To investigate the kinetic effect on enzyme activity in solution, physical and covalent immobilization. First, the enzyme was immobilized covalently and physically on a chitosan-zinc oxide hybrid nano-substrate. Then, concentrations of 50, 100, 150, 200, and 250 μl of substrate were applied to the covalently immobilized enzyme and incubated for 15 minutes at 30°C. Then, they were boiled for 5 minutes to stop the reaction. After centrifugation, their absorbance was read at a wavelength of 410 nm. The results of the Arrhenius diagram show that the activation values ​​in the soluble enzyme, physical immobilization of the enzyme, and covalent immobilization of the enzyme are 53.38, 57.54, and 57, respectively. This means that if we want to heat the enzyme and inactivate it, we need a larger amount of thermal energy for covalent immobilization. A clear and precise statement of the advantages of the claimed invention over prior inventions. Compared to free enzyme, immobilized enzyme on chitosan-zinc oxide nanohybrid substrate is economical due to its greater stability, continuous enzyme application, enzyme recovery, smaller reaction volume, increased flexibility in contact with enzyme substrate, better control and higher reaction efficiency, higher product purity, and reduced environmental pollution. The purpose of immobilization is to increase the enzyme resistance under extreme conditions of temperature, pH, and organic solvents, and to recycle and reuse the enzyme. Despite these advantages, its industrial applications are limited due to substrate costs, mass transfer limitations, changes in properties (specific performance), and loss of activity during immobilization. The most prominent advantage of enzyme immobilization technology is the increase in enzyme thermal and pH stability, which allows for the reuse of a specific enzyme multiple times and also easy separation of the enzyme from the reaction mixture. The most important factor affecting immobilization is the selection of the appropriate substrate type and immobilization method.In the stabilization process, substrates are efficient if their nanoparticles have suitable physical and chemical properties, high surface area to volume ratio, and suitable biocompatibility; therefore, considering the above, in this study, we used two nanoparticles of chitosan and zinc oxide to synthesize the nanohybrid substrate. After synthesizing the hybrid substrate and examining it with a DLS device, the substrate charge (mV) was reported to be -0.6, and considering that the enzyme base was also reported to be negative, it was necessary to make the substrate positive with amine-containing particles for electrostatic binding of the enzyme to the substrate and better stabilization. So, we performed the amination process and the charge on the substrate reached +5. After immobilizing the enzyme on the substrate and examining its optimal temperature compared to the enzyme in solution, we found that the enzyme in solution has the best activity at 35 °C; but during stabilization, in addition to increasing activity, the enzyme's peak temperature also increases.It is also worth noting that the optimum pH in solution for the lipase enzyme is 8, and at this pH the enzyme has the highest activity; but after stabilization, the pH peak range has become wider and often during the stabilization process, the pH of the enzyme moves towards neutralization. The stabilization of the lipase enzyme showed that the enzyme has maintained its catalytic efficiency in physical and covalent stabilization at about 90 and 96 percent, respectively. At the same time, the stability of the enzyme has increased. For example, the half-life of the enzyme at 35 degrees showed that the half-life has increased by 7% in physical stabilization and 15% in covalent stabilization. By stabilizing the enzyme on the substrate, the enzyme can be reused several times, and in this study, the enzyme was able to continue to be active up to 14 times with a simple centrifugation and separation of the enzyme from the product; but with the difference that in physical stabilization, the enzyme was able to maintain 50 percent of its activity until the ninth cycle; but in covalent stabilization, it was able to maintain 50 percent of its activity until the twelfth cycle.The storage stability of the enzyme in physical immobilization at 25 degrees Celsius after 35 days has increased by 200% compared to the solution conditions and by 320% in covalent immobilization. The enzyme has more than 50% activity in physical and covalent immobilization up to the tenth cycle and maintains more than 80% activity in the eighth cycle, which is important because it saves energy in industrial applications. The study of thermodynamic parameters showed that the activation energy of the enzyme in the solution state, physical immobilization and covalent immobilization is 38.53, 54.57 and 57 mol / j, respectively. This means that we must heat the enzyme in the immobilization state more to deactivate the enzyme. In terms of entropy, if we want to examine it according to the tables mentioned, the amount of structural disorder in immobilization has decreased significantly because the enzyme is attached to the substrate. At the same time, the delta-starr binding also increased during stabilization, indicating that more energy must be expended to denature the enzyme and cause loss of enzyme activity.This enzyme is used to remove fat waste in industrial environments such as wastewater. Also, if other wastes such as salt are present in the environment, by examining the effect of salts on enzyme activity in the physically and covalently immobilized state, the type of immobilization can be determined to determine which type of immobilization is better; because the effect of salts on enzyme activity is not the same and sometimes increasing the salt concentration increases or decreases the enzyme activity in physical and covalent immobilization. For example, KCl salt was able to be active in covalent immobilization by increasing the concentration up to 180%. Also, the immobilized enzyme was able to play a protective role against solvents, detergents, and chelators because the synthesized substrate protects the enzyme against detergents and organic solvents. In kinetic studies, the km value of the free enzyme decreased compared to physical and covalent immobilization; that is, the affinity of the enzyme for the substrate increased.In fact, in studies from previous articles, we had concluded that in dihybrid substrates, the km value decreases, and fortunately, the kcat value increased; therefore, the results of lipase enzyme immobilization showed that the enzyme maintained its catalytic efficiency in physical and covalent immobilization at about 90 and 96 percent, respectively, while the stability of the enzyme increased. In fact, a small cost of kinetic parameters has been paid to increase the stability of the enzyme; therefore, it can be concluded that performing the immobilization process in industries is very valuable. It is hoped that, given the many applications of the psychrophilic lipase enzyme and the use of the mentioned method, investment will be made for mass production and the chitosan-zinc oxide substrate will be used in the stabilization process; because this substrate, in addition to being easily accessible, is safe and inexpensive, and considering the above-mentioned points, it has performed exceptionally well and can be used well in industrial applications. Description of at least one implementation method for implementing the invention According to the detailed process of the invention, the produced enzyme can be lyophilized after the stabilization process and marketed along with a catalog of how to use it. Offering this process to commercial markets is very cost-effective: - Cryophilic lipase enzymes are used in wastewater treatment, bioremediation in cold oil-contaminated environments, and soil bioremediation industries. - In leather processing, lipases are used to remove the natural fat present in animal skin, and due to environmental pollution by chemicals and the implementation of global environmental regulations in the leather industry, lipase enzymes are at the forefront of use. - Cleaning starch stains from clothes was done by immobilizing the heat-resistant amylase enzyme inside agarose beads; therefore, using a pyrophilic enzyme in the detergent industry is cost-effective because it reduces energy and increases performance, and creates environmental goals. - Another industrial biocatalyst is related to phenylacetone dehydrogenase, the stabilization process of which was successfully carried out and led to the treatment of wastewater contaminated with textile dyes. - In food processing production, oil and fat modification is one of the important areas that economically requires green technologies and is a very important component of food. Changing the location of the fatty acid chain of lipases allows us to modify the value of lipids in glycerides and exchange one of them for new ones. In this way, a relatively cheap and low-value fat can be upgraded to a higher-value fat. - The applications of this enzyme in the food industry are many and varied, from texturing to flavoring. Lipases are used for the hydrolysis of milk fat, cheese ripening, flavor enrichment and butter fat lipolysis in the dairy industry. Lipase obtained from Lactobacillus plantarum is used in the synthesis of various fermented food products such as sourdough, olives, vegetable sausages and cheese. Pseudomonas lipase is considered useful in food processing and oil production. Also, the lipase enzyme leads to the release of fats even in cold environments, which can be edible fats, fats found in plants or fats found in the juice industry. Other cases have also been mentioned in the previous research section. Explicit mention of the industrial application of the invention Given that we immobilized the psychrophilic lipase enzyme on a chitosan-zinc oxide substrate, which increased the stability of the enzyme, and the components of this substrate have been approved by the Food and Drug Administration, the immobilized enzyme can be used in the food industry. Lipase enzymes, which are more or less common in food applications, are applied in processed food products as processing agents upstream of the final product. Several advances have been made in optimizing enzymes for existing applications and using recombinant protein production to provide efficient single-component enzymes that do not have the potential. Lipase obtained from Lactobacillus plantarum is used in the synthesis of various fermented food products such as sourdough, olives, vegetable sausages, and cheese. Pseudomonas lipase was considered useful in food processing and oil production. Castor oil is a unique vegetable oil that contains high amounts (90%) of the hydroxy unsaturated fatty acid called ricinoleic acid. This industrially important acid can be obtained by hydrolysis of castor oil using a more efficient lipase-catalyzed process.Cocoa butter equivalents are produced with the help of lipase catalysts from natural triglycerides such as palm or sunflower mid-fat. In addition, lipase can be used to increase the shelf life, taste and rheological properties of juices, soups, sauces, cheeses and baked goods. The shelf life of various bakery products and their softness can be improved with lipases. The softness of noodles can also be increased with the help of lipases. Phospholipases found in egg yolk are also used to produce mayonnaise and various emulsifiers. Acinetobacter. Lipase EH28 sp. is used to make flavor esters such as ethyl butyrate, ethyl valerate and ethyl caprylate in organic solvents. Ethyl aprilate, which has a fruity-floral aroma, is used to give various fruit flavors such as peach, apple, banana and pineapple. One of the important applications of lipase is in the hydrolysis of vegetable oil to produce free acids, which are mostly used in the food, soap, and biomedical industries.Various esters such as monoglycerides, isoamyl acetate (banana flavor), valerate and hexyl acetate (pear flavor), and butyl acetate (pineapple flavor) are synthesized using immobilized staphylococcal lipase. Monoglycerides are nonionic surfactants that have both hydrophilic and hydrophobic regions and are used in food emulsifiers for bakery products, margarine, dairy products, and sauces. Low-value lipids can be converted into high-value products by the action of lipase enzymes. These enzymes change the location of the fatty acid chain in the glycerides and replace them with a new enzyme. Pseudomonas, Alcaligenes, and Achromobacter lipases are known to resist pasteurization and affect flavor development during cheese ripening. Lipolytic lactic acid bacteria also play a role in vegetable fermentation. The use of phospholipases to remove phospholipids from vegetables (demugning) is an environmentally friendly process. In order to preserve heat-sensitive micronutrients and macronutrients in food, the food industry prefers reactions that occur at low temperatures; therefore, cold-active enzymes are widely used in the food industry instead of traditional chemical processes. The use of the cryogenic lipase Fluorescens.p38 in the synthesis of the flavor compound, butyl caprylate, in n-heptane at low temperatures was reported. Lipases from C. antarctica (CAL-B), Hansinuela lanuginosa, Pseudomonas sp, and G. candidum were used in the esterification of functionalized phenols to form antioxidants for use in sunflower oil. Lipases are widely used in the dairy industry to hydrolyze milk fat, modify the chain length of fatty acids, and enhance the flavor of cheese. They are also currently used to accelerate the ripening of cheese and lipolysis of fat, butter, and cream. Microbial lipases are used to produce a wide range of quality cheeses. Enzyme modified cheese (EMC) is produced by incubating cheese at high temperatures in the presence of the enzyme. Compared to regular cheese, EMC has a 10-fold higher fat concentration and is used as an ingredient in other products such as dips, sauces, soups, and snacks. Acetoacetates, beta-keto acids, flavor esters, methyl ketones, and lactone flavorings are synthesized from free fatty acids by initiating simple chemical reactions. Several types of cheese such as cheddar cheese, provolone, and cheddar cheese are used to accelerate ripening and improve flavor. The rate of fatty acid release increases after the addition of lipase, which improves the taste. The release of fatty acids was significantly increased with the addition of calf lipase.The total amount of short-chain fatty acids (C4 to C6) released during preparation is significant for improving the flavor of cheddar cheese as observed. Highly soluble proteins and free fatty acids have a better flavor within 3 months. In cheese, milk lipase is an intrinsic component of unpasteurized milk that significantly affects the lipolytic activity. Blue-vein and Camembert cheeses are lipolytic and produce lipase by culture and secondary microflora such as P. roqueforti and P. camembertii. Parmesan, provolone, and Romano are Italian cheeses that generally become more palatable after the addition of lipases. Lipases release fatty acids from triglycerides, which contribute to the flavor of the cheese. In dairy foods, the general investigation of conjugated linoleic acid (CLA) has been made possible by the immobilization of lipases. Both lipases and proteases accelerate the ripening of cheeses individually. Thus, the enzymes may be added or encapsulated.Lipases, proteases, and lactase enzymes hydrolyze lipids, proteins, and lactose, respectively, to enhance the levels of flavor components and / or primary flavor ingredients. Lipases have specific applications in industry and medicine due to their activity in aqueous and non-aqueous environments. Microbial lipases are of great importance due to their wide variety of biochemical activities and ease of isolation and production. Among microbial lipases, bacterial enzymes are usually preferred over fungal enzymes due to their higher stability and higher activity at neutral or alkaline pH. Bacterial cells have simpler nutritional requirements, shorter production times, and easier genetic manipulation; therefore, bacterial enzymes are preferred. Due to these properties, bacterial lipases have many applications in the chemical, food, detergent, pulp and paper, leather, environmental management, and pharmaceutical industries. Among bacterial lipases, Pseudomonas lipases have received much attention due to their heat resistance and activity at alkaline pH, which is not common among lipases produced by other microorganisms.Thermal stability of lipases is considered the most important property for industrial applications, but low thermal stability is useful for some applications. Cold-active enzymes can be easily inactivated by heat treatment at relatively low temperatures after use for food and other materials processing. One of the useful properties of cold-active enzymes is their ability to catalyze reactions at low or moderate temperatures. Given these two properties, cold-active lipases are now gaining attention and have various applications in the detergent industry (cold washing), bioremediation of cold oil-contaminated environments, and bioconversion reactions of heat-sensitive compounds. Although bacteria cannot grow at temperatures above 30°C, the optimum temperature of purified lipase was 45°C and it showed very little activity at the optimum temperature of bacterial growth, i.e. 25°C. Chemical compounds of detergents are dangerous to humans and cause environmental pollution, so lipases are used as a substitute for these harmful substances. Currently, most companies are producing enzyme-based detergents.Lipase-based detergents digest lipid molecules from soiled substrates, are active at ambient temperatures. Cold-active lipases are used as additives in detergents, which can be used in laundry detergents for washing clothes at low temperatures and in the organic synthesis of chiral intermediates. The alkaline and thermostable lipase produced by Pseudomonas aeruginosa strain BUP2 has high specific activity and is effectively used in the detergent industry. Lipases from Bacillus flexus XJU, Bacillus licheniformis, Bacillus licheniformis VSG, Bacillus pumilus SG, Bacillus subtilis JPBW, Geobacillus sp. Pseudomonas aeruginosa san-ai are frequently used.

Claims

Claims What is claimed: Claim 1) A method for immobilizing lipase enzyme on a chitosan-zinc oxide dihybrid substrate modified with amine-containing compounds, comprising the steps of: a- Purifying lipase enzyme from Escherichia coli bacteria; b- Preparing a crosslinked chitosan-zinc oxide dihybrid substrate; c- Modifying the substrate obtained in step b with thionyl chloride; d- Modifying the substrate obtained in step c with ethylene diamine to bind the lipase enzyme to prepare an amine-containing chitosan-zinc oxide dihybrid substrate; and e- immobilization of the purified lipase enzyme on a chitosan-zinc oxide dihybrid substrate ferritically and covalently, such that immobilization of the purified lipase enzyme on a chitosan-zinc oxide dihybrid substrate ferritically includes the following steps: I. Mixing the amine substrate with the purified enzyme in a buffer with a pH of 8; and II. Immobilizing the purified enzyme on the amine substrate in a shaking incubator such that the ratio of amine substrate to purified enzyme is 1:1.Thus, the immobilization of the purified lipase enzyme on the covalently amined chitosan-zinc oxide dihybrid substrate includes the following steps: I. Preparation of glutaraldehyde stock; II. Adding glutaraldehyde to the amined substrate for two hours at room temperature; III. Centrifuging the substrate and washing it to remove excess glutaraldehyde; IV. Drying the substrate; and V. Immobilizing the purified enzyme on the amined substrate in a shaking incubator for one hour so that the ratio of amined substrate to purified enzyme is 1:

5. Claim 2) The method according to claim 1, wherein the purification of lipase enzyme from Escherichia coli bacteria comprises the steps of constructing a carrier plasmid by inserting the lipase enzyme gene into the plasmid; introducing the carrier plasmid into bacterial cells to express the lipase enzyme in the bacteria; inducing and expressing the lipase enzyme using IPTG; and purifying the lipase enzyme with a nickel-agarose purification chromatography column to adsorb the lipase enzyme via binding to nickel ions. Claim 3) The method according to claim 1, wherein the preparation of the crosslinked chitosan-zinc oxide dihybrid substrate comprises the steps of dissolving nanochitosan in an acetic acid solution; adding zinc oxide to the nanochitosan and acetic acid solution to prepare a chitosan-zinc oxide mixture; stirring the chitosan-zinc oxide mixture continuously; adding an alkaline solution to the chitosan-zinc oxide mixture to form a precipitate; and washing the precipitate with distilled water and then drying it to prepare a crosslinked chitosan-zinc oxide dihybrid substrate. Claim 4) The method according to claim 3, wherein the alkaline solution is 1 molar sodium hydroxide. Claim 5) The method according to claims 3 and 4, wherein the addition of an alkaline solution to the chitosan-zinc oxide mixture is aimed at creating an ether bond between chitosan and zinc oxide. Claim 6) The method according to claim 1, wherein the modification of the crosslinked chitosan-zinc oxide dihybrid substrate with thionyl chloride comprises the steps of dissolving the substrate obtained in step b in dimethylformamide at a temperature of 80°C; adding thionyl chloride to the substrate dissolved in dimethylformamide at a temperature of 80°C for 4 hours; adjusting the pH to within 7 with ammonia to prepare a chlorinated precipitate; and washing the chlorinated precipitate with distilled water and then drying at a temperature of 25°C to prepare a chlorinated chitosan-zinc oxide dihybrid substrate. Claim 7) The method according to claim 1, wherein the modification of the chitosan-zinc oxide dihybrid substrate with ethylene diamine for binding the lipase enzyme comprises the steps of adding the chlorinated chitosan-zinc oxide substrate to ethylene diamine to prepare the amined precipitate; and freeze-drying the amined precipitate to prepare the amined chitosan-zinc oxide dihybrid substrate. Claim 8) The method according to claim 1, wherein the concentration of glutaraldehyde is 1-5%. Claim 9) The method according to claims 1 and 8, wherein the concentration of glutaraldehyde is 2.5%. Claim 10) A chitosan-zinc oxide dihybrid matrix modified with amine compounds, such that nanochitosan is attached to zinc oxide via an ether bond, for physically or covalently stabilizing the purified lipase enzyme. Claim 11) A method for immobilizing lipase enzyme on a chitosan-zinc oxide dihybrid substrate modified with amine-containing compounds, comprising: a- Purifying lipase enzyme from Escherichia coli bacteria; b- Preparing a crosslinked chitosan-zinc oxide dihybrid substrate, comprising the steps of:  Dissolving 12 mg of nanochitosan in 25 ml of 1% v / v acetic acid;  Adding 350 mg of zinc oxide to the nanochitosan / acetic acid solution and stirring the mixture for 30 minutes;  Adding 25 ml of 1 M sodium hydroxide to the mixture to form a precipitate and centrifuging it; and  Washing the precipitate with distilled water and drying it at 100°C for 3 hours to prepare a crosslinked chitosan-zinc oxide dihybrid substrate.C- Preparation of the crosslinked chitosan-zinc oxide dihybrid substrate, including the steps of: Dissolving the substrate obtained from step b in 4 mL of dimethylformamide (DMF) at 80°C; Adding 700 μL of thionyl chloride to the substrate solution in DMF for 4 hours at 80°C; Adjusting the pH to 7 with ammonia to prepare a chlorinated precipitate; and Washing and drying the chlorinated precipitate in an oven at 25°C to prepare a chlorinated chitosan-zinc oxide dihybrid substrate. D-Modification of the chitosan-zinc oxide dihybrid substrate with ethylene diamine for binding the lipase enzyme, including the steps of: Adding 0.2 g of chlorinated substrate to 11 ml of ethylene diamine and placing in a water and oil bath for 3 hours to prepare the amined precipitate; and Centrifuging the solution and washing the amined precipitate with water, then drying in a freeze-dryer to prepare the amined chitosan-zinc oxide dihybrid substrate.E- Immobilization of the purified lipase enzyme on a chitosan-zinc oxide dihybrid substrate ferritically and covalently, such that the immobilization of the purified lipase enzyme on a chitosan-zinc oxide dihybrid substrate ferritically includes the steps: I. Mixing the amine substrate with the purified enzyme in a buffer with pH 8 in a ratio of 1:1; and II. Immobilization of the purified enzyme on the amine substrate in a shaking incubator with shaking at 150 rpm at 4 ° C. Such that the immobilization of the purified lipase enzyme on a chitosan-zinc oxide dihybrid substrate ferritically includes the steps: I. Preparing a glutaraldehyde stock with a concentration of 10%; II. Adding glutaraldehyde at concentrations of 1, 2.5, and 5% to the aminated substrate for 2 hours at room temperature; III. Centrifuging the substrate and washing it to remove excess glutaraldehyde; IV. Drying the substrate in an oven at 25°C; and V. Immobilizing the purified enzyme on the aminated substrate in a shaking incubator for 1 hour so that the ratio of aminated substrate to purified enzyme is 1:5.

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