An immobilized enzyme carrier and immobilized enzyme

Abstract

The invention provides a rice bran immobilized carrier, which has at least one of the following characteristics: a) natural lipase hydrolysis activity of 50-600 U / Kg; b) oil content of 5-15 wt%; c) starch content of 8-25%; d) water holding capacity of 220-320 ml / 100 g; and e) oil holding capacity of 90-150 ml / 100 g. The invention also provides a preparation method of the rice bran immobilized carrier and an immobilized enzyme prepared by using the rice bran immobilized carrier. The invention also provides a method for catalytically preparing biodiesel by using the immobilized enzyme prepared by using the rice bran immobilized carrier. The invention also provides a method for improving the catalytic esterification activity of the lipase and / or the tolerance to short-chain alcohols.

Description

Technical Field

[0001] The invention relates to the field of immobilized enzymes, and in particular to an immobilized carrier and application thereof. Background Art

[0002] As a biocatalyst, lipase can catalyze the hydrolysis of oils and fats, and can also catalyze esterification, transesterification, acidolysis and alcoholysis under non-aqueous phase conditions. Lipase has the advantages of mild catalytic conditions, catalytic diversity and strong substrate specificity, and is widely used in food, oil, papermaking, leather, washing, pharmaceutical and other industries.

[0003] Thermomyces lanuginosus is a widely distributed fungus with a high upper growth temperature limit. It can produce a heat-stable lipase with important industrial value, namely Thermomyces lanuginosus lipase (TL lipase for short). The molecular weight of TL lipase is about 31.7kDa, the molecular size is 3.5Å×4.5Å×5.0Å, and the isoelectric point is 4.4. Compared with other types of lipases, TL lipase has mature fermentation technology, commercial production, low price, good immobilization stability, and has a good application prospect in the enzymatic preparation of biodiesel. Patent CN107012136A uses a cross-linking agent and a macroporous adsorption resin to oscillate and cross-link the immobilized Lipozyme TL 100L enzyme solution. The immobilized TL lipase catalyzes the methyl esterification of soybean oil deodorization distillate, which is reused for 20 batches, and the reaction conversion rate is above 90%. Patent CN100552003C uses commercial enzyme Lipozyme TL IM to catalyze the production of biodiesel from acid-containing oils in a solvent-free or organic solvent, and the esterification rate is about 70%. Patent CN102839166B provides a method for preparing immobilized TL lipase, which uses polystyrene non-polar macroporous adsorption resin as a carrier to immobilize TI lipase. The obtained immobilized TL lipase has high activity and stability in catalyzing transesterification reactions.

[0004] Generally speaking, after the TL lipase is immobilized, it shows more catalytic transesterification ability, but weak catalytic esterification ability, especially when it is fixed on some cheap carriers, such as silica, white carbon black, zeolite, cellulose, bentonite, etc.; when some organic polymers such as macroporous resins and ion exchange resins are used as carriers, the immobilized TL lipase can obtain better esterification activity by adjusting the carrier functional groups and immobilization methods, but the cost of the resin carrier is relatively expensive, and the immobilization cost increases. Obviously, the above technical methods are not very suitable for applying TL lipase to the reaction of catalyzing high acid value oil raw materials to prepare biodiesel. The existing technical methods still lack a low-cost immobilized TL lipase product with high catalytic esterification activity.

[0005] Rice bran refers to the by-product produced in the whitening step of brown rice in the rice processing process, and its main components are pericarp, husk, germ, endosperm, etc. Patent CN104651341B provides a method for immobilizing biological enzymes using rice bran as a carrier, but it is only used as an auxiliary agent in the water treatment process, and does not involve improving the enzyme's tolerance to short-chain alcohols in the process of immobilizing lipase. Summary of the invention

[0006] The first object of the present invention is to provide an immobilized enzyme carrier, wherein the immobilized enzyme carrier contains rice bran or consists of rice bran.

[0007] In a specific embodiment, the immobilized enzyme carrier contains rice bran.

[0008] In a specific embodiment, the immobilized enzyme carrier consists of rice bran.

[0009] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 50-600 U / Kg.

[0010] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 55-580 U / Kg.

[0011] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 55-420 U / Kg.

[0012] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 130-280 U / Kg.

[0013] In a specific embodiment, based on the mass of rice bran, the oil content in the rice bran is 5-15wt%.

[0014] In a specific embodiment, based on the mass of rice bran, the oil content in the rice bran is 6-15wt%.

[0015] In a specific embodiment, based on the mass of rice bran, the oil content in the rice bran is 6-14wt%.

[0016] In a specific embodiment, based on the mass of rice bran, the oil content in the rice bran is 9-11wt%.

[0017] In a specific embodiment, based on the mass of rice bran, the starch content in the rice bran is 8-25wt%.

[0018] In a specific embodiment, based on the mass of rice bran, the starch content in the rice bran is 8-24wt%.

[0019] In a specific embodiment, based on the mass of rice bran, the starch content in the rice bran is 8-17wt%.

[0020] In a specific embodiment, based on the mass of rice bran, the starch content in the rice bran is 10-13wt%.

[0021] In a specific embodiment, the water holding capacity of the rice bran is 220-320 ml / 100 g.

[0022] In a specific embodiment, the water holding capacity of the rice bran is 240-317 ml / 100 g.

[0023] In a specific embodiment, the water holding capacity of the rice bran is 260-290 ml / 100 g.

[0024] In a specific embodiment, the oil holding capacity of the rice bran is 90-150 ml / 100g.

[0025] In a specific embodiment, the oil holding capacity of the rice bran is 91-150 ml / 100g.

[0026] In a specific embodiment, the oil holding capacity of the rice bran is 91-130 ml / 100g.

[0027] In a specific embodiment, the oil holding capacity of the rice bran is 100-115 ml / 100g.

[0028] In a specific embodiment, the hydrolysis activity of the natural lipase in the immobilized enzyme carrier is 50-600 U / Kg.

[0029] In a specific embodiment, the hydrolysis activity of the natural lipase in the immobilized enzyme carrier is 55-580 U / Kg.

[0030] In a specific embodiment, the hydrolysis activity of the natural lipase in the immobilized enzyme carrier is 55-420 U / Kg.

[0031] In a specific embodiment, the hydrolysis activity of the natural lipase in the immobilized enzyme carrier is 130-280 U / Kg.

[0032] In a specific embodiment, based on the mass of the immobilized enzyme carrier, the oil content in the immobilized enzyme carrier is 5-15wt%.

[0033] In a specific embodiment, based on the mass of the immobilized enzyme carrier, the oil content in the immobilized enzyme carrier is 6-15wt%.

[0034] In a specific embodiment, based on the mass of the immobilized enzyme carrier, the oil content in the immobilized enzyme carrier is 6-14wt%.

[0035] In a specific embodiment, based on the mass of the immobilized enzyme carrier, the oil content in the immobilized enzyme carrier is 9-11 wt %.

[0036] In a specific embodiment, based on the mass of the immobilized enzyme carrier, the starch content in the immobilized enzyme carrier is 8-25wt%.

[0037] In a specific embodiment, based on the mass of the immobilized enzyme carrier, the starch content in the immobilized enzyme carrier is 8-24 wt %.

[0038] In a specific embodiment, based on the mass of the immobilized enzyme carrier, the starch content in the immobilized enzyme carrier is 8-17 wt %.

[0039] In a specific embodiment, based on the mass of the immobilized enzyme carrier, the starch content in the immobilized enzyme carrier is 10-13 wt %.

[0040] In a specific embodiment, the water holding capacity of the immobilized enzyme carrier is 220-320 ml / 100 g.

[0041] In a specific embodiment, the water holding capacity of the immobilized enzyme carrier is 240-317 ml / 100 g.

[0042] In a specific embodiment, the water holding capacity of the immobilized enzyme carrier is 260-290 ml / 100 g.

[0043] In a specific embodiment, the oil holding capacity of the immobilized enzyme carrier is 90-150 ml / 100 g.

[0044] In a specific embodiment, the oil holding capacity of the immobilized enzyme carrier is 91-150 ml / 100 g.

[0045] In a specific embodiment, the oil holding capacity of the immobilized enzyme carrier is 91-130 ml / 100 g.

[0046] In a specific embodiment, the oil holding capacity of the immobilized enzyme carrier is 100-115 ml / 100 g.

[0047] In a specific embodiment, the immobilized enzyme carrier further contains at least one of white carbon black, silica gel, activated carbon, diatomaceous earth, magnesium silicate, cellulose, and resin.

[0048] In a specific embodiment, the immobilized enzyme carrier further contains at least one of white carbon black, silica gel, activated carbon, diatomaceous earth, and magnesium silicate.

[0049] In a specific embodiment, the carrier other than rice bran in the immobilized enzyme carrier accounts for less than 10% of the total mass of the immobilized carrier, preferably less than 6%.

[0050] In a specific embodiment, the immobilized enzyme carrier consists of rice bran.

[0051] A second aspect of the present invention is to provide an immobilized enzyme, wherein the immobilized enzyme contains rice bran.

[0052] In a specific embodiment, based on the mass of the immobilized enzyme, the fat content in the immobilized enzyme is 5-15wt%.

[0053] In a specific embodiment, based on the mass of the immobilized enzyme, the fat content in the immobilized enzyme is 6-15wt%.

[0054] In a specific embodiment, based on the mass of the immobilized enzyme, the fat content in the immobilized enzyme is 6-14 wt %.

[0055] In a specific embodiment, based on the mass of the immobilized enzyme, the fat content in the immobilized enzyme is 9-11 wt %.

[0056] In a specific embodiment, based on the mass of the immobilized enzyme, the starch content in the immobilized enzyme is 8-25wt%.

[0057] In a specific embodiment, based on the mass of the immobilized enzyme, the starch content in the immobilized enzyme is 8-24 wt %.

[0058] In a specific embodiment, based on the mass of the immobilized enzyme, the starch content in the immobilized enzyme is 8-17 wt %.

[0059] In a specific embodiment, based on the mass of the immobilized enzyme, the starch content in the immobilized enzyme is 10-13 wt %.

[0060] In a specific embodiment, the water holding capacity of the immobilized enzyme is 220-320 ml / 100 g.

[0061] In a specific embodiment, the water holding capacity of the immobilized enzyme is 240-317 ml / 100 g.

[0062] In a specific embodiment, the water holding capacity of the immobilized enzyme is 260-290 ml / 100 g.

[0063] In a specific embodiment, the oil holding capacity of the immobilized enzyme is 90-150 ml / 100 g.

[0064] In a specific embodiment, the oil holding capacity of the immobilized enzyme is 91-150 ml / 100 g.

[0065] In a specific embodiment, the oil holding capacity of the immobilized enzyme is 91-130 ml / 100 g.

[0066] In a specific embodiment, the immobilized enzyme has an oil holding capacity of 100-115 ml / 100 g.

[0067] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 50-600 U / Kg.

[0068] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 55-580 U / Kg.

[0069] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 55-420 U / Kg.

[0070] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 130-280 U / Kg.

[0071] In a specific embodiment, based on the mass of rice bran, the oil content in the rice bran is 5-15wt%.

[0072] In a specific embodiment, based on the mass of rice bran, the oil content in the rice bran is 6-15wt%.

[0073] In a specific embodiment, based on the mass of rice bran, the oil content in the rice bran is 6-14wt%.

[0074] In a specific embodiment, based on the mass of rice bran, the oil content in the rice bran is 9-11wt%.

[0075] In a specific embodiment, based on the mass of rice bran, the starch content in the rice bran is 8-25wt%.

[0076] In a specific embodiment, based on the mass of rice bran, the starch content in the rice bran is 8-24wt%.

[0077] In a specific embodiment, based on the mass of rice bran, the starch content in the rice bran is 8-17wt%.

[0078] In a specific embodiment, based on the mass of rice bran, the starch content in the rice bran is 10-13wt%.

[0079] In a specific embodiment, the water holding capacity of the rice bran is 220-320 ml / 100 g.

[0080] In a specific embodiment, the water holding capacity of the rice bran is 240-317 ml / 100 g.

[0081] In a specific embodiment, the water holding capacity of the rice bran is 260-290 ml / 100 g.

[0082] In a specific embodiment, the oil holding capacity of the rice bran is 90-150 ml / 100g.

[0083] In a specific embodiment, the oil holding capacity of the rice bran is 91-150 ml / 100g.

[0084] In a specific embodiment, the oil holding capacity of the rice bran is 91-130 ml / 100g.

[0085] In a specific embodiment, the oil holding capacity of the rice bran is 100-115 ml / 100g.

[0086] In a specific embodiment, the immobilized enzyme carrier further contains at least one of white carbon black, silica gel, activated carbon, diatomaceous earth, magnesium silicate, cellulose, and resin.

[0087] In a specific embodiment, the immobilized enzyme carrier further contains at least one of white carbon black, silica gel, activated carbon, diatomaceous earth, and magnesium silicate.

[0088] In a specific embodiment, the mass of the carrier other than rice bran in the immobilized enzyme accounts for less than 10% of the total mass of the immobilized carrier, preferably less than 6%.

[0089] In a specific embodiment, the carrier of the immobilized lipase is selected from the carrier described in the first aspect.

[0090] In a particular embodiment the enzyme is selected from lipase, phospholipase, protease and / or amylase.

[0091] In a particular embodiment the enzyme is selected from lipase.

[0092] In a specific embodiment, the lipase is selected from at least one of TL lipase, TL lipase, CALB lipase, RM lipase or ROL lipase.

[0093] In a specific embodiment, the TL lipase is selected from: a fermentation product of Thermomyces lanuginosus and / or a TL lipase obtained by genetic engineering or protein engineering and / or a commercially available TL lipase.

[0094] In a specific embodiment, the commercially available TL lipase is selected from lipozyme TL 100L.

[0095] The third aspect of the present invention aims to provide a method for preparing rice bran or the rice bran described in the first or second aspect:

[0096] a) treating defatted rice bran A with amylase to obtain defatted rice bran B;

[0097] b) mixing defatted rice bran B with fresh full-fat rice bran to obtain mixed rice bran C; and

[0098] c) contacting the mixed rice bran C with a soluble calcium salt solution, and then separating to obtain the rice bran;

[0099] In a specific embodiment, the amylase in step a) is selected from α-amylase, β-amylase, glucoamylase, and isoamylase.

[0100] In a particular embodiment the amylase is selected from alpha-amylases.

[0101] In a particular embodiment the alpha-amylase is selected from the group consisting of high temperature amylases.

[0102] In a specific embodiment, the contact temperature between the amylase and the defatted rice bran A in step a) is 80-90°C.

[0103] In a specific embodiment, the contact time is 30 minutes.

[0104] In a specific embodiment the contacting pH is 5.0-6.6.

[0105] In a specific embodiment, in step b), the particle size of the mixed rice bran C obtained is adjusted to 140-700 meshes.

[0106] In a specific embodiment, the particle size of the defatted rice bran B and / or the fresh full-fat rice bran is adjusted to 140-700 meshes before the step b) mixing.

[0107] In a specific embodiment, the particle size adjustment method adopts at least one of dry pulverization, wet pulverization, ultrafine pulverization and low temperature pulverization.

[0108] In a specific embodiment, the mass ratio of the defatted rice bran B to the fresh full-fat rice bran is 1:4 to 4:1.

[0109] In a specific embodiment, the soluble calcium salt in step c) is selected from at least one of calcium chloride, calcium nitrate, calcium hydrogen sulfate and calcium hydrogen carbonate.

[0110] In a specific embodiment, the soluble calcium salt is selected from calcium chloride.

[0111] In a specific embodiment, the concentration of the soluble calcium salt solution is 10%-80% (w / v).

[0112] In a specific embodiment, the volume dosage of the soluble calcium salt solution is at least 1 mL / g based on the mass of the mixed rice bran C.

[0113] In a specific embodiment, the immersion time is 1-2 hr.

[0114] In a specific embodiment the immersion temperature is 25-35°C.

[0115] In a specific embodiment, the impregnation is followed by drying to a moisture content of 5-10%.

[0116] In a specific embodiment, the drying method adopts at least one of freeze drying, low-temperature vacuum drying, natural drying, and low-temperature fluidized drying.

[0117] In a specific embodiment, in the step a), the method further comprises: a1) contacting the defatted rice bran A with a buffer solution at a pH of 5.0-6.6.

[0118] In a specific embodiment, before the defatted rice bran A is treated with amylase, the defatted rice bran A is contacted with a buffer solution.

[0119] In a specific embodiment, the contact temperature between the defatted rice bran A and the buffer solution is 80-120°C.

[0120] In a specific embodiment, the buffer solution is selected from at least one of a citric acid buffer, a phosphate buffer, a potassium dihydrogen phosphate-sodium hydroxide buffer, a tris-hydrochloric acid buffer, and a PBS buffer.

[0121] In a specific embodiment, the buffer solution is selected from citrate buffer.

[0122] In a specific embodiment, the contact time is 20-40 minutes.

[0123] In a specific embodiment, the concentration of the citric acid buffer solution is 0.01-0.1 mol / L.

[0124] The fourth aspect of the present invention aims to provide rice bran prepared by the method according to the third aspect of the present invention, wherein the rice bran has at least one of the following characteristics:

[0125] 1) The hydrolysis activity of the natural lipase in the rice bran is 50-600 U / Kg;

[0126] 2) The oil content in the rice bran is 5-15wt%;

[0127] 3) The starch content in the rice bran is 8-25%;

[0128] 4) The water holding capacity of the rice bran is 220-320 ml / 100 g;

[0129] 5) The oil holding capacity of the rice bran is 90-150 ml / 100g.

[0130] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 50-600 U / Kg.

[0131] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 55-580 U / Kg.

[0132] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 55-420 U / Kg.

[0133] In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 130-280 U / Kg.

[0134] In a specific embodiment, the oil content in the rice bran is 5-15wt%.

[0135] In a specific embodiment, the oil content in the rice bran is 6-15wt%.

[0136] In a specific embodiment, the oil content in the rice bran is 6-14wt%.

[0137] In a specific embodiment, the oil content in the rice bran is 9-11 wt %.

[0138] In a specific embodiment, the starch content in the rice bran is 8-25wt%.

[0139] In a specific embodiment, the starch content in the rice bran is 8-24 wt %.

[0140] In a specific embodiment, the starch content in the rice bran is 8-17 wt %.

[0141] In a specific embodiment, the starch content in the rice bran is 10-13 wt %.

[0142] In a specific embodiment, the water holding capacity of the rice bran is 220-320 ml / 100 g.

[0143] In a specific embodiment, the water holding capacity of the rice bran is 240-317 ml / 100 g.

[0144] In a specific embodiment, the water holding capacity of the rice bran is 260-290 ml / 100 g.

[0145] In a specific embodiment, the oil holding capacity of the rice bran is 90-150 ml / 100g.

[0146] In a specific embodiment, the oil holding capacity of the rice bran is 91-150 ml / 100g.

[0147] In a specific embodiment, the oil holding capacity of the rice bran is 91-130 ml / 100g.

[0148] In a specific embodiment, the oil holding capacity of the rice bran is 100-115 ml / 100g.

[0149] The fifth aspect of the present invention aims to provide an immobilized enzyme containing the immobilized enzyme carrier according to the first aspect of the present invention.

[0150] In a particular embodiment the enzyme is selected from lipase, phospholipase, protease and / or amylase.

[0151] In a particular embodiment the enzyme is selected from lipase.

[0152] In a specific embodiment, the lipase is selected from at least one of TL lipase, TL lipase, CALB lipase, RM lipase or ROL lipase.

[0153] In a specific embodiment, the TL lipase is selected from: a fermentation product of Thermomyces lanuginosus and / or a TL lipase obtained by genetic engineering or protein engineering and / or a commercially available TL lipase.

[0154] In a specific embodiment, the commercially available TL lipase is selected from lipozyme TL 100L.

[0155] In a specific embodiment, the immobilized enzyme further contains at least one carrier selected from the group consisting of white carbon black, silica gel, activated carbon, diatomaceous earth, magnesium silicate, cellulose, and resin.

[0156] In a specific embodiment, the immobilized enzyme further contains at least one carrier selected from the group consisting of white carbon black, silica gel, activated carbon, diatomaceous earth, and magnesium silicate.

[0157] In a specific embodiment, the mass of the carrier other than rice bran in the immobilized enzyme accounts for less than 10% of the total mass of the immobilized carrier, preferably less than 6%.

[0158] The sixth aspect of the present invention provides a method for preparing the immobilized enzyme as described in the fifth aspect of the present invention, characterized in that the immobilized enzyme carrier as described in the first aspect of the present invention is contacted with the enzyme.

[0159] In a specific embodiment, the immobilized enzyme is prepared by at least one of adsorption, covalent binding, embedding and cross-linking methods.

[0160] The object of the seventh aspect of the present invention is to provide an application of the immobilized enzyme as described in the second aspect of the present invention or the fifth aspect of the present invention in at least one reaction selected from catalyzing esterification, transesterification, alcoholysis, acidolysis and / or hydrolysis.

[0161] The eighth aspect of the present invention is to provide a use of the immobilized enzyme as described in the second aspect of the present invention or the fifth aspect of the present invention in the preparation of biodiesel.

[0162] The ninth aspect of the present invention aims to provide a method for improving the lipase-catalyzed esterification activity and / or short-chain alcohol tolerance, characterized in that the immobilized carrier described in the first aspect of the present invention is contacted with the enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0163] Figure 1 The present invention relates to a biodiesel preparation process in which the immobilized TL lipase catalyzes the esterification of oleic acid and methanol. The present invention relates to a biodiesel preparation process in which the immobilized TL lipase is catalyzed by the immobilized TL lipase ...

[0164] Figure 2 The present invention relates to an immobilized TL lipase. The present invention relates to an immobilized TL lipase. The present invention relates to an immobilized TL lipase. The present invention relates to an immobilized TL lipase. The present invention relates to an immobilized TL lipase. The present invention relates to an immobilized TL lipase. The present invention relates to an immobilized TL lipase. The present invention relates to an immobilized TL lipase. The present invention relates to an immobilized TL lipase. The present invention relates to an immobilized TL lipase. DETAILED DESCRIPTION

[0165] The following examples are used to specifically illustrate the embodiments and technical effects of the present invention, but the present invention is not limited to the following examples.

[0166] raw material:

[0167] Defatted rice bran source: Yihai Kerry Group Jiamusi Factory

[0168] Full fat rice bran source: Yihai Kerry Group Jiamusi Factory

[0169] Source of high temperature amylase: purchased from Hebei Pengyu Biotechnology Co., Ltd.

[0170] Lipase source: lipozymeTL 1OOL, purchased from Novozymes

[0171] Calcium chloride: purchased from Sinopharm Group

[0172] Citric acid source: purchased from Sinopharm Group

[0173] Crushing equipment: Tester high-speed universal crusher FW100

[0174] Freeze Dryer: Guansen Biotechnology Shanghai Company

[0175] Oil source: Wilmar Oil Technology (Shanghai) Co., Ltd.

[0176] Methanol source: purchased from Sinopharm Group

[0177] Ethanol source: purchased from Sinopharm Group

[0178] Biodiesel testing methods:

[0179] Oleic acid conversion rate = (initial acid value of oil phase - acid value of oil phase after reaction) / initial acid value of oil phase × 100%, where the determination method of oil phase acid value refers to "GB / T 5530-2005 Determination of acid value and acidity of animal and vegetable oils and fats"

[0180] Rice bran detection method:

[0181] The method for determining the hydrolysis activity of natural lipase in rice bran refers to the literature "Anita V. Prabhu, Shreehas P. Tambe, et al. Rice bran lipase: extraction, activity and stability. Biotechnol. Prog. 1999, 15, 1083-1089.".

[0182] The method for determining the oil content in rice bran refers to "GB / T 6433-2006 Determination of crude fat in feeds".

[0183] The method for determining the starch content in rice bran refers to "GB / T 20194-2018 Determination of starch content in animal feed - Polarimetry".

[0184] The method for determining the water-holding capacity and oil-holding capacity of rice bran is as follows: Weigh 100 g of rice bran sample into a 1 L beaker, pour in 500 mL of distilled water (or soybean oil), place the beaker in a 20°C water bath to absorb water (or oil) at a constant temperature for 2 hours, then invert the water-absorbed (or oil-absorbed) rice bran on the filter cloth until no continuous droplets drip, measure the volume V of the flowing liquid, and the water-holding capacity (or oil-holding capacity) = 500 – V. Example 1

[0185] Weigh 50g defatted rice bran in a 1L beaker, add 250mL 0.01moL / L pH5.0 citric acid-sodium citrate buffer, boil in boiling water for 30min, lower the temperature to 80°C, add 0.2wt% (based on the weight of defatted rice bran) of high-temperature α-amylase, stir for 30min, filter, and then mix evenly with 200g fresh rice bran in a ratio of 1:4 (based on the weight of defatted rice bran), grind to a particle size of 140-700 mesh, add 500mL CaCl2 saturated solution, stir and soak at 25°C for 2h, filter, pre-freeze with liquid nitrogen, and freeze-dry to a moisture content of 5%; then add 125mL TL lipase enzyme solution to the pretreated rice bran carrier, and add 250mL 0.01 mol / L sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 was added and stirred for 2 h, and then 10 mL of 0.5% glutaraldehyde solution was added for cross-linking reaction for 1 h; the rice bran part was separated by filtration and naturally dried at room temperature to a moisture content of 10% to obtain immobilized TL lipase 1. Example 2

[0186] Weigh 50g defatted rice bran in a 1L beaker, add 250mL 0.03mol / L pH5.4 citric acid-sodium citrate buffer, boil in boiling water for 30min, reduce the temperature to 90℃, add 0.3wt% (based on the weight of defatted rice bran) of high temperature α-amylase, stir for 30min, filter, and then mix with 100g fresh rice bran in a ratio of 1:2 (based on the weight of defatted rice bran), grind to a particle size of 140-700 mesh, add 300mL of 10% mass concentration CaCl2 solution, stir and soak at 30℃ for 1h, filter and pre-freeze with liquid nitrogen, and freeze-dry to a moisture content of 7%; then add 150mL TL lipase enzyme solution to the pretreated rice bran carrier, and add 150mL 0.01 mol / L sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 was added and stirred for 3 h, and then 8 mL of 0.5% glutaraldehyde solution was added for cross-linking reaction for 0.5 h; the rice bran part was separated by filtration and vacuum dried at 40°C to a moisture content of 8% to obtain immobilized TL lipase 2. Example 3

[0187] Weigh 50g defatted rice bran in a 1L beaker, add 250mL 0.06moL / L citric acid-sodium citrate buffer with pH 5.8, boil in boiling water for 30min, lower the temperature to 85°C, add 0.4wt% (based on the weight of defatted rice bran) of high-temperature α-amylase, stir for 30min, filter, and then mix evenly with 50g fresh rice bran in a ratio of 1:1 (based on the weight of defatted rice bran), grind to a particle size of 140-700 mesh, add 100mL of 40% mass concentration CaCl2 solution, stir and soak at 35°C for 1.5h, filter, pre-freeze with liquid nitrogen, and freeze-dry to a moisture content of 10%; then add 200mL TL lipase enzyme solution to the pretreated rice bran carrier, and add 100mL 0.01 mol / L sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 was added, stirred for 4 hours, and then 6 mL of 0.5% glutaraldehyde solution was added for cross-linking reaction for 0.5 hours; the rice bran part was separated by filtration and freeze-dried to a moisture content of 5% to obtain immobilized TL lipase 3. Example 4

[0188] Weigh 50g defatted rice bran in a 1L beaker, add 250mL 0.1moL / L pH6.0 citric acid-sodium citrate buffer, boil in boiling water for 30min, lower the temperature to 85°C, add 0.3wt% (based on the weight of defatted rice bran) of high-temperature α-amylase, stir for 30min, filter, and then mix evenly with 25g fresh rice bran in a ratio of 2:1 (based on the weight of defatted rice bran), grind to a particle size of 140-700 mesh, add 75mL of 60% mass concentration CaCl2 solution, stir and soak at 30°C for 1.5h, filter, pre-freeze with liquid nitrogen, and freeze-dry to a moisture content of 8%; then add 225mL TL lipase enzyme solution to the pretreated rice bran carrier, and add 75mL 0.01 mol / L sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 was added and stirred for 3 h, and then 3 mL of 0.5% glutaraldehyde solution was added for cross-linking reaction for 1 h; the rice bran part was separated by filtration and vacuum dried at 35°C to a moisture content of 7% to obtain immobilized TL lipase 4. Example 5

[0189] Weigh 100g defatted rice bran in a 1L beaker, add 500mL 0.01moL / L pH6.6 citric acid-sodium citrate buffer, boil in boiling water for 30min, reduce the temperature to 80°C, add 0.3wt% (based on the weight of defatted rice bran) of high-temperature α-amylase, stir for 30min, filter, and then mix evenly with 25g fresh rice bran in a ratio of 4:1 (based on the weight of defatted rice bran), grind to a particle size of 140-700 mesh, add 125mL of 80% mass concentration CaCl2 solution, stir and soak at 35°C for 2h, filter, pre-freeze with liquid nitrogen, and freeze-dry to a moisture content of 5%; then add 375mL TL lipase enzyme solution to the pretreated rice bran carrier, and add 125mL 0.01 mol / L sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 was added and stirred for 3 h, and then 5 mL of 0.5% glutaraldehyde solution was added for cross-linking reaction for 1 h; the rice bran part was separated by filtration and naturally dried at room temperature to a moisture content of 9% to obtain immobilized TL lipase 5. Example 6

[0190] Weigh 70g defatted rice bran in a 1L beaker, add 350mL 0.01moL / L pH6.6 citric acid-sodium citrate buffer, boil in boiling water for 30min, lower the temperature to 80℃, add 0.3wt% (based on the weight of defatted rice bran) of high-temperature α-amylase, stir for 30min, filter, add 30g white carbon black, and then mix it with 400g fresh rice bran in a ratio of 1:4 (based on the total weight of defatted rice bran and white carbon black), grind to a particle size of 140-700 mesh, add 500mL CaCl2 saturated solution, stir and soak at 25℃ for 2h, filter, pre-freeze with liquid nitrogen, and freeze-dry to a moisture content of 5%; then add 125mL TL lipase enzyme solution to the above-mentioned pretreated carrier, and add 250mL 0.01 mol / L sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 was added and stirred for 2 h, and then 10 mL of 0.5% glutaraldehyde solution was added for cross-linking reaction for 1 h; the rice bran part was separated by filtration and naturally dried at room temperature to a moisture content of 10% to obtain immobilized TL lipase 6.

[0191] Comparative Example 1

[0192] Fresh full-fat rice bran was directly used as the immobilization carrier, and the immobilization conditions and drying conditions were exactly the same as those in Example 1.

[0193] Comparative Example 2

[0194] Stabilized defatted rice bran was directly used as the immobilization carrier, and the immobilization conditions and drying conditions were exactly the same as those in Example 1.

[0195] Comparative Example 3

[0196] Defatted and destarched rice bran was directly used as the immobilization carrier, and the immobilization conditions and drying conditions were exactly the same as those in Example 1.

[0197] Determination of relevant indicators of rice bran carrier:

[0198]

[0199] Experimental Results

[0200] 1. Esterification activity assay

[0201] Weigh 20 g of oleic acid (oleic acid purity is 75%) into a 50 mL round-bottom flask, add 3% immobilized TL lipase (based on the mass of the oil phase substrate), add 6 mL of methanol three times within 2 h, react at 40 ° C for 4 h, and measure the oleic acid conversion rate.

[0202] Oleic acid conversion rate = (initial acid value of oil phase - acid value of oil phase after reaction) / initial acid value of oil phase × 100%;

[0203]

[0204] From the experimental results, it can be seen that the oleic acid conversion rate of the rice bran immobilized TL lipase of the present invention in the catalytic esterification reaction of oleic acid and methanol is more than 93%, which is more than 20% higher than the conversion rate of the commercial enzyme Lipozyme TL IM (TL is immobilized on a silica carrier). In Comparative Example 1, fresh rice bran is used for immobilizing TL lipase, and fresh rice bran itself lacks pores and has a relatively dense structure, which is not conducive to the deposition and adsorption of TL lipase. Therefore, the loading amount of TL on fresh rice bran is low, and the esterification activity is also the lowest; in Comparative Examples 2 and 3, defatted rice bran or defatted and destarched rice bran is used for immobilizing TL lipase, which is conducive to the loading and adsorption of TL lipase, but due to the lack of natural rice bran lipase and the pre-activation effect of rice bran lipid hydrolysis on TL lipase, the activity of the immobilized TL lipase in Comparative Example 2 is not significantly improved.

[0205] 2. Methanol tolerance test

[0206] Weigh 20g of oleic acid (oleic acid purity is 75%) into a 50mL round-bottom flask and add 3% Figure 1 or Figure 2 The immobilized TL lipase (based on the mass of the oil phase substrate) was added at once with 6 mL and 12 mL of methanol (the molar ratio of oleic acid to methanol was 1:2 and 1:4, respectively) and reacted for 4 h. After the reaction, the immobilized TL lipase was filtered out and used for the next batch of reactions. Figure 1 or Figure 2 The indicated number of batches were reacted.

[0207] Since short-chain alcohols such as methanol have a strong inactivating effect on lipase, methanol is often added in batches or in a flow-feed manner during the preparation of biodiesel. Figure 2 or Figure 1 As shown, although the above method can reduce the inactivation of enzymes to a certain extent, it also greatly reduces the reaction efficiency. Compared with the resin-immobilized TL lipase, the rice bran-immobilized TL lipase of the present invention has better methanol tolerance, especially in a reaction system with high methanol concentration, the inactivation rate of the rice bran-immobilized TL lipase is significantly slower than that of the resin-immobilized lipase.

Claims

1. An immobilized enzyme carrier, characterized in that: The immobilized enzyme carrier is composed of rice bran; The hydrolysis activity of the natural lipase in the rice bran is 50-600 U / Kg; The starch content in the rice bran is 8-25wt% based on the mass of the rice bran; The water holding capacity of the rice bran is 220-320 ml / 100 g; The oil holding capacity of the rice bran is 90-150 ml / 100g; Described rice bran preparation method is as follows: a) treating defatted rice bran A with amylase to obtain defatted rice bran B; b) mixing defatted rice bran B with fresh full-fat rice bran to obtain mixed rice bran C; and c) after the mixed rice bran C is contacted with a soluble calcium salt solution, separating the rice bran to obtain; Before step a), the method further comprises contacting the defatted rice bran A with a buffer solution at a pH of 5.0-6.6; the contact temperature of the defatted rice bran A with the buffer solution is 80-120° C. The amylase in step a) is α-amylase; The mass ratio of the defatted rice bran B to the fresh full-fat rice bran in step b) is 1:4 to 4:1; The concentration of the soluble calcium salt solution in step c) is 10%-80% (w / v).

2. The immobilized enzyme carrier according to claim 1, characterized in that The rice bran has at least one of the following characteristics: The hydrolysis activity of the natural lipase in the rice bran is 55-580 U / Kg; The starch content in the rice bran is 8-24wt% based on the mass of the rice bran; The water holding capacity of the rice bran is 240-317 ml / 100 g; The oil holding capacity of the rice bran is 91-150 ml / 100g.

3. The immobilized enzyme carrier according to claim 1, characterized in that The rice bran has at least one of the following characteristics: The hydrolysis activity of the natural lipase in the rice bran is 55-420 U / Kg; The starch content in the rice bran is 8-17wt% based on the mass of the rice bran; The water holding capacity of the rice bran is 260-290 ml / 100 g; The oil holding capacity of the rice bran is 91-130 ml / 100g.

4. The immobilized enzyme carrier according to claim 1, characterized in that The rice bran has at least one of the following characteristics: The hydrolysis activity of the natural lipase in the rice bran is 130-280 U / Kg; The starch content in the rice bran is 10-13wt% based on the mass of the rice bran; The oil holding capacity of the rice bran is 100-115 ml / 100g.

5. The immobilized enzyme carrier according to any one of claims 1 to 4, characterized in that The rice bran has the following characteristics: Calculated by the mass of rice bran, the oil content in the rice bran is 5-15wt%.

6. The immobilized enzyme carrier according to any one of claims 1 to 4, characterized in that The immobilized enzyme carrier has the following characteristics: Calculated by the mass of rice bran, the oil content in the rice bran is 6-15wt%.

7. The immobilized enzyme carrier according to any one of claims 1 to 4, characterized in that The immobilized enzyme carrier has the following characteristics: Calculated by the mass of rice bran, the oil content in the rice bran is 6-14wt%.

8. The immobilized enzyme carrier according to any one of claims 1 to 4, characterized in that The immobilized enzyme carrier has the following characteristics: Calculated by the mass of rice bran, the oil content in the rice bran is 9-11wt%.

9. An immobilized enzyme, characterized in that The immobilized enzyme comprises the immobilized enzyme carrier according to any one of claims 1 to 8 and TL lipase, and the TL lipase is a commercially available TL lipase.

10. The immobilized enzyme according to claim 9, characterized in that The commercially available TL lipase is selected from lipozyme TL100L.

11. A method for preparing an immobilized enzyme carrier, characterized in that: a) treating defatted rice bran A with amylase to obtain defatted rice bran B; b) mixing defatted rice bran B with fresh full-fat rice bran to obtain mixed rice bran C; and c) contacting the mixed rice bran C with a soluble calcium salt solution, and then separating to obtain the rice bran; The immobilized enzyme carrier is composed of rice bran; Before step a), the method further comprises contacting the defatted rice bran A with a buffer solution at a pH of 5.0-6.6; the contact temperature of the defatted rice bran A with the buffer solution is 80-120° C. The amylase in step a) is α-amylase; The mass ratio of the defatted rice bran B to the fresh full-fat rice bran in step b) is 1:4 to 4:1; The concentration of the soluble calcium salt solution in step c) is 10%-80% (w / v).

12. The preparation method according to claim 11, characterized in that: The method has at least one of the following characteristics: In step a), the contact temperature of the amylase and the defatted rice bran A is 80-90° C., or the contact time is 30 min, or the contact pH is 5.0-6.6; In the step b), the particle size of the mixed rice bran C obtained is adjusted to 140-700 mesh; or In step c), the soluble calcium salt is selected from at least one of calcium chloride, calcium nitrate, calcium hydrogen sulfate and calcium hydrogen carbonate.

13. The preparation method according to claim 12, characterized in that: The amylase is selected from high temperature amylase; or Before step b) of mixing, the particle size of the defatted rice bran B and / or the fresh full-fat rice bran is adjusted to 140-700 meshes.

14. The preparation method according to claim 13, characterized in that: The particle size adjustment method adopts at least one of dry pulverization, wet pulverization, ultrafine pulverization and low temperature pulverization.

15. The preparation method according to claim 14, characterized in that: In the step c), The volume dosage of the soluble calcium salt solution is at least 1 mL / g based on the mass of the mixed rice bran C; or the immersion time is 1-2 hr; or the immersion temperature is 25-35° C.; or after immersion, the solution is dried to a moisture content of 5-10%.

16. The preparation method according to claim 15, characterized in that The drying method adopts at least one of freeze drying, low-temperature vacuum drying, natural drying, and low-temperature fluidized drying.

17. The preparation method according to any one of claims 11 to 16, characterized in that: The buffer solution is selected from at least one of a citric acid buffer, a phosphate buffer, and a potassium dihydrogen phosphate-sodium hydroxide buffer; or the contact time is 20-40 minutes.

18. The preparation method according to claim 17, characterized in that: The concentration of the citric acid buffer solution is 0.01-0.1 mol / L.

19. An immobilized enzyme carrier prepared by the method according to any one of claims 11 to 18, characterized in that: The immobilized enzyme carrier has at least one of the following characteristics; 1) The hydrolysis activity of the natural lipase in the rice bran is 50-600 U / Kg; 2) The rice bran has a fat content of 5-15wt% based on the mass of the rice bran; 3) The rice bran has a starch content of 8-25% based on the mass of the rice bran; 4) The water holding capacity of the rice bran is 220-320 ml / 100 g; or 5) The oil holding capacity of the rice bran is 90-150 ml / 100g.

20. The immobilized enzyme carrier according to claim 19, characterized in that The rice bran has at least one of the following characteristics; 1) The hydrolysis activity of the natural lipase in the rice bran is 55-580 U / Kg; 2) The rice bran has a fat content of 6-15wt% based on the mass of the rice bran; 3) The rice bran has a starch content of 8-24 wt % based on the mass of the rice bran; 4) The water holding capacity of the rice bran is 240-317 ml / 100 g; or 5) The oil holding capacity of the rice bran is 91-150 ml / 100g.

21. The immobilized enzyme carrier according to claim 19, characterized in that The rice bran has at least one of the following characteristics; 1) The hydrolysis activity of the natural lipase in the rice bran is 55-420 U / Kg; 2) The rice bran has a fat content of 6-14wt% based on the mass of the rice bran; 3) The rice bran has a starch content of 8-17 wt % based on the mass of the rice bran; 4) The water holding capacity of the rice bran is 260-290 ml / 100 g; or 5) The oil holding capacity of the rice bran is 91-130 ml / 100g.

22. The immobilized enzyme carrier according to claim 19, characterized in that The rice bran has at least one of the following characteristics; 1) The hydrolysis activity of the natural lipase in the rice bran is 130-280 U / Kg; 2) The rice bran has a fat content of 9-11 wt % based on the mass of the rice bran; 3) The rice bran has a starch content of 10-13 wt % based on the mass of the rice bran; or 4) The oil holding capacity of the rice bran is 100-115 ml / 100g.

23. A method for preparing the immobilized enzyme according to any one of claims 9 to 10, characterized in that: The immobilized enzyme carrier according to any one of claims 1 to 8 is contacted with an enzyme.

24. The method of claim 23, wherein: The immobilized enzyme is prepared by at least one of adsorption method, covalent binding method, embedding method and cross-linking method.

25. Use of the immobilized enzyme according to any one of claims 9 to 10 in catalyzing at least one reaction selected from esterification, transesterification, alcoholysis, acidolysis and hydrolysis.

26. Use of the immobilized enzyme according to any one of claims 9 to 10 in the preparation of biodiesel.

27. A method for improving the catalytic esterification activity and / or short-chain alcohol tolerance of TL lipase, characterized in that: The immobilized enzyme carrier according to any one of claims 1 to 8 is used to contact the enzyme.

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