CN1944629A - Full cell catalyst of intracellular secretory lipase type and its preparing method - Google Patents

Full cell catalyst of intracellular secretory lipase type and its preparing method Download PDF

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CN1944629A
CN1944629A CN 200610124855 CN200610124855A CN1944629A CN 1944629 A CN1944629 A CN 1944629A CN 200610124855 CN200610124855 CN 200610124855 CN 200610124855 A CN200610124855 A CN 200610124855A CN 1944629 A CN1944629 A CN 1944629A
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thalline
yeast
nah
hpo
damping fluid
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CN100455656C (en
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江木兰
胡小加
晏立英
张银波
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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Abstract

The present invention relates to one kind of full cell catalyst with lipase activity and its preparation process. The intracellular lipase secreting full cell catalyst is prepared with the materials including saccharomycetes 0.7-1.2 wt%, diatomite 2.1-3.6 wt%, sodium alginate 2.0-2.3 wt%, calcium dichloride 0.5-0.8 wt%, and pH 7.7 Na2HPO4-NaH2PO4 buffer solution of 92.1-94.7 wt%, with the saccharomycetes to diatomite weight ratio being 1 to 3. The intracellular lipase secreting full cell catalyst is used to replace lipase in producing biological diesel oil, and has simple preparation process, easy product recovery, reusability and low production cost.

Description

Full cell catalyst of intracellular secretory lipase type and preparation method thereof
Technical field
The present invention relates to a kind of whole-cell catalyst and preparation method thereof with lipase activity.
Background technology
In the production of biofuel, lipase is the production cost height of lipase as the biggest obstacle of the large-scale production of catalyzer, is difficult to realize large-scale industrial production and application.The biological full cell that produces lipase is as catalyzer (abbreviation whole-cell catalyst), both the microorganism strains of yielding lipase has been carried out fermentation culture after, collect thalline and replace lipase through after the simple process, participate in the reaction of synthetic mono fatty acid methyl esters.Whole-cell catalyst has saved the complicated procedures of forming such as separation, extraction, purifying and immobilization of lipase, is to overcome one of present lipase production, effective way that application cost is high.
About the report of the biological whole-cell catalyst of yielding lipase seldom, and be in conceptual phase.Present rarely seen abroad report from Japanese scientist.First report is the yeast saccharomyces cerevisiae that Matsumoto etc. utilizes great expression Rhizopus oryzae lipase in the cell, adopt freeze thawing or air-dry the grade to handle the back and participate in and react, but yeast cell can not recycling.The 2nd report be with the Rhizopus oryzae cell fixation in the polyurethane foam particle, handle the back with glutaraldehyde cross-linking and participate in reaction, can use repeatedly 6 times.Nearest Matsumoto etc. tests with the yeast of exocytosis lipase again.They have made up a new yeast cell surface, and as FS albumen or the proteic cell walls of FL anchorage zone, the recombinant lipase albumen of yeast expression can merge with FS or FL albumen, and fusion rotein is distributed in the cell surface of new structure.Achieve success as whole-cell catalyst with this cell, but also do not have reusable report.Domestic rarely seen Tsing-Hua University once waits quietly with the directly participation reaction practice of the dry and freezing back of mould, but does not see reusable report.
Summary of the invention
The object of the present invention is to provide that a kind of production process is simple, cost is low, repeatedly used full cell catalyst of intracellular secretory lipase type and preparation method thereof.
To achieve these goals, technical scheme of the present invention is: full cell catalyst of intracellular secretory lipase type is characterized in that: it mainly is 7.7 Na by yeast thalline, diatomite, sodium alginate, U-Ramin MC and pH value 2HPO 4-NaH 2PO 4The damping fluid feedstock production forms, and the per-cent of the shared gross weight of each raw material is:
Yeast thalline 0.7-1.2, diatomite 2.1-3.6,
Sodium alginate 2.0-2.3, U-Ramin MC 0.5-0.8,
The pH value is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid 92.1-94.7; Wherein yeast thalline and diatomaceous weight ratio=1: 3, the shared total weight percent sum of each raw material is 100.
The optimized percentage of the shared gross weight of each raw material is:
Yeast thalline 1.0, diatomite 3.0,
Sodium alginate 2.0, U-Ramin MC 0.56,
The pH value is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid 93.44.
The preparation method of above-mentioned full cell catalyst of intracellular secretory lipase type is characterized in that it comprises the steps:
1). choosing of raw material: the per-cent by the shared gross weight of each raw material is: yeast thalline 0.7-1.2, diatomite 2.1-3.6, sodium alginate 2.0-2.3, U-Ramin MC 0.5-0.8, pH value are 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid 92.1-94.7, wherein yeast thalline and diatomaceous weight ratio=1: 3, the shared total weight percent sum of each raw material is 100, chooses yeast thalline, diatomite, sodium alginate, U-Ramin MC and pH value and be 7.7 Na 2HPO 4-NaH 2PO 4The damping fluid raw material, standby;
2). yeast body and function pH value is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid washed twice, pH value are 7.7 Na 2HPO 4-NaH 2PO 4The weight consumption of damping fluid is the twice of yeast thalline, lyophilize;
3). the immobilization first time of thalline: the thalline of lyophilize gained adds diatomite (fixation support press yeast thalline and diatomaceous weight ratio=1: 3 adding diatomite for the first time), in container, break up thalline and with the diatomite mixing; The pH value that adds yeast body weight twice is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid, after stirring 1 hour under 25 ℃ of conditions, centrifugal collection solid with collected solids washed with acetone 2 times, disperses carrier granule, lyophilize once more, time immobilized whole-cell of winning;
4). the immobilization second time of thalline: sodium alginate (the 2nd fixation support) is made into water solution A, is 7.7 Na in addition with remaining pH value 2HPO 4-NaH 2PO 4The damping fluid preparation first time, immobilized full cell became the solution B of 14.5-15.5%, merged two solution, fully stirred, and final sodium alginate concentration is 2%, gets mixing solutions; With syringe this mixing solutions is dropwise joined the aseptic Ca of 0.05mol/L 2In the Cl solution, solidify after 1 hour and take out, centrifugal collection solid is with distillation washing 2 times, to remove the unnecessary Ca in immobilized particlesization surface 2Cl, vacuum-drying promptly gets product.The full cell of cell internal secretion type yeast of moulding is a particulate state, and particle diameter is about 1mm.
The preparation of described yeast thalline:
1). choosing of bacterial classification and substratum: (1) bacterial classification: by dilution-plate method oily factory waste oil is inserted and to separate with carrying out separation and Culture in the substratum, separating with substratum is the potato sucrose substratum, (separation method is seen " agriculture Experiment on Microbiology technology " 69-72 page or leaf must to produce the yeast (Candidalipolytica Candida lipolytica) of emiocytosis type lipase, Chinese agriculture press, in May, 1996, the 1st edition); Wherein the potato sucrose substratum is: the 200g potato be cut into small pieces and be boiled into juice, potato juice and 20g sucrose are dissolved in the 1L water, and add 20g agar, 121 ℃ of high-temperature sterilization 15min, the potato sucrose substratum; (2) fermentation is used substratum: soybean oil 29.0g/L, sucrose 1.0g/L, peptone 70.0g/L, NaNO 31.0g/L, KH 2PO 41.0g/L, MgSO 47H 2O 0.5g/L sterilized 20 minutes for 121 ℃;
2). fermentation culture: the fermentation substratum of packing in the sterile chamber and accounting for volume 1/3, inoculation produces the barms of emiocytosis type lipase; Put 28 ℃, 150r/min cultivated the liquid after must fermenting 72 hours;
3). collect thalline: centrifugal 10 minutes of the liquid 4700r/min after will fermenting, collect thalline, get the yeast thalline.
The present invention successfully replaces lipase as the production of biodiesel catalyzer, and can repeatedly use repeatedly after adopting 2 kinds of carriers of different nature and secondary fixation method that the yeast thalline is fixed.Its preparation method is simple, and finished product easily reclaims, and is convenient to repeatedly use, and has reduced production cost.
The invention has the beneficial effects as follows:
1. make simple: production process of the present invention is simple, does not need complex apparatus; After strains tested carried out fermentation culture, collect thalline and carry out drying, fixing and freeze thawing is existing, be trapped in intracellular lipase and be equivalent to be fixed.
2. reduce cost: the form with the whole-cell biological catalyzer is utilized lipase, separation and Extraction, purifying and the operation such as fixing of lipase had both been saved, also avoid the enzyme loss in this process, also save great deal of investment, working cost, in suitability for industrialized production, had higher cost efficiency.
3. advanced technology: the full cell of the yeast of cell internal secretion lipase replaces lipase to be used for the transesterification catalyzer, though successfully report has been arranged, not seeing has the immobilization of the full cell of yeast manufacturing technology and nonexpondable report; The present invention adopts macromolecular carrier and 2 fixation methods, and the yeast cell after fixing is collected easily and had a lipase activity.
4. use for many times: adopts the rate of recovery after the yeast whole-cell catalyst use that 2 fixation methods make higher by 10% than the rate of recovery of other method; In grease-methyl alcohol reactive system, use 6 active nothings obviously to descend at least.
Embodiment
In order to understand the present invention better, further illustrate content of the present invention below in conjunction with embodiment, but content of the present invention not only is confined to the following examples.
Embodiment 1:
The preparation method of full cell catalyst of intracellular secretory lipase type, it comprises the steps:
1). choosing of bacterial classification and substratum: (1) bacterial classification: by dilution-plate method oily factory waste oil is inserted and to separate with carrying out separation and Culture in the substratum, separating with substratum is the potato sucrose substratum, (separation method is seen " agriculture Experiment on Microbiology technology " 69-72 page or leaf must to produce the yeast (Candidalipolytica Candida lipolytica) of emiocytosis type lipase, Chinese agriculture press, in May, 1996, the 1st edition); Wherein the potato sucrose substratum is: the 200g potato be cut into small pieces and be boiled into juice, potato juice and 20g sucrose are dissolved in the 1L water, and add 20g agar, 121 ℃ of high-temperature sterilization 15min, the potato sucrose substratum; (2) fermentation is used substratum: soybean oil 29.0g/L, sucrose 1.0g/L, peptone 70.0g/L, NaNO 31.0g/L, KH 2PO 41.0g/L, MgSO 47H 2O 0.5g/L sterilized 20 minutes for 121 ℃;
2). fermentation culture: the fermentation substratum of packing in the sterile chamber and accounting for volume 1/3, inoculation produces the barms of emiocytosis type lipase; Put 28 ℃, 150r/min cultivated the liquid after must fermenting 72 hours;
3). collect thalline: centrifugal 10 minutes of the liquid 4700r/min after will fermenting, collect thalline, get the yeast thalline;
4). choosing of raw material: the per-cent of the shared gross weight of each raw material is: yeast thalline 1.0, diatomite 3.0, sodium alginate 2.0, U-Ramin MC 0.56, pH value are 7.7 Na 2HPO 4-NaH 2PO 4It is 7.7 Na that damping fluid 93.44 is chosen yeast thalline, diatomite, sodium alginate, U-Ramin MC, pH value 2HPO 4-NaH 2PO 4The damping fluid raw material, standby;
5). yeast body and function pH value is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid washed twice, pH value are 7.7 Na 2HPO 4-NaH 2PO 4The weight consumption of damping fluid is the twice of yeast thalline, lyophilize;
6). the immobilization first time of thalline: the thalline of lyophilize gained adds diatomite (fixation support press yeast thalline and diatomaceous weight ratio=1: 3 adding diatomite for the first time), in container, break up thalline and with the diatomite mixing; The pH value that adds yeast body weight twice is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid, after stirring 1 hour under 25 ℃ of conditions, centrifugal collection solid with collected solids washed with acetone 2 times, disperses carrier granule, lyophilize once more, time immobilized whole-cell of winning;
7). the immobilization second time of thalline: sodium alginate (fixation support for the second time) is made into water solution A, is 7.7 Na in addition with remaining pH value 2HPO 4-NaH 2PO 4Damping fluid preparation immobilized full cell for the first time becomes 15% solution B, merges two solution, fully stirs, and final sodium alginate concentration is 2%, gets mixing solutions; With syringe this mixing solutions is dropwise joined the aseptic Ca of 0.05mol/L 2In the Cl solution, solidify after 1 hour and take out, centrifugal collection solid is with distillation washing 2 times, to remove the unnecessary Ca in immobilized particlesization surface 2Cl, vacuum-drying promptly gets product.The full cell of cell internal secretion type yeast of moulding is a particulate state, and particle diameter is about 1mm.
Embodiment 2:
The preparation method of full cell catalyst of intracellular secretory lipase type, it comprises the steps:
1). choosing of bacterial classification and substratum: (1) bacterial classification: by dilution-plate method oily factory waste oil is inserted and to separate with carrying out separation and Culture in the substratum, separating with substratum is the potato sucrose substratum, (separation method is seen " agriculture Experiment on Microbiology technology " 69-72 page or leaf must to produce the yeast (Candidalipolytica Candida lipolytica) of emiocytosis type lipase, Chinese agriculture press, in May, 1996, the 1st edition); Wherein the potato sucrose substratum is: the 200g potato be cut into small pieces and be boiled into juice, potato juice and 20g sucrose are dissolved in the 1L water, and add 20g agar, 121 ℃ of high-temperature sterilization 15min, the potato sucrose substratum; (2) fermentation is used substratum: soybean oil 29.0g/L, sucrose 1.0g/L, peptone 70.0g/L, NaNO 31.0g/L, KH 2PO 41.0g/L, MgSO 47H 2O 0.5g/L sterilized 20 minutes for 121 ℃;
2). fermentation culture: the fermentation substratum of packing in the sterile chamber and accounting for volume 1/3, inoculation produces the barms of emiocytosis type lipase; Put 28 ℃, 150r/min cultivated the liquid after must fermenting 72 hours;
3). collect thalline: centrifugal 10 minutes of the liquid 4700r/min after will fermenting, collect thalline, get the yeast thalline;
4). choosing of raw material: the per-cent of the shared gross weight of each raw material is: yeast thalline 0.7, diatomite 2.1, sodium alginate 2.0, U-Ramin MC 0.5, pH value are 7.7 Na 2HPO 4-NaH 2PO 4It is 7.7 Na that damping fluid 94.7 is chosen yeast thalline, diatomite, sodium alginate, U-Ramin MC, pH value 2HPO4-NaH 2PO 4The damping fluid raw material, standby;
5). yeast body and function pH value is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid washed twice, pH value are 7.7 Na 2HPO 4-NaH 2PO 4The weight consumption of damping fluid is the twice of yeast thalline, lyophilize;
6). the immobilization first time of thalline: the thalline of lyophilize gained adds diatomite (fixation support press yeast thalline and diatomaceous weight ratio=1: 3 adding diatomite for the first time), in container, break up thalline and with the diatomite mixing; The pH value that adds yeast body weight twice is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid, after stirring 1 hour under 25 ℃ of conditions, centrifugal collection solid with collected solids washed with acetone 2 times, disperses carrier granule, lyophilize once more, time immobilized whole-cell of winning;
7). the immobilization second time of thalline: sodium alginate (fixation support for the second time) is made into water solution A, is 7.7 Na in addition with remaining pH value 2HPO 4-NaH 2PO 4Damping fluid preparation immobilized full cell for the first time becomes 15% solution B, merges two solution, fully stirs, and final sodium alginate concentration is 2%, gets mixing solutions; With syringe this mixing solutions is dropwise joined the aseptic Ca of 0.05mol/L 2In the Cl solution, solidify after 1 hour and take out, centrifugal collection solid is with distillation washing 2 times, to remove the unnecessary Ca in immobilized particlesization surface 2Cl, vacuum-drying promptly gets product.The full cell of cell internal secretion type yeast of moulding is a particulate state, and particle diameter is about 1mm.
Embodiment 3:
The preparation method of full cell catalyst of intracellular secretory lipase type, it comprises the steps:
1). choosing of bacterial classification and substratum: (1) bacterial classification: by dilution-plate method oily factory waste oil is inserted and to separate with carrying out separation and Culture in the substratum, separating with substratum is the potato sucrose substratum, (separation method is seen " agriculture Experiment on Microbiology technology " 69-72 page or leaf must to produce the yeast (Candidalipolytica Candida lipolytica) of emiocytosis type lipase, Chinese agriculture press, in May, 1996, the 1st edition); Wherein the potato sucrose substratum is: the 200g potato be cut into small pieces and be boiled into juice, potato juice and 20g sucrose are dissolved in the 1L water, and add 20g agar, 121 ℃ of high-temperature sterilization 15min, the potato sucrose substratum; (2) fermentation is used substratum: soybean oil 29.0g/L, sucrose 1.0g/L, peptone 70.0g/L, NaNO 31.0g/L, KH 2PO 41.0g/L, MgSO 47H 2O 0.5g/L sterilized 20 minutes for 121 ℃;
2). fermentation culture: the fermentation substratum of packing in the sterile chamber and accounting for volume 1/3, inoculation produces the barms of emiocytosis type lipase; Put 28 ℃, 150r/min cultivated the liquid after must fermenting 72 hours;
3). collect thalline: centrifugal 10 minutes of the liquid 4700r/min after will fermenting, collect thalline, get the yeast thalline;
4). choosing of raw material: the per-cent of the shared gross weight of each raw material is: yeast thalline 1.2, diatomite 3.6, sodium alginate 2.3, U-Ramin MC 0.8, pH value are 7.7 Na 2HPO 4-NaH 2PO 4It is 7.7 Na that damping fluid 92.1 is chosen yeast thalline, diatomite, sodium alginate, U-Ramin MC, pH value 2HPO 4-NaH 2PO 4The damping fluid raw material, standby;
5). yeast body and function pH value is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid washed twice, pH value are 7.7 Na 2HPO 4-NaH 2PO 4The weight consumption of damping fluid is the twice of yeast thalline, lyophilize;
6). the immobilization first time of thalline: the thalline of lyophilize gained adds diatomite (fixation support press yeast thalline and diatomaceous weight ratio=1: 3 adding diatomite for the first time), in container, break up thalline and with the diatomite mixing; The pH value that adds yeast body weight twice is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid, after stirring 1 hour under 25 ℃ of conditions, centrifugal collection solid with collected solids washed with acetone 2 times, disperses carrier granule, lyophilize once more, time immobilized whole-cell of winning;
7). the immobilization second time of thalline: sodium alginate (fixation support for the second time) is made into water solution A, is 7.7 Na in addition with remaining pH value 2HPO 4-NaH 2PO 4Damping fluid preparation immobilized full cell for the first time becomes 15% solution B, merges two solution, fully stirs, and final sodium alginate concentration is 2%, gets mixing solutions; With syringe this mixing solutions is dropwise joined the aseptic Ca of 0.05mol/L 2In the Cl solution, solidify after 1 hour and take out, centrifugal collection solid is with distillation washing 2 times, to remove the unnecessary Ca in immobilized particlesization surface 2Cl, vacuum-drying promptly gets product.The full cell of cell internal secretion type yeast of moulding is a particulate state, and particle diameter is about 1mm.
Embodiment 4:
The preparation method of full cell catalyst of intracellular secretory lipase type, it comprises the steps:
1). choosing of raw material: the per-cent of the shared gross weight of each raw material is: yeast thalline 1.0, diatomite 3.0, sodium alginate 2.0, U-Ramin MC 0.56, pH value are 7.7 Na 2HPO 4-NaH 2PO 4It is 7.7 Na that damping fluid 93.44 is chosen yeast thalline, diatomite, sodium alginate, U-Ramin MC, pH value 2HPO 4-NaH 2PO 4The damping fluid raw material, standby; The yeast thalline adopts existing yeast thalline;
2). yeast body and function pH value is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid washed twice, pH value are 7.7 Na 2HPO 4-NaH 2PO 4The weight consumption of damping fluid is the twice of yeast thalline, lyophilize;
3). the immobilization first time of thalline: the thalline of lyophilize gained adds diatomite (fixation support press yeast thalline and diatomaceous weight ratio=1: 3 adding diatomite for the first time), in container, break up thalline and with the diatomite mixing; The pH value that adds yeast body weight twice is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid, after stirring 1 hour under 25 ℃ of conditions, centrifugal collection solid with collected solids washed with acetone 2 times, disperses carrier granule, lyophilize once more, time immobilized whole-cell of winning;
4). the immobilization second time of thalline: sodium alginate (fixation support for the second time) is made into water solution A, is 7.7 Na in addition with remaining pH value 2HPO 4-NaH 2PO 4The damping fluid preparation first time, immobilized full cell became the solution B of 14.5-15.5%, merged two solution, fully stirred, and final sodium alginate concentration is 2%, gets mixing solutions; With syringe this mixing solutions is dropwise joined the aseptic Ca of 0.05mol/L 2In the Cl solution, solidify after 1 hour and take out, centrifugal collection solid is with distillation washing 2 times, to remove the unnecessary Ca in immobilized particlesization surface 2Cl, vacuum-drying promptly gets product.The full cell of cell internal secretion type yeast of moulding is a particulate state, and particle diameter is about 1mm.
Embodiment 5:
The preparation method of full cell catalyst of intracellular secretory lipase type, it comprises the steps:
1). choosing of raw material: the per-cent of the shared gross weight of each raw material is: yeast thalline 0.7, diatomite 2.1, sodium alginate 2.0, U-Ramin MC 0.8, pH value are 7.7 Na 2HPO 4-NaH 2PO 4It is 7.7 Na that damping fluid 94.4 is chosen yeast thalline, diatomite, sodium alginate, U-Ramin MC, pH value 2HPO 4-NaH 2PO 4The damping fluid raw material, standby; The yeast thalline adopts existing yeast thalline; The yeast thalline adopts existing yeast thalline;
2). yeast body and function pH value is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid washed twice, pH value are 7.7 Na 2HPO 4-NaH 2PO 4The weight consumption of damping fluid is the twice of yeast thalline, lyophilize;
3). the immobilization first time of thalline: the thalline of lyophilize gained adds diatomite (fixation support press yeast thalline and diatomaceous weight ratio=1: 3 adding diatomite for the first time), in container, break up thalline and with the diatomite mixing; The pH value that adds yeast body weight twice is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid, after stirring 1 hour under 25 ℃ of conditions, centrifugal collection solid with collected solids washed with acetone 2 times, disperses carrier granule, lyophilize once more, time immobilized whole-cell of winning;
4). the immobilization second time of thalline: sodium alginate (fixation support for the second time) is made into water solution A, is 7.7 Na in addition with remaining pH value 2HPO 4-NaH 2PO 4The damping fluid preparation first time, immobilized full cell became the solution B of 14.5-15.5%, merged two solution, fully stirred, and final sodium alginate concentration is 2%, gets mixing solutions; With syringe this mixing solutions is dropwise joined the aseptic Ca of 0.05mol/L 2In the Cl solution, solidify after 1 hour and take out, centrifugal collection solid is with distillation washing 2 times, to remove the unnecessary Ca in immobilized particlesization surface 2Cl, vacuum-drying promptly gets product.The full cell of cell internal secretion type yeast of moulding is a particulate state, and particle diameter is about 1mm.

Claims (4)

1. full cell catalyst of intracellular secretory lipase type is characterized in that: it mainly is 7.7 Na by yeast thalline, diatomite, sodium alginate, U-Ramin MC and pH value 2HPO 4-NaH 2PO 4The damping fluid feedstock production forms, and the per-cent of the shared gross weight of each raw material is:
Yeast thalline 0.7-1.2, diatomite 2.1-3.6,
Sodium alginate 2.0-2.3, U-Ramin MC 0.5-0.8,
The pH value is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid 92.1-94.7; Wherein yeast thalline and diatomaceous weight ratio=1: 3, the shared total weight percent sum of each raw material is 100.
2. full cell catalyst of intracellular secretory lipase type according to claim 1 is characterized in that: the per-cent of the shared gross weight of each raw material is:
Yeast thalline 1.0, diatomite 3.0,
Sodium alginate 2.0, U-Ramin MC 0.56,
The pH value is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid 93.44.
3. the preparation method of full cell catalyst of intracellular secretory lipase type as claimed in claim 1 is characterized in that it comprises the steps:
1). choosing of raw material: the per-cent by the shared gross weight of each raw material is: yeast thalline 0.7-1.2, diatomite 2.1-3.6, sodium alginate 2.0-2.3, U-Ramin MC 0.5-0.8, pH value are 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid 92.1-94.7, wherein yeast thalline and diatomaceous weight ratio=1: 3, the shared total weight percent sum of each raw material is 100, chooses yeast thalline, diatomite, sodium alginate, U-Ramin MC and pH value and be 7.7 Na 2HPO 4-NaH 2PO 4The damping fluid raw material, standby;
2). yeast body and function pH value is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid washed twice, pH value are 7.7 Na 2HPO 4-NaH 2PO 4The weight consumption of damping fluid is the twice of yeast thalline, lyophilize;
3). the immobilization first time of thalline: the thalline of lyophilize gained adds diatomite, in container, break up thalline and with the diatomite mixing; The pH value that adds yeast body weight twice is 7.7 Na 2HPO 4-NaH 2PO 4Damping fluid, after stirring 1 hour under 25 ℃ of conditions, centrifugal collection solid with collected solids washed with acetone 2 times, disperses carrier granule, lyophilize once more, time immobilized whole-cell of winning;
4). the immobilization second time of thalline: sodium alginate is made into water solution A, is 7.7 Na in addition with remaining pH value 2HPO 4-NaH 2PO 4Damping fluid preparation immobilized full cell for the first time becomes solution B, merges two solution, fully stirs, and final sodium alginate concentration is 2%, gets mixing solutions; With syringe this mixing solutions is dropwise joined the aseptic Ca of 0.05mol/L 2In the Cl solution, solidify after 1 hour and take out, centrifugal collection solid is with distillation washing 2 times, to remove the unnecessary Ca in immobilized particlesization surface 2Cl, vacuum-drying promptly gets product.
4. the preparation method of full cell catalyst of intracellular secretory lipase type according to claim 3 is characterized in that: the preparation of described yeast thalline:
1). choosing of bacterial classification and substratum: (1) bacterial classification: by dilution-plate method oily factory waste oil is inserted and to separate with carrying out separation and Culture in the substratum, separating with substratum is the potato sucrose substratum, must produce the yeast of emiocytosis type lipase---Candida lipolytica Candida lipolytica; Wherein the potato sucrose substratum is: the 200g potato be cut into small pieces and be boiled into juice, potato juice and 20g sucrose are dissolved in the 1L water, and add 20g agar, 121 ℃ of high-temperature sterilization 15min, the potato sucrose substratum; (2) fermentation is used substratum: soybean oil 29.0g/L, sucrose 1.0g/L, peptone 70.0g/L, NaNO 31.0g/L, KH 2PO 41.0g/L, MgSO 47H 2O 0.5g/L sterilized 20 minutes for 121 ℃;
2). fermentation culture: the fermentation substratum of packing in the sterile chamber and accounting for volume 1/3, inoculation produces the barms of emiocytosis type lipase; Put 28 ℃, 150r/min cultivated the liquid after must fermenting 72 hours;
3). collect thalline: centrifugal 10 minutes of the liquid 4700r/min after will fermenting, collect thalline, get the yeast thalline.
CNB2006101248550A 2006-10-26 2006-10-26 Full cell catalyst of intracellular secretory lipase type and its preparing method Expired - Fee Related CN100455656C (en)

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CN103540535A (en) * 2013-05-17 2014-01-29 贵州大学 Intracellular lipase producing strain as well as application, screening method and using method thereof
CN103789364A (en) * 2013-11-19 2014-05-14 南宁市新科健生物技术有限责任公司 Method for preparing biodiesel from pichia pastoris whole-cell lipase catalysis
CN103789364B (en) * 2013-11-19 2016-11-30 南宁市新科健生物技术有限责任公司 The method of Pichia sp. Whole-cell lipase catalysis for preparing biodiesel oil
CN114990091A (en) * 2022-06-10 2022-09-02 中牛集团有限公司 Preparation method and application of biological enzyme preparation for ecological leather

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CN1101931A (en) * 1994-08-15 1995-04-26 任春山 Formulation for producing diesel oil and gasoline by biological method using anthracite and its preparation method
ES2345973T3 (en) * 2001-07-11 2010-10-07 Cognis Ip Management Gmbh LIPASE / ACILTRANSPHERASE FROM CANDIDA PARPSYLOSIS.
CN1238469C (en) * 2004-01-16 2006-01-25 清华大学 Novel process for preparing biological diesel oil from grease catalyzed by lipase in the reaction system with organic substrate as medium
CN100375781C (en) * 2005-11-09 2008-03-19 中国科学院广州能源研究所 Production of biological diesel oil by fixed enzyme method

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103540535A (en) * 2013-05-17 2014-01-29 贵州大学 Intracellular lipase producing strain as well as application, screening method and using method thereof
CN103789364A (en) * 2013-11-19 2014-05-14 南宁市新科健生物技术有限责任公司 Method for preparing biodiesel from pichia pastoris whole-cell lipase catalysis
CN103789364B (en) * 2013-11-19 2016-11-30 南宁市新科健生物技术有限责任公司 The method of Pichia sp. Whole-cell lipase catalysis for preparing biodiesel oil
CN114990091A (en) * 2022-06-10 2022-09-02 中牛集团有限公司 Preparation method and application of biological enzyme preparation for ecological leather

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