CN104277989A - Bread yeast and application thereof in producing coenzyme I by fermenting - Google Patents
Bread yeast and application thereof in producing coenzyme I by fermenting Download PDFInfo
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- CN104277989A CN104277989A CN201410495185.8A CN201410495185A CN104277989A CN 104277989 A CN104277989 A CN 104277989A CN 201410495185 A CN201410495185 A CN 201410495185A CN 104277989 A CN104277989 A CN 104277989A
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Abstract
The invention discloses bread yeast and an application thereof in producing a coenzyme I by fermenting. The bread yeast is prepared through the steps of after original starting strains sc-00 are mutated by implanting a low-energy nitrogen ion beam, carrying out preliminary screening on the strains, fermenting in a shake flask, and carrying out secondary screening; and the bread yeast is classified and named Saccharomycescerevisiae SC-Z180, and preserved in China Center for Type Culture Collection CCTCC, and the Preservation Number is CCTCCM2014317. The bread yeast can be used for greatly increasing the components of the coenzyme I in the process of fermentation, and reducing other components; and through carrying out fermentation and secondary screening on obtained strains, the strains can be used for significantly improving the problem that the fermentation yield is low, and in a fermentation tank with a volume of 5 L, the yield of the coenzyme I is increased by 45.8% in comparison with that of original starting strains.
Description
Technical field
The present invention relates to a strain bread yeast and the application in fermentative production nadide thereof, belong to biological technical field.
Background technology
Nadide (nicotinamide adenine dinucleotide, NAD+) is a kind of important biochemical reagents, often extracts from bread yeast.It is the important coenzyme of many reductase enzymes in organism, for the dehydrogenation reaction of lactic acid, isocitric acid, α mono-ketoisocaproic, oxysuccinic acid, β mono-hydroxyl ester acyl coenzyme A etc. in life entity intracellular metabolite process, play in biological oxidation process and pass hydrogen effect, activate various enzyme system, promote synthesis and the metabolism of nucleic acid, protein, polysaccharide, increase substance transportation and regulable control, improve metabolic function.Clinically be used for coronary heart disease as assisting therapy, the symptoms such as uncomfortable in chest, stenocardia can be improved.Nadide itself there is no katalysis, but participates in the effect of redox or acyl carrier, if lose this cofactor, desaturase will lose catalytic capability.The desaturase effect of two hydrogen atoms through taking NAD as coenzyme on many metabolic intermediates, makes NAD be reduced to NADH, then through nadh dehydrogenase effect, sloughs hydrogen.This is an electron transport route, and the effect of NAD in respiratory chain is the electronics accepting to be taken off by desaturase.
Along with the development of China's economy, the significantly improving of living standards of the people, the dietary structure of people also there occurs larger change, grain, potato, the beans ratio in food configuration obviously declines, the intake of animal and lipid then obviously increases, and causes the body constitution of people day by day to decline.Recent years, various geriatric disease is obvious ascendant trend as arteriosclerosis, myocardial infarction, hyperlipidemia, Parkinson's disease etc.At present in China, it is higher to suffer from Parkinsonian ratio, just has 6 people ill in about 1500 people.Therefore, for can be used for the treatment of ischemic heart disease, hepatopathy, a variety of causes metabolism disorder and can as the Coenzyme Complex preparation of chemotherapy ancillary drug, its market requirement is very huge.
Because the cause of numerous disease is because sugar, fat and protein metabolism decline or badly to cause in body mostly; and nadide is the key coenzymes participating in acetylization reaction in body, redox reaction and energy metabolism, sugar, fat and protein metabolism in body are played an important role.How to strengthen above-mentioned sugar, fat and protein metabolism and become one of medicament research and development main direction.
Preparing nadide mainly through following several method now both at home and abroad, is extraction method, fermentation method, biological synthesis process, reinforcement and organic synthesis method etc. respectively.But these methods are not all gone into operation on a large scale.The biochemical institute in domestic Chinese Academy of Sciences Shanghai once spent ion exchange resin get from yeast NAD fresh yeast per ton (weight in wet base) about 70 grams, purity is 70 %, productive rate and purity lower.Domestic follow always this method produce use enzymatic clarification NADH again.Microbe fermentation method is the new nadide production method of in recent years rising, and because microorganism cells is easy to large scale culturing growth, product is entirely natural alltrans configuration, and bioavailability is high.And safe relative to chemical synthesis, efficient, in product, almost non-toxic harmful chemical residue, is easy to separation and purification, and clinical efficacy is better, is the production method having DEVELOPMENT PROSPECT most.
Summary of the invention
The technical problem to be solved in the present invention is to provide a strain bread yeast and the application in fermentative production nadide thereof, and this bread yeast passes through simply, mutagenesis screening method acquisition safely and efficiently, significantly can improve the content of nadide in tunning.
In order to achieve the above object, the technical solution used in the present invention is:
A kind of bread yeast (Saccharomyces cerevisiae) SC-Z180, be preserved in China typical culture collection center CCTCC on July 3rd, 2014, address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M 2014317.
The described application of bread yeast SC-Z180 in fermentative production nadide.
The application of described bread yeast in fermentative production nadide, comprises the steps:
1) slat chain conveyor: bread yeast SC-Z180 is connected on plate culture medium and cultivates, culture temperature is 28-34 DEG C, incubation time 1-2d;
2) slant culture: the bread yeast SC-Z180 that step 1) middle plateform is cultivated is transferred on slant medium, incubation time 18-20h, culture temperature 28-34 DEG C;
3) plant liquid to cultivate: by step 2) in the bread yeast SC-Z180 of slant culture receive in seed culture medium and cultivate, incubation time 8-11h, culture temperature 28-34 DEG C, shaking speed is 150-200r/min;
4) fermentation culture: be transferred in fermention medium by the kind liquid in step 3), inoculum size is 2-10% (V/V), and incubation time is 36-38 h, and culture temperature is 28-34 DEG C, and shaking speed is 160-220r/min.
Step 1) and 2) in flat board and slant medium comprise following composition by mass percentage: carbon source 2-3%, nitrogenous source 2-4%, agar 1.5-2%, surplus is water; Wherein said carbon source is one or both in glucose or W-Gum, and described nitrogenous source is one or both of peptone or yeast extract paste.
Step 3) seed culture medium comprises following component by mass percentage: carbon source 5%, nitrogenous source 0.5-1%, inorganic salt 0.1-0.2%, and surplus is water, pH=5.5, and wherein carbon source is at least one in glucose or molasses; Nitrogenous source is one or both in yeast extract paste or ammonium sulfate; Inorganic salt are at least one in magnesium sulfate or Secondary ammonium phosphate.
Step 4) fermention medium comprises following component by mass percentage: carbon source 2-6%, nitrogenous source 1-2%, inorganic salt 0.2-0.5%, somatomedin 0.1-0.6%, pH=5.5, and wherein carbon source is at least one in glucose or molasses; Nitrogenous source is at least one in yeast extract paste or ammonium sulfate, and inorganic salt are Secondary ammonium phosphate; Somatomedin is at least one of tryptophane or nicotinamide.
The screening method of bread yeast SC-Z180 of the present invention is: the original starting strain sc-00 of bread yeast injects after mutagenesis through ion beam of low energy N+, is sieved again obtain the starting strain of the high bacterial strain of nadide output as next round mutagenesis by primary dcreening operation, shake flask fermentation.Repeat above-mentioned steps, finally obtain aimed strain SC-Z180; Concrete mutagenesis steps is as follows:
A) preparation of monospore suspension: it is 1 × 10 that bread yeast starting strain spore is made concentration
6individual/ml suspension;
B) N~+ implantation mutagenesis: get 100 μ L step a) spore suspensions, even spread, on aseptic empty flat board, dries up with sterile wind, is 18 Kev at energy, and implantation dosage is 170 × 10
14ions/cm
2, target chamber vacuum tightness is 1.5 × 10
-3pa carries out Nitrogen ion mutagenesis, after ion implantation, carries out wash-out in an aseptic environment with 1ml sterilized water;
C) primary dcreening operation of mutagenic strain: by the elutriant of step b) aseptically even spread on plate culture medium, at 28-34 DEG C, cultivate 1-2d;
D) the multiple sieve of mutagenic strain: on bacterial strain access slant medium step c) filtered out, cultivate 18h at 28-34 DEG C, then inclined-plane proceeds in seed culture medium, cultivates 8-10h at 28-34 DEG C.Get seed liquor by inoculum size 2-10%(v/v) access in fermention medium and carry out shake flask fermentation cultivation, leavening temperature 28-34 DEG C, shaking speed is 160-220r/min, the content of nadide is detected after fermentation 36h, select the starting strain of the highest bacterial strain of nadide content as next round mutagenesis screening, repeat above-mentioned steps until filter out the high bread yeast SC-Z180 of nadide output.
Plate culture medium in step c) and step d) and slant medium are: carbon source 2-3%, nitrogenous source 2-4%, agar 1.5-2%, and surplus is water, and pH nature, each component by mass percentage.Wherein said carbon source is one or both in glucose or W-Gum, and described nitrogenous source is one or both of peptone or yeast extract paste.
The seed culture medium of step d) is: carbon source 5%, nitrogenous source 0.5-1%, inorganic salt 0.1-0.2%, and surplus is water, pH=5.5, and by mass percentage, wherein carbon source is one in glucose or molasses or mixture to each component; Nitrogenous source is one or both in yeast extract paste or ammonium sulfate; Inorganic salt are magnesium sulfate or Secondary ammonium phosphate.
The fermention medium of step d) is: carbon source 2-6%, nitrogenous source 1-2%, inorganic salt 0.2-0.5%, somatomedin 0.1-0.6%, and surplus is water, pH=5.5, and by mass percentage, wherein carbon source is one or both in glucose or molasses to each component; Nitrogenous source is one or both in yeast extract paste, ammonium sulfate, and inorganic salt are Secondary ammonium phosphate; Somatomedin is one or both of tryptophane or nicotinamide.
Bread yeast of the present invention (
saccharomyces cerevisiae) morphology of SC-Z180 and physiochemical properties:
Colony colour: oyster white
Aerobic mode: amphimicrobian
Bacterium colony size: 1-4 μm
Growth temperature: 28-32 DEG C
Optimal pH: 5.4-6.5
Colonial morphology: oval
Mode of reproduction: budding
Gramstaining: Gram-positive
Beneficial effect: the invention provides a strain bread yeast bacterial strain, it is very capable that this bacterial strain produces nadide, present invention employs low energy N
+inject induced-mutation technique, final screening obtains strain nadide Producing Strain Zhu bread yeast SC-Z180, this bacterial strain significantly can improve the component of nadide in fermenting process, and reduce other components, the bacterial strain obtained carries out the multiple sieve that ferments, this bacterial strain can improve the low problem of fermentation production rate greatly, and in 5L fermentor tank, nadide output improves 45.8% than the original bacterium that sets out.
Embodiment
Be described further the present invention below in conjunction with concrete embodiment, the present invention may be better understood.But those skilled in the art will readily understand, concrete material proportion, processing condition and result thereof described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1 the present embodiment illustrates the method for bread yeast starting strain being carried out mutagenesis screening object bacterial strain.
With the bread yeast SC-00 of Laboratories Accession for starting strain carries out mutagenic treatment, concrete steps are as follows:
A) preparation of monospore suspension: get cultured bread yeast slant strains one, add 10ml sterilized water, gently the spore of agar surface is scraped, this spore suspension is placed in sterilizing 50ml triangular flask, places several sterile glass balls in bottle in advance, filter with the absorbent cotton of sterilizing after shake well, and with aseptic water washing filter residue 2 ~ 3 times, finally make filtrate volume reach 10ml, filtrate counts with blood counting chamber, and adjustment spore concentration is 1 × 10
6individual/ml;
B) N~+ implantation mutagenesis: get 100 μ L step a) spore suspensions, even spread, on aseptic empty flat board, dries up with sterile wind, is 18 Kev at energy, and implantation dosage is 170 × 10
14ions/cm
2, target chamber vacuum tightness is 1.5 × 10
-3pa carries out Nitrogen ion mutagenesis, after ion implantation, carries out wash-out in an aseptic environment with 1ml sterilized water;
C) screening of mutagenic fungi: by the elutriant of step b) aseptically even spread on plate culture medium, at 28-34 DEG C, cultivate 1-2d;
D) the multiple sieve of mutagenic strain: on bacterial strain access slant medium step c) filtered out, cultivate 18h at 28-34 DEG C, then inclined-plane proceeds in seed culture medium, cultivates 8-10h at 28-34 DEG C.Get seed liquor by inoculum size 2-10%(v/v) access in fermention medium and carry out shake flask fermentation cultivation, leavening temperature 28-34 DEG C, shaking speed is 160-220r/min, the content of nadide is detected after fermentation 36h, select the starting strain of the highest bacterial strain of nadide content as next round mutagenesis screening, repeat above-mentioned steps until filter out the high bread yeast SC-Z180 of nadide output.
Plate culture medium used in step c) is: glucose 2%, Tryptones 2%, agar 2%, yeast extract paste 2%, and all the other are water.
Seed culture medium used in step d) is: glucose 5%, yeast extract paste 0.5%, ammonium sulfate 1%, Secondary ammonium phosphate 0.1%, pH5.5, and wherein glucose needs to divide disappear (dividing being meant to of disappearing to separate sterilizing).
Fermention medium used in step d) is: glucose 4%, yeast extract paste 0.1%, ammonium sulfate 1%, Secondary ammonium phosphate 0.5%, tryptophane 0.2%, PH5.5, wherein glucose needs point to disappear.
Slant medium used in step d) is: glucose 2%, Tryptones 2%, agar 2%, yeast extract paste 2%, and all the other are water.
Embodiment 2
The present embodiment illustrates biology morphology and the genetic stability of mutagenic fungi bread yeast SC-Z180
The morphology of bread yeast SC-Z180 of the present invention and physiochemical properties:
Colony colour: oyster white
Aerobic mode: amphimicrobian;
Bacterium colony size: 1-4 μm;
Growth temperature: 28-32 DEG C;
Optimal pH: 5.4-6.5;
Colonial morphology: oval;
Mode of reproduction: budding;
Gramstaining: Gram-positive.
The fermentation test result that goes down to posterity is as shown in table 1.
The genetic stability of table 1 bread yeast SC-Z180
| Passage number | Nadide output (g/L) |
| 1 | 4.36 |
| 2 | 4.45 |
| 3 | 4.20 |
| 4 | 4.31 |
| 5 | 4.49 |
| 6 | 3.89 |
From genetic stability interpretation, through 6 tests of going down to posterity, it is basicly stable that nadide is produced in mutant strain bread yeast SC-Z180 fermentation, has good genetic stability, can as researching and developing industrialization bacterial strain further.
Embodiment 3
This example illustrates the production technique of nadide Producing Strain bacterial strain bread yeast SC-Z180 fermentative production nadide
Described in the present embodiment, culture medium prescription is as follows:
Plate culture medium (g/L): glucose 20, fish meal protein peptone 20, agar 20, yeast extract paste 20, all the other are water, pH nature.
Slant medium (g/L): glucose 20, fish meal protein peptone 20, agar 20, yeast extract paste 20, all the other are water, pH nature.
Seed culture medium (g/L): glucose 45, yeast extract paste 8, magnesium sulfate 0.1, ammonium sulfate 2, Secondary ammonium phosphate 1, with 1mol/L hydrochloric acid adjust pH be 5.5, wherein grape sugar disappears.
Fermention medium (g/L): glucose 50, yeast extract paste 10, magnesium sulfate 0.1, ammonium sulfate 2, Secondary ammonium phosphate 1, tryptophane 3, adjust pH to be 5.5 with 1mol/L hydrochloric acid, all the other are water, and wherein grape sugar disappears.
Saccharomyces Cerevisiae in S C-Z180 is inoculated into plate culture medium, culture temperature 30 DEG C, proceed to slant medium after cultivating 36h, at 30 DEG C, cultivate 18h, then inclined-plane proceeds in seed culture medium, cultivates 9 h at 30 DEG C.Get seed liquor by 10%(v/v) in inoculum size access fermention medium, shaking flask liquid amount 20%, shaking speed is 200r/min, leavening temperature 30 DEG C, fermentation 36h, measures the content of nadide in fermented liquid and reaches 4.82g/L, improve 40.1% than original starting strain.
Embodiment 4
This example illustrates the production technique of nadide Producing Strain bacterial strain bread yeast SC-Z180 fermentative production nadide
Described in the present embodiment, culture medium prescription is as follows:
Plate culture medium (g/L): glucose 20, fish meal protein peptone 20, agar 20, yeast extract paste 20, all the other are water, pH nature.
Slant medium (g/L): glucose 20, fish meal protein peptone 20, agar 20, yeast extract paste 20, all the other are water, pH nature.
Seed culture medium (g/L): glucose 45, yeast extract paste 8, magnesium sulfate 0.1, ammonium sulfate 2, Secondary ammonium phosphate 1, with 1mol/L hydrochloric acid adjust pH be 5.5, wherein grape sugar disappears.
Fermention medium (g/L): molasses 47, yeast extract paste 10, magnesium sulfate 0.1, ammonium sulfate 2, Secondary ammonium phosphate 1, tryptophane 3, adjust pH to be 5.5 with 1mol/L hydrochloric acid, all the other are water, and wherein grape sugar disappears.
Saccharomyces Cerevisiae in S C-Z180 is inoculated into plate culture medium, culture temperature 30 DEG C, proceed to slant medium after cultivating 36h, at 30 DEG C, cultivate 18h, then inclined-plane proceeds in seed culture medium, cultivates 9 h at 30 DEG C.Get seed liquor by 10%(v/v) in inoculum size access fermention medium, shaking flask liquid amount 20%, shaking speed is 200r/min, leavening temperature 30 DEG C, fermentation 36h, measures the content of nadide in fermented liquid and reaches 4.73g/L and improve 37.5% than original starting strain.
Embodiment 5
This example illustrates that bread yeast SC-Z180 produces nadide in 5L fermentation cylinder for fermentation
Described in the present embodiment, culture medium prescription is as follows:
Plate culture medium (g/L): glucose 20, fish meal protein peptone 20, agar 20, yeast extract paste 20, all the other are water, pH nature.
Slant medium (g/L): glucose 20, fish meal protein peptone 20, agar 20, yeast extract paste 20, all the other are water, pH nature.
Seed culture medium (g/L): glucose 45, yeast extract paste 8, magnesium sulfate 0.1, ammonium sulfate 2, Secondary ammonium phosphate 1, with 1mol/L hydrochloric acid adjust pH be 5.5, wherein grape sugar disappears.
Fermention medium (g/L): glucose 50, yeast extract paste 10, magnesium sulfate 0.1, ammonium sulfate 2, Secondary ammonium phosphate 1, tryptophane 3, all the other are water, pH 5.5.
Saccharomyces Cerevisiae in S C-Z180 is inoculated into plate culture medium, culture temperature 30 DEG C, proceed to slant medium after cultivating 36h, at 30 DEG C, cultivate 18h, then inclined-plane proceeds in seed culture medium, cultivates 9 h at 30 DEG C.Get seed liquor by 10%(v/v) in inoculum size access 5L fermentor tank, fermentor tank stirring velocity is 200r/min, leavening temperature 30 DEG C, measures the content of nadide in fermented liquid and reach 5.06g/L after fermentation 36h.
Embodiment 6 examples illustrate the detection method of nadide content in yeast.
Plant and instrument: high performance liquid chromatograph, whizzer, ultrasonic cleaning machine, millipore filtration etc.
Reagent: chromatogram methyl alcohol, pure water, dipotassium hydrogen phosphate, potassium primary phosphate
The phosphate buffer solution pH6.5 of testing conditions: moving phase: 0.5mol and methyl alcohol 10% aqueous solution, adjust pH to be 6.5 with TBAH; Flow velocity 0.8ml/min, determined wavelength 254nm, sample introduction 20 μ L, temperature 10 DEG C.
Concrete operation step:
Preparation standard product: accurately take nadide standard substance 10mg, shake up constant volume with pure water in 50mL volumetric flask, get 10 mL standard solutions, at 4 DEG C, under 10000 rpm, centrifugal 5 min, remove supernatant liquor, add 0.3mol/L HCl 3mL, in 55 DEG C of water-bath 10 min, then be placed on rapidly in ice and be cooled to 0 DEG C, then slowly drip 0.3 mol/L NaOH and neutralize, at 4 DEG C, under 10000 rpm, centrifugal 15 min, get supernatant liquor and save backup.
Prepare sample: get 10 mL fermented liquids, at 4 DEG C, under 10000 rpm, centrifugal 5 min, remove supernatant liquor, add 0.3mol/L HCl 3mL, in 55 DEG C of water-bath 10 min, then be placed on rapidly in ice and be cooled to 0 DEG C, then slowly drip 0.3 mol/L NaOH and neutralize, at 4 DEG C, centrifugal 15 min under 10000 rpm, get nadide content to be measured in supernatant liquor to another test tube.
Cubage method:
Nadide content (g/L)=(sample peak area/standard substance peak area) × standard concentration.
Claims (6)
1. a strain bread yeast, its Classification And Nomenclature is bread yeast (Saccharomyces cerevisiae) SC-Z180, is preserved in China typical culture collection center CCTCC, deposit number: CCTCC M 2014317.
2. the application of bread yeast according to claim 1 in fermentative production nadide.
3. the application of bread yeast according to claim 2 in fermentative production nadide, is characterized in that comprising the steps:
1) slat chain conveyor: bread yeast SC-Z180 is connected on plate culture medium and cultivates, culture temperature is 28-34 DEG C, incubation time 1-2d;
2) slant culture: the bread yeast SC-Z180 that step 1) middle plateform is cultivated is transferred on slant medium, incubation time 18-20h, culture temperature 28-34 DEG C;
3) plant liquid to cultivate: by step 2) in the bread yeast SC-Z180 of slant culture receive in seed culture medium and cultivate, incubation time 8-11h, culture temperature 28-34 DEG C, shaking speed is 150-200r/min;
4) fermentation culture: be transferred in fermention medium by the kind liquid in step 3), inoculum size is 2-10% (V/V), and incubation time is 36-38h, and culture temperature is 28-34 DEG C, and shaking speed is 160-220r/min.
4. the application of bread yeast according to claim 3 in fermentative production nadide, it is characterized in that: step 1) and 2) in flat board and slant medium comprise following composition by mass percentage: carbon source 2-3%, nitrogenous source 2-4%, agar 1.5-2%, surplus is water; Wherein said carbon source is one or both in glucose or W-Gum, and described nitrogenous source is one or both in peptone or yeast extract paste.
5. the application of bread yeast according to claim 3 in fermentative production nadide, it is characterized in that: step 3) seed culture medium comprises following component by mass percentage: carbon source 5%, nitrogenous source 0.5-1%, inorganic salt 0.1-0.2%, surplus is water, pH=5.5, wherein carbon source is at least one in glucose or molasses; Nitrogenous source is one or both in yeast extract paste or ammonium sulfate; Inorganic salt are at least one in magnesium sulfate or Secondary ammonium phosphate.
6. the application of bread yeast according to claim 3 in fermentative production nadide, it is characterized in that: step 4) fermention medium comprises following component by mass percentage: carbon source 2-6%, nitrogenous source 1-2%, inorganic salt 0.2-0.5%, somatomedin 0.1-0.6%, pH=5.5, wherein carbon source is at least one in glucose or molasses; Nitrogenous source is at least one in yeast extract paste or ammonium sulfate, and inorganic salt are Secondary ammonium phosphate; Somatomedin is at least one of tryptophane or nicotinamide.
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| CN105463007A (en) * | 2016-01-08 | 2016-04-06 | 南京工业大学 | Method for producing coenzyme I by constructing recombinant escherichia coli based on ion beam mediated technology |
| CN105463007B (en) * | 2016-01-08 | 2018-07-31 | 苏州至汇生物科技有限公司 | The method for producing Coenzyme I based on Ion Beam Mediated Technique structure recombination bacillus coli |
| CN106755210A (en) * | 2016-12-12 | 2017-05-31 | 安徽翠鸟生物技术有限公司 | Coenzyme mixed solution and preparation method thereof |
| CN111733198A (en) * | 2020-07-24 | 2020-10-02 | 开封康诺药业有限公司 | Fermentation method for increasing output of coenzyme I |
| CN111733198B (en) * | 2020-07-24 | 2024-03-26 | 开封康诺药业有限公司 | Fermentation method for improving coenzyme I yield |
| CN112322511A (en) * | 2020-11-13 | 2021-02-05 | 湖州颐盛生物科技有限公司 | Saccharomyces cerevisiae for producing coenzyme I |
| CN112322511B (en) * | 2020-11-13 | 2022-11-29 | 湖州颐盛生物科技有限公司 | Saccharomyces cerevisiae for producing coenzyme I |
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