AU2021102566A4

AU2021102566A4 - Co-production process of pyruvic acid and Levodopa and use thereof

Info

Publication number: AU2021102566A4
Application number: AU2021102566A
Authority: AU
Inventors: Wanhe Chen; Yue Cheng; Xiaohe Chu; Mingshan Fang; Ting LIN; Pengfu Liu; Jian Shen; Yingtao Sheng; Junjie Sun; Licheng WU; Shunqing Xu; Wei Yu
Original assignee: Zhejiang Lvchuang Biotechnology Co Ltd; Zhejiang University of Technology ZJUT
Current assignee: Zhejiang Lvchuang Biotechnology Co Ltd; Zhejiang University of Technology ZJUT
Priority date: 2021-05-14
Filing date: 2021-05-14
Publication date: 2021-07-01
Anticipated expiration: 2029-05-14

Abstract

OF THE DISCLOSURE The present disclosure relates to a co-production process of pyruvic acid and levodopa and use thereof. The following technical solution is adopted: step 1, fermenting Torulopsis glabrata to obtain a pyruvic acid fermentation broth, removing cell from the fermentation broth by a ceramic membrane, removing proteins, nucleic acids, pigments and other macromolecules through an ultrafiltration membrane, and concentrating a resulting filtrate to a pyruvic acid concentration of 150-300 g/L; step 2, preparing the enzyme-catalyzed substrate solution by adding a certain amount of pyruvic acid concentrated solution, catechol, ammonium acetate, ethylene diamine tetraacetic acid (EDTA), and sodium sulfite to a resulting pyruvic acid concentrate to adjust to pH 7.0-9.0, and formulating into an; step 3, using genetically engineered tyrosine phenol lyase (TPL) strain fermentation to obtain a TPL bacterial suspension, centrifuging to collect cells, breaking cells on a high-pressure homogenizer, centrifuging, and collecting an enzyme extract; and step 4, adding a certain amount of substrate solution to the enzyme extract, stirring well, sealing at 25°C, and reacting under shaking. The substrate solution is prepared by the pyruvic acid concentrate, catechol concentration is controlled at 0-10 g/L by feeding the substrate solution, when a product reaches above 120 g/L, stop feeding the substrate solution, and when the content of catechol in the reaction mixture is less than 0.2 g/L, stop the reaction.

Description

CO-PRODUCTION PROCESS OF PYRUVIC ACID AND LEVODOPA AND USE THEREOF TECHNICAL FIELD

[01] The present disclosure relates to a co-production process of pyruvic acid and levodopa and use thereof, and belongs to the field of fermentation and enzyme catalytic processes.

BACKGROUND ART

[02] The chemical name of levodopa (L-DOPA) is 3,4-dihydroxyphenylalanine, and the structural formula thereof is:

OH H-N

[03] H0

[04] As an important bioactive substance, L-DOPA is an important intermediate in the biochemical metabolic pathway from L-tyrosine to catechol or melanin.

[05] In the 1960s, a plurality of international scholars had begun on studies of microbial enzymatic synthesis of L-DOPA. In order to improve the yield and substrate conversion rate of L-DOPA, researchers conducted a plurality of studies on the process of microbial enzymatic synthesis of L DOPA.

[061 Tyrosine phenol lyase (TPL, E.C. 4.1.99.2) is a tetrameric enzyme with a molecular weight of about 200 kDa. TPL uses pyridoxal-phosphate (PLP) as a coenzyme. TPL can catalyze the p elimination of L-tyrosine to produce phenol, pyruvic acid, and ammonia. Since this reaction is reversible, after catechol is substituted for phenol, L-DOPA can be produced from catechol, pyruvic acid and ammonia under the catalysis of TPL. The precursor of levodopa inhibits the enzyme activity at higher concentrations. In addition to strong inhibition, catechol and pyruvate can cause irreversible inactivation of the enzyme, so that the reaction conditions are difficult to control, leading to more by products and low yield of L-DOPA.

[07] R. Krishnaveni, Vandana Rathod, et al. from India used the fungus Acremonium rutilum to transform L-tyrosine to L-DOPA with a tyrosinase specific activity of 1,095 U/mg. However, the current production capacity is as low as 0.89 g/L only.

[08] Ikram-Ul-Haq et al. from Pakistan performed UV mutagenesis on Aspergillus oryzae producing tyrosinase, and the maximum yield of mutant strain was 1.28 g/L.

[09] Doaa A. R. Mahmoud and Magda A. El Bendary from Egypt used the tyrosinase of Egyptian halophilic black yeast to produce L-DOPA with a yield of 66 [g/ml.

[10] When Korean researchers used tyrosinase as a catalytic agent, they used electro-substituted reducing reagents to reduce DOPA quinone to L-DOPA, resulting in a conversion rate of 95.9 and an enhanced productivity of 47.27 mg/L*h.

[11] Some researchers used natural bacteria, such as Escherichia,Proteus, Stizolobium hassjoo, and Erwinia, to synthesize L-DOPA, for example, a review of levodopa enzymatic synthesis reported that genetically engineered Escherichiacoli strain expressing TPL could produce 29.6 g/L L-DOPA in 30 h. As another example, Jang-Young Lee et al. cloned p-hydroxyphenylacetate 3-hydroxylase (PHAH) from Escherichiacoli W (ATCC 11105) and converted L-tyrosine to L-DOPA. The product could be accumulated to 10 g/L. The researchers found that although the expression level of TPL was higher in these recombinant strains than in wild strains, the final L-DOPA synthetic ability was not significantly improved or even lower than that of wild strains. In order to obtain the higher L-DOPA synthetic ability, in addition to the high activity of TPL, the strain with better substrate and product transport channels and higher substrate tolerance were also necessary. In general, strict reaction conditions, the unstable TPL enzyme, and plenty of by-products, led to low yield of L-DOPA.

[12] Pyruvic acid is also known as 2-oxopropanoic acid or a-ketopropionic acid. Pyruvic acid is one of the most important organic acids and has a wide range of applications in the fields of pharmaceuticals, food, chemicals, agrochemicals, and scientific research.

[13] Pyruvic acid is used in the pharmaceutical industry for the synthesis of new drugs for the treatment of hypertension, angiotensin II, a series of protease inhibitors, sedatives, anti-inflammatory analgesic cinchophen, medicament phosphoenolpyruvate, 4-metazoline formic acid, antitubercular agent isoniazid calcium pyruvinate, antipyretic 2-phenylquinoline-4-carboxylic acid, and thiadazole drugs.

[14] Pyruvic acid is an important intermediate in the glucose metabolism and synthesis of amino acids and saccharides thereof in vivo. Researchers found that supplementation with pyruvic acid could accelerate the TCA cycle by stimulating mitochondria, thereby accelerating fat consumption for weight loss. Thus, up to 48% of fat is metabolized, and at the same time, the protein loss caused by the low-fat diet can be reduced, and pyruvic acid is highly safe to the human body. In recent years, developed countries have utilized such characteristic of pyruvic acid and calcium salt thereof as a slimming health care medicine.

[15] Industrial production methods of pyruvic acid mainly include chemical synthesis method, enzymatic conversion method, and microbial fermentation method.

[16] Chemical synthesis method:

[17] In 1977, the Japan Research Institute firstly realized the industrialization of the production of pyruvic acid by the chemical synthesis of tartaric acid as raw material. In the liquid or gas phase, tartaric acid (or lactate) is oxidized to pyruvate and hydrolyzed to pyruvic acid, but the method is heavily polluted, high-cost and lack of competitiveness.

[18] Enzymatic conversion method:

[19] Enzyme systems in microbial cells are used for dehydrogenation and oxidation of lactic acid to pyruvic acid. Carsten Schinsche and Helmut Simon found that Proteus vulgaris and Proteus mirabilis could effectively hydrolyze (R)-lactate to produce pyruvic acid; Ping Xu et al. further screened strain KY6 belonging to Edwardisellatarda biogroup 1 from the soil, which could convert DL-lactate to pyruvate. Dariush Hekmat et al. used an enzyme-membrane reactor to convert lactate to pyruvate from a crude extract of Proteus vulgaris cells. This method has high conversion rate but high substrate cost and does not achieve industrial production.

[20] Microbial fermentation method:

Since 1950s, people have begun to explore bio-fermentation way to produce pyruvic acid by the microbes including yeasts, basidiomycetes, actinomycetes, and bacteria. After more than 40 years of research, Japanese scholars have successfully bred strains with high yield of pyruvic acid, and achieved industrialized fermented production in 1989. The level of pyruvic acid production was as high as 67.8 g/L, and the conversion rate was 0.494 g/g. The fermentation method has the following advantages: the method can use sustainable and low-cost glucose as main raw material, can obtain high production and yield, is environmentally friendly, and can avoid some by-products, such as H2 0 2 produced by enzymatic reaction. The fermentation method is a promising industrial way for producing pyruvic acid. At present, a plurality of researchers in China are also focus on doveloping the production of pyruvic acid by fermentation, and have accumulated a plurality of research results. LI Yin, CHEN Jian, et al. had reported and bred a TorulopsisglabrataWSH-IP303 strain, which was subjected to fed-batch fermentation in a 2.5 L fermentor for 56 h, and the yield and conversion rate of pyruvate could reach 69.4 g/L and 0.636 g/g, respectively; the strain was subjected to fed-batch fermentation in a 5 L fermentor for 56 h, and pyruvate yield could reach up to 77.8 g/L, with a glucose conversion rate of 0.651 g/g. XU Qinglong, LIU Liming, et al. reported that Torulopsis glabrata CCTCCM 202019 was fermented in a 7 L fermentor by glucose feeding process, after 83 h fermentation, the yield and conversion rate of pyruvate reached 83.1 g/L and 0.621 g/g, respectively.

High pyruvic acid producing strain, TorulopsisglabrataTP19 bred by GAO Nianfaet al. from Tianjin University of Science and Technology, was fermented in a 5 L fermentor for 48 h, resulting in a maximum pyruvate production of 65.1 g/L, a conversion rate of 58.6%, and a production intensity of 1.35 g/L/h. GAO Nianfa et al. subsequently bred Torulopsis glabrataTP204, the yield of pyruvate in the 5 L fermenter could reach 71.23 g/L. JIANG Ning, WANG Qinhong, et al. from the Institute of Microbiology, Chinese Academy of Sciences bred Torulopsis glabrata IF0005-36, which was fermented in a 5 L fermentor for 52 h, with a pyruvate yield of 82.2 g/L.

SUMMARY

[21] Preferred embodiments of the present invention seek to co-produce pyruvic acid by fermentation and levodopa by enzyme catalysis. The pyruvic acid after crude purification is used for production of levodopa under the catalysis of tyrosine phenol lyase (TPL), reducing the cost of levodopa production.

[22] To this end, the present disclosure adopts the following technical solution:

[23] step 1, using Torulopsis glabrata fermentation to obtain a pyruvic acid fermentation broth, removing the cell from the fermentation broth by a ceramic membrane, removing proteins, nucleic acids, pigments through an ultrafiltration membrane, and concentrating a resulting filtrate to a pyruvic acid concentration of 150-300 g/L;

[24] step 2, preparing the enzyme-catalyzed substrate solution by adding a certain amount of catechol, ammonium acetate, ethylene diamine tetraacetic acid (EDTA), and sodium sulfite into the concentrated pyruvic acid solution, and adjusting pH to 7.0-9.0;

[25] step 3, fermenting genetically engineered Escherichiacoli strain expressing tyrosine phenol lyase (TPL) to obtain a TPL bacterial suspension, centrifuging to collect cells, breaking cells on a high-pressure homogenizer, centrifuging, and collecting cell extract as enzyme solution; and

[26] step 4, adding a certain amount of substrate solution to the cell extract, sealing and stirring at °C; where catechol concentration in reaction solution is controlled at 0-10 g/L by feeding the feed solution, and the product reaches above 120 g/L.

[27] Stop feeding the feed solution; when the content of catechol in the reaction mixture is less than 0.2 g/L, the reaction is ended.

[28] The present disclosure has the following beneficial effects: By directly using a crude extract of pyruvic acid fermentation broth as a TPL-catalyzed substrate, the present disclosure may reduce raw material costs and the three wastes (waste gas, waste water, and industrial residue), and have the application value of industrial production.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[29] The present disclosure will be further described below with reference to examples.

[30] Example 1: Fermentation to obtain pyruvic acid

[31] Activation medium (YPD broth) included: 1% yeast extract paste, 2% peptone, and 2% glucose.

[32] Inoculum medium included: 10% glucose, 3% fish peptone, 0.1% K112 P0 4 , 0.05% MgSO4-7H20, 4 mg/L nicotinic acid, 400 [g/L pyridoxal, 20 g/L biotin, and 4% CaCO3, at pH 5.5.

[33] Fermentation medium included: 10% glucose, 0.1% soy peptone, 0.6% (NH4 ) 2 SO 4 , 0.1% KH 2 PO4 , 0.05% MgSO4-7H20, 8 mg/L nicotinic acid, 1 mg/L pyridoxal, 20 [g/L biotin, 20 [g/L thiamine, and 4% CaCO3, at pH 5.5.

[34] Activated culture: A strain stored in a -80°C cryogenic vial was inoculated into a 30 ml threaded-mouth bottle containing 5 ml of YPD broth using a pipette tip, and incubated at 220 r/min and 30°C or 40°C for approximately 12 h, until cells grew to logarithmic phase for transfer culture.

[35] Inoculum culture: The activated bacterial suspension was inoculated into a 500 ml shake flask containing a certain volume (10, 15, and 25 ml) of inoculum medium, and cultured at 220 r/min and °C or 40°C for 24 h.

[36] Fermentation culture: The inoculum fermentation broth was transferred to 500 ml shake flasks containing a certain volume (10, 15, and 25 ml) of fermentation medium at an inoculum size of 10%, and cultured at 220 r/min and 30°C and 40°C for 0-48 h, respectively.

[37] Fermentor fermentation: 15% glucose was added without CaCO3, and other components were the same as those of fermentation medium. The pH was adjusted to 5.5 with 5 M NaOH, rotational speed and oxygen flux were set at 500 and 1:1; without controlling the dissolved oxygen, fermentation was conducted at the corresponding temperature; after culturing for 50-72 h, the fermentation was stopped until pyruvic acid reached above 75 g/L and the product no longer increased.

[38] Crude extraction of pyruvic acid: The fermentation broth passed through a ceramic membrane to remove cells, and the filtrate passed through an ultrafiltration membrane to remove macromolecules such as proteins, nucleic acids, and pigments to obtain an aqueous solution of crude pyruvic acid. The pyruvic acid solution was diluted to 12 g/L, and other substrates were added to prepare a substrate solution; the pyruvic acid solution was concentrated to 120 g/L through a reverse osmosis membrane, and substrates such as catechol were added to prepare a feed solution.

[39] Example 2: Fermentation to produce TPL

[40] LB broth included: 10 g/L tryptone, 0.5 g/L yeast extract, 10 g/L sodium chloride, and pure water.

[41] Fermentation medium included: 12 g/L tryptone, 24 g/L yeast extract, 5 g/L glycerol, 2.31 g/L potassium dihydrogen phosphate, 16.43 g/L dipotassium hydrogen phosphate trihydrate, and pure water.

[42] 1) A single colony was picked and inoculated into a test tube with 4 ml of LB broth supplemented with kanamycin (50 mg/L), and cultured at 37°C and 220 rpm for 12 h to obtain primary inoculum;

[43] 2) The primary inoculum was inoculated into a 100 ml shake flask with fermentation medium, cultured at 37°C and 220 rpm for 4 h, mixed with isopropyl-p-D-thiogalactoside (IPTG) to a final concentration of 1 mM, and cultured at 25°C and 220 rpm for 12 h;

[44] 3) The bacterial suspension in step (2) was centrifuged to collect cells, and the cells were stored in a refrigerator at -20°C.

[45] Example 3: Extraction of TPL

[46] 1) The cells were mixed with 3 times the volume of water, and broken in a high-pressure homogenizer;

[47] 2) A supernatant enzyme extract was obtained by high speed centrifugation;

[48] Example 4: Catalytic reaction of TPL to produce L-DOPA

[49] 1) 0.5 L substrate solution: Crude extraction of pyruvic acid was diluted to a pyruvic acid concentration of 12 g/L; 10 g/L catechol, 60 g/L ammonium acetate, 2 g/L sodium sulfite, and 1 g/L EDTA were added to adjust to pH 8.0;

[50] 2) 0.5 L feed solution: After membrane filtration, the fermented pyruvic acid was concentrated to 120 g/L through a reverse osmosis membrane, and catechol was added to 120 g/L.

[51] 3) 10-100 g of enzyme extract was added to 0.5 L of substrate solution, mixed with pyridoxal triphosphate to 100 mg/L, sealed and stirred at 25°C;

[52] 4) The feed solution was added, and the concentrations of two substrates were controlled at not higher than 10 g/L;

[53] 5) When the product concentration reached above 120 g/L, feeding of feed solution was stopped;

[54] 6) When residual concentration of catechol was less than 0.2 g/L, the reaction was stopped, and L-DOPA concentration would accumulated to over 130 g/L.

[55] 7) The reaction mixture was acidified with dilute sulfuric acid or dilute hydrochloric acid to dissolve crystallized L-DOPA; the cells were removed by centrifugation, and L -DOPA in resulting supernatant was crystallized by adding dilute aqueous ammonia; crystals were collected by centrifugation;

[56] 8) The crude crystals were recrystallized once to obtain the pure L-DOPA product.

[57] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[58] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

WHAT IS CLAIMED IS:

1. A co-production process of pyruvic acid and levodopa, comprising the following steps: step 1, fermenting Torulopsis glabratato obtain a pyruvic acid fermentation broth, removing the cell from the fermentation broth by a ceramic membrane, removing proteins, nucleic acids, pigments through an ultrafiltration membrane, and concentrating a resulting filtrate to a pyruvic acid concentration of 150-300 g/L; step 2, preparing the enzyme-catalyzed substrate solution by adding a certain amount of pyruvic acid concentrated solution, catechol, ammonium acetate, ethylene diamine tetraacetic acid (EDTA), and sodium sulfite and adjusting pH to 7.0-9.0; step 3, fermenting genetically engineered Escherichiacoli strain expressing tyrosine phenol lyase (TPL) , centrifuging to collect cells, breaking cells on a high-pressure homogenizer, recentrifuging, and collecting cell extract containing TPL enzyme; and step 4, adding a certain amount of substrate solution to the cell extract, sealing and stirring at 25°C; wherein catechol concentration in the reaction solution is controlled at 0-10 g/L by feeding the feed solution, when a product reaches above 120 g/L, feeding of the feed solution is stopped, and when the content of catechol in the reaction mixture is less than 0.2 g/L, the reaction is ended.

2. The co-production process of pyruvic acid and levodopa according to claim 1, wherein a fermentor fermentation process in step 1 is as follows: adding 15% glucose without CaCO3, and other components being the same as those of fermentation medium; adjusting pH to 5.5 with 5 M NaOH, setting rotational speed at 500 and oxygen flux at 1 vvm; without controlling the dissolved oxygen, fermenting at the corresponding temperature for 50-72 h, and stopping the fermentation until the pyruvic acid reaches above 75 g/L and the product no longer increases.

3. The co-production process of pyruvic acid and levodopa according to claim 1, wherein in step 3, Luria-Bertani (LB) broth comprises: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride, and pure water; the fermentation medium comprises: 12 g/L tryptone, 24 g/L yeast extract, 5 g/L glycerol, 2.31 g/L potassium dihydrogen phosphate, 16.43 g/L dipotassium hydrogen phosphate trihydrate, and pure water.

4. The co-production process of pyruvic acid and levodopa according to claim 3, wherein a single colony is picked and inoculated into a test tube with 4 ml of LB broth supplemented with kanamycin

(50 mg/L), and cultured at 37°C and 220 rpm for 12 h to obtain a primary inoculum; the primary inoculum is inoculated into a 100 ml shake flask with fermentation medium, cultivated at 37°C and 220 rpm for 4 h, adding isopropyl-p-D-thiogalactoside (IPTG) to a final concentration of 1 mM, and continued to incubate at 25°C and 220 rpm for 12 h; the cells was collected by centrifugation and stored in a refrigerator at -20°C.

5. The co-production process of pyruvic acid and levodopa according to claim 4, wherein the TPL enzyme is prepared by breaking cells with 3 times volume of water in a high-pressure homogenizer; a supernatant cell extract is obtained by high speed centrifugation;

wherein in the step 4, 10-100 g of the enzyme extract is added to 0.5 L of the substrate solution, mixed with pyridoxal triphosphate to 100 mg/L, sealed and stirred at 25°C;

wherein in step 4, when residual concentration of catechol is less than 0.2 g/L, the reaction is stopped, and levodopa concentration is accumulated to above 130 g/L; the reaction mixture is acidified with dilute sulfuric acid or dilute hydrochloric acid to dissolve crystals; the cells are removed by centrifugation, and L-DOPA in resulting supernatant is crystallized by adding dilute aqueous ammonia; crystals of L-DOPA are collected by centrifugation, and crude crystals are recrystallized once by acid dissolution and alkaline precipitation to obtain the pure product.