[0001] The following specification describes the invention and the manner in which it is to be performed:

DESCRIPTION OF THE INVENTION Technical field of the invention [0002] The present invention relates to a biochemical method for producing vanillin and related phenylpropanoids and derivatives. More particularly, the invention discloses a method of producing vanillin using the native enzyme with specific modification or supplementation to derive commercially attractive yield.

Background of the invention [0003] Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a phenolic aldehyde with the molecular formula CsHsCb having functional groups aldehyde, hydroxyl, and ether. It is the primary component of the extract of the vanilla bean and is used widely in the industry as a flavoring agent. Natural vanilla extract is a mixture of several hundred different compounds in addition to vanillin, such as piperonal/heliotropine (1,3- benzodioxole-5-carbaldehyde), anisaldehyde (4-methoxybenzaldehyde), Veratraldehyde (3,4-Dimethoxybenzaldehyde), acetylvanillin (4-Formyl-2-methoxyphenyl acetate), 4- hydroxybenzaldehyde, O-Benzylvanillin (4-Benzyloxy-3-Methoxybenzaldehyde), etc. However, due to the scarcity and expense of natural vanilla extract, synthetic preparation of its predominant component has been in use in the current scenario. [0004] The synthesis of vanillin by chemical derivatives including guaiacol, a byproduct from petroleum refinery and lignin, derived from agricultural wastes is widely used over natural vanilla extract as a flavoring agent in foods, beverages, pharmaceuticals and related industries. However, there has been a huge surge in the exploration of more environmentally friendly biosynthetic procedures to make natural flavors. Vanillin derived using plant derived raw material (eugenol, ferulic acid, curcumin, glucose etc.) with natural biochemical reaction processes.

[0005] The use of synthetic vanillin for flavoring is a cost effective and affordable substitute for vanilla pod-derived vanilla flavor. The commercial synthesis of vanillin as with the more readily available natural compound eugenol (4-allyl-2-methoxyphenol), a colorless to pale yellow, aromatic oily liquid extracted from certain essential oils especially from clove oil, nutmeg, cinnamon, basil and bay leaf. Currently, artificial vanillin is generally synthesized either from guaiacol (CeH) or lignin. Lignin is a class of complex organic polymers that form key structural materials in the support tissues of vascular plants and some algae and lignin is available in abundance in the form of agriculture waste. The method of extraction of these flavoring agents plays a very important role to determine quality of the final product.

[0006] Various potential biotechnological methods are available for production of these high-value flavor compounds, as an alternative to the vanilla orchid derived vanilla flavor agents. Eugenol, isoeugenol, ferulic acid, chavicol, isochavicol, methyleugenol, saffrole, methyl isoeugenol, estragole, anethole, propenylphenol are the major phenylpropene compounds, formed by terpene synthesis in plants and these compounds are present in reasonable quantities in various plant-materials and essential oils.

[0007] Phenylpropanoids also known as cinnamic acids are relatively simple secondary metabolites that are derived from the shikimic acid pathway through phenylalanine and tyrosine in some plants. The phenylpropanoid pathway serves as a rich source of metabolites in plants, being required for the biosynthesis of lignin, and serve as a starting point for the production of many other important compounds, such as the flavonoids, coumarins, and lignans. These phenylpropanoid pathway components either in purified or crude form have often been attempted as the starting materials to produce vanillin and vanillin related high-value aromatic flavor compounds using enzymatic or microbial biotransformation.

[0008] Although lots of research has taken place with respect to production of vanillin and related phenylaldehyde compounds using microorganisms, yet, the requirement of sophisticated bioreactors, sterilized media-conditions, dependence on the microbial culture growth and associated limitations such as lower yields or conversion ratios, lead to a complex and costly processes. As a result, the price of the biotransformed vanillin is high and cost-effective method for synthesis of “natural” vanillin is still a challenge.

[0009] The process of bioconversions to produce natural vanillin using recombinant- technology derived enzymes and genetically modified microbes promise attractive conversion yields. For industrial scale processes, it is often necessary to produce one or several functionally modified: mutant or engineered enzymes in a recombinant microbe. However, Genetically Modified Organism (GMO) products have less consumer acceptance and necessitates specific labelling or declaration requirements, which makes the overall process complicated even though functionally GMO or GMO derived engineered enzymes may ensure better yield of the product.

[0010] Specific microbial strains or genetically engineered microbial strains can successfully be used for biotransformation and biosynthesis of vanillin and related phenyl aldehydes. However, making and maintaining sterile media, healthy culture conditions to grow microbes and susceptibility of microbes to substrate toxicity and phage/contamination makes the production complex and costly, especially at the industrial level.

[0011] As an alternate to microbial culture, directly using the purified enzymes responsible for specific transformation reaction may be considered as an alternate option. However, as most of these pure enzymes are derived from recombinant hosts and for commercial production, such pure enzyme based processes pose limitations due to low protein-stability, cofactor-requirements and low activity rates. [0012] The Patent Application No. US5358861A entitled “Process for the preparation of phenyl aldehydes ” discloses a process for the enzymatic preparation of phenylaldehydes, in particular vanillin using a soybean lipoxygenase for conversion of isoeugenol to vanillin, in the presence of specific metal ion chelators high enzymatic transformation rates could be further achieved.

[0013] The Patent Application No WO2019185926 entitled “ METHOD FOR PRODUCING VANILLIN ’ discloses a novel method of producing vanillin and/or derivatives thereof by applying improved biocatalysts. The invention also discloses the expression systems for preparing said improved biocatalysts. In addition, novel enzyme mutants, corresponding coding sequences and vectors applicable in the biochemical production of vanillin are also disclosed. The invention further provides recombinant host cells or organisms genetically modified for improved functional expression of biocatalysts, as well as recombinant host cells or organisms useful to produce vanillin.

[0014] The Patent Application No. CN108795889A entitled “A kind of mutant and mutant strain of trans-anethole oxygenase ” discloses a kind of mutant and mutant strain of trans-anethole oxygenase, synthesize p-anisaldehyde, veratraldehyde, heliotropin, vanillic aldehyde, it is more suitable for industrial production application, cost can be reduced, production efficiency is improved.

[0015] However, the available processes or methods could only partially address the above-mentioned challenges associated with biotransformation technique to derive vanillin and related phenyl aldehydes.

[0016] Hence, there is a need to have plant- and/or microbial-culture derived native purified enzymes as an alternative to bio-convert plant-derived natural phenylpropene compounds with high yield.

Summary of the invention

[0017] The present invention overcomes the drawbacks existing prior arts. The present invention relates to a method for production of vanillin and related phenylpropanoids and derivatives. The present invention relates to the method of production of vanillin by the catalysis of isoeugenol by mini lipoxygenase (LOX) enzyme.

[0018] The enzyme LOX is extracted from soybean seeds. LOX catalyzes the hydroperoxidation of polyunsaturated fatty acids and esters. LOX comprises catalytically inactive N-terminal domain, catalytically active C-terminal domain, iron and substrate binding site. LOX exhibits decreased efficiency due to low catalytic activity and product inhibition. The proteolytic truncation of N-terminal domain of LOX results in increased catalytic efficiency and reduced substrate specificity, which exhibits in increased yield.

[0019] The biochemical method of catalysis of isoeugenol to vanillin comprises extraction and purification of lipoxygenase from soybean seed extract followed by truncation of N-terminal of LOX by tryptic cleavage resulting in the formation of mini- LOX. The truncation of LOX is detected by Sodium Dodecyl Sulphate- Poly Acrylamide gel electrophoresis (SDS-PAGE).

[0020] The enzyme mini-LOX is mixed with isoeugenol for the catalysis of the reaction. Vanillin is obtained as a product during the course of the reaction by sequential solvent extraction. The crude vanillin extract is purified and crystallized for further use.

[0021] The biochemical method of catalysis of isoeugenol to vanillin by mini-LOX was analyzed by performing high performance liquid chromatography (HPLC). The yield of vanillin obtained by Mini-LOX enzyme was 48%-50% higher when compared with native LOX enzyme. The peaks in the chromatogram indicated the presence of vanillin and isoeugenol.

[0022] The production of mini-LOX for the catalysis of isoeugenol to vanillin is also achieved by the overexpression of mini-LOX by recombinant E. coli. The synthetic gene encoding N-terminal domain truncated LOX was cloned into pETRXVD-TOPO vector. The vector was transformed into E. coli cells and the recombinant cells were cultured. The cells were harvested and autolyzed. The autolyzed cells were centrifuged to obtain the cell lysate. The cell lysate comprising mini-LOX enzyme was mixed with isoeugenol. Crude vanillin extract was obtained which was further purified and crystallized. [0023] The present invention further relates to the method of production of related phenylpropanoids. Veratraldehye belongs to the class of phenylpropanoids. The catalysis of methyl isoeugenol to veratraldehyde is also achieved by mini-LOX enzyme. The yield of veratraldehyde was analyzed by HPLC. The yield of veratraldehyde obtained by Mini- LOX enzyme was 90% higher when compared with native LOX enzyme. The peaks in the chromatogram indicated the presence of veratraldehyde and methyl isoeugenol.

[0024] The present invention relates to the biochemical method of production of vanillin and related phenylpropanoids. The method involves proteolytic truncation of soybean LOX to produce mini-LOX. The mini-LOX enzyme exhibits increased catalytic efficiency and yield. The method is simple, cost effective and natural. Vanillin has a distinct aroma and flavor and is majorly used in food, beverage industries, agriculture, pharmaceuticals and personal care products. It is also used as chemical intermediate in several reactions.

Brief description of the drawings

[0025] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.

[0026] FIG 1 illustrates the flowchart for the production of vanillin from the catalysis of isoeugenol by mini- lipoxygenase (mini-LOX).

[0027] FIG 2 illustrates the flowchart for the preparation of mini-LOX from soybean seed extract.

[0028] FIG 3 illustrates the schematic representation of the catalysis of isoeugenol to vanillin by mini-LOX.

[0029] FIG 4 illustrates the recombinant E. coli construct with soybean mini-LOX gene. [0030] FIG 5 illustrates the flowchart for the method of overexpression of mini-LOX by recombinant E. coli.

[0031] FIG 6 illustrates the SDS-PAGE gel visualization of mini-LOX protein.

[0032] FIG 7 illustrates the yield of vanillin obtained by the catalysis of isoeugenol by native-LOX and mini-LOX.

[0033] FIG 8 illustrates the chromatogram of vanillin and isoeugenol.

[0034] FIG 9 illustrates the schematic representation of catalysis of methyl isoeugenol to valeraldehyde by mini-LOX.

[0035] FIG 10 illustrates the chromatogram of veratraldehyde and methyl isoeugenol.

Detailed description of the invention

[0036] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.

[0037] The term “Genetically Modified Organism (GMO)” are living organisms whose genetic material has been artificially manipulated in a laboratory through genetic engineering.

[0038] The term “Phenylpropanoids” refers to a diverse family of organic compounds that are synthesized by plants from the amino acids phenylalanine and tyrosine.

[0039] The term “Vanillin ” refers to an organic compound with the molecular formula C ₈ H ₈ O ₃ . It is a phenolic aldehyde. Its functional groups include aldehyde, hydroxyl, and ether.

[0040] The present invention relates to a biochemical method of producing vanillin and related phenylpropanoids and derivatives. The invention discloses a method of using native enzyme with specific modification and supplementation to derive commercially attractive yields of vanillin in order to overcome the existing problem of conversion-rate, co-factor or substrate-specificity etc. The enzymatic method results in higher conversion rate and broad substrate specificity and cofactor stability. The enzyme is adaptable for using various commonly available plant-derived substrates for biosynthesis/transformation to multiple high-value flavor compounds. The method is simple and overcomes requirement of GMO, sterile-media conditions, microbial contamination. Further, the enzymes involved are derived through common food plant or microbe sources. In addition, the substrate, enzymatic reaction and purification components employed in, the method involves food-grade ingredients.

[0041] The biochemical method of producing vanillin relates to enzymatic catalysis of isoeugenol substrate to vanillin. Lipoxygenase is extracted from soybean seeds to catalyze isoeugenol for the production of vanillin.

[0042] Lipoxygenase is an iron-containing dioxygenase having a 30kDa N-terminal domain and a catalytically active 60kDa C-terminal domain with iron and substrate binding site. Lipoxygenase exhibits low enzyme catalytic activity and product inhibition. The truncation of catalytically inactive N-terminal domain by tryptic digestion results in significant increase in catalytic activity and increased yield due to decreased substrate specificity.

[0043] FIG 1 illustrates the flowchart for the production of vanillin from the catalysis of isoeugenol by mini- lipoxygenase (mini-LOX). The method (100) of vanillin production from isoeugenol begins at step (101) where the enzyme lipoxygenase is extracted and purified from soybean extract. The soybean seeds are ground, defatted and homogenized in tris-HCl buffer. The homogenized mixture is centrifuged at 20,000g for 30 minutes and filtered to obtain crude enzyme extract which is further fractionated and purified. The lipoxygenase comprises 30kDa N-terminal domain and 60kDa C-terminal domain. At step (102), the proteolytic digestion of N-terminal domain results in truncated mini-LOX. The proteolytic digestion is carried out using trypsin and LOX at a ratio of 5: 1 where the reaction is allowed for 1 -2 hours followed by termination of the reaction by the addition of trypsin inhibitors. At step (103), the truncation of N-terminal domain of LOX is analyzed by running SDS-PAGE.

[0044] At step (104), the oxidative cleavage of isoeugenol to vanillin is catalyzed by mini-LOX. The substrate isoeugenol at a concentration range of 20mM-100mM dispersed in buffer is mixed with 10% mini-LOX catalyst in 0.2M borate buffer. The reaction mixture is agitated at 180rpm, at a temperature of 30°C and >80% oxygen saturation. At step (105), the catalysis of isoeugenol to vanillin by mini-LOX is subjected for 72 hours. Upon completion of the catalysis, crude vanillin extract is obtained. The concentration of the crude vanillin extract is analyzed at regular time intervals. At step (106), the excess isoeugenol is separated from the reaction mixture by extraction twice with equal volumes of hexane. At step (107), the crude vanillin extract is subjected to sequential solvent extraction with ethyl acetate as solvent. At step (108), the crude vanillin extract obtained through solvent extraction is further dehydrated and concentrated by rotary evaporation. At step (109), the concentrated vanillin extract is mixed with distilled water and heated at a temperature in a range between 50°C - 80° C. At step (110), the vanillin extract is subjected to crystallization at 4° C. The brownish-pink vanillin crystals are obtained with 70%-90% purity.

[0045] The enzyme lipoxygenase (LOX) is extracted from soybean seeds. LOX exhibits reduced catalytic activity and product inhibition due to increased substrate specificity. In order to overcome said problem, the catalytically inactive N-terminal domain of LOX is truncated to produce mini-LOX, which exhibits increased enzyme catalytic activity due to decreased substrate specificity.

[0046] FIG 2 illustrates the flowchart for a method of preparation of mini-LOX from soybean seeds. The method (200) of preparation of mini-LOX from soybean begins at a step of (201) where 50g of soybean seeds are ground and the ground mixture is defatted by petroleum ether. At step (202), the ground soybean mixture is homogenized with 50mM Tris-HCL buffer in the ratio of 1:10 at 4°C overnight. At step (203), the homogenized soybean mixture is centrifuged at 20,000 g for 20 minutes. At step (204), the supernatant obtained after centrifuging is filtered through a 0.2pm membrane filter. At step (205), the filtered supernatant is subjected to ammonium sulphate fractionation to obtain lipoxygenase enzyme pellet. At step (206), the obtained lipoxygenase enzyme pellet is stored in borate buffer at 4°C. At step (207), lipoxygenase enzyme pellet is added with 10ml of 0.2M borate buffer and 0.5ml of EDTA to obtain lipoxygenase enzyme solution. At step (208), the N-terminal domain of lipoxygenase enzyme fraction is digested with trypsin in the ratio of 5:1 for a duration of about 1 hour. At step (209), the tryptic digestion is terminated by adding al -antitrypsin. The tryptic digestion results in truncated mini-LOX enzyme. At step (210), truncated mini-LOX enzyme mixture is subjected to membrane filtration and purification. The 30kDa N-terminal domain is filtered separated. At step (211), the purified retentate fraction comprising mini-LOX is washed and concentrated with buffer.

[0047] The truncation of 30kDa N-terminal domain of lipoxygenase is analyzed by Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS-PAGE). The LOX enzyme comprises a 30-kDa catalytically inactive N-terminal domain and a catalytically active 60-kDa C-terminal domain with substrate binding sites. Upon tryptic digestion of LOX, the catalytically inactive N-terminal domain is cleaved and 60kDa C-terminal domain is retained. FIG 6 illustrates the SDS-PA gel electrophoresis of mini-LOX protein. The bands on SDS-PA gel electrophoresis indicates the presence of enriched purification fraction with 60kDa mini-LOX fragment.

[0048] FIG 3 illustrates the schematic representation of the catalysis of isoeugenol to vanillin by mini-LOX. The enzyme mini-LOX catalyzes the oxidative cleavage of isoeugenol to vanillin. The enzyme comprises iron-containing dioxygenase which catalyzes the hydroperoxidation of polyunsaturated fatty acids and esters. The oxidized iron-containing mini-LOX binds to the isoeugenol substrate at the C-terminal. The reaction leads to formation of several intermediates including free radical intermediate, hydroperoxide intermediate and dioxetane intermediate. The catalysis of isoeugenol by mini-LOX results in formation of vanillin and acetaldehyde as by-product.

[0049] The present invention further discloses the catalysis of isoeugenol to vanillin by overexpression of mini-LOX using recombinant E. coli. The gene encoding mini-LOX is induced in a vector and transformed into E. coli cells. The recombinant cells produce mini-LOX enzyme, which is extracted from the cells. The substrate isoeugenol is mixed with mini-LOX to obtain vanillin. The obtained vanillin is purified and crystallized.

[0050] FIG 4 illustrates the recombinant E. coli construct with soybean mini-LOX gene. A synthetic gene encoding the N-terminal domain truncated versions of Soy mini-LOX is designed and cloned into pET 100/D TOPO vector. The mini-LOX amino acid sequence is illustrated as SEQ ID NO: 1 and comprises 562 amino acids.

[0051] FIG 5 illustrates the flowchart for the method of overexpression of mini-LOX by recombinant E. coli. The method (500) of overexpression of mini-LOX by recombinant E. coli begins at a step of (501) where synthetic gene encoding N-terminal truncated soy lipoxygenase for prokaryotic expression is synthesized in pET 100/D TOPO vector. At step (502), the chemically competent E. coli strains are subjected to transformation by calcium chloride method. At step (503), the transformed E. coli cells are cultured on Luria-Bertani agar (LB agar) media comprising ampicillin. At step (504), protein expression of recombinant mini-LOX protein is induced by the addition of 0.5mM isopropyl-P-d-thiogalactoside (IPTG). At step (505), the transformed recombinant E. coli cells are cultured for 12 hours. The cultured cells are harvested at 18° C. At step (506), the harvested cells are washed with phosphate buffer saline (PBS). The washed cells are resuspended in Tris buffer. At step (507), the suspended cells are frozen at -80° C and stored in liquid nitrogen. At step (508), the frozen cells are thawed. The thawed cells are autolyzed and ultrasonicated for 5-10 minutes to obtain the cell lysate with mini-LOX enzyme. At step (509), the cell debris is removed by centrifugation at 10,000g for 30 min at 4°C. Upon centrifugation, crude enzyme lysate is obtained. At step (510), the crude enzyme lysate along with additives are added to isoeugenol in borate buffer and the mixture is agitated at 180 rpm and at a temperature of 25-30 °C. At step (511), the sample comprising vanillin in the supernatant is centrifuged and analyzed by high performance liquid chromatography (HPLC).

[0052] The truncation of N-terminal domain of LOX overcomes product inhibition and reduced catalytic activity. The catalysis of isoeugenol to vanillin by truncated mini-LOX yields in increased catalytic activity due to decreased substrate specificity. FIG 7 illustrates the yield of vanillin obtained by the catalysis of isoeugenol by native-LOX and mini-LOX. The catalysis of isoeugenol to vanillin by native LOX is hindered due to product inhibition and low enzyme activity. The truncation of catalytically inactive C- terminal domain yielded in enhanced catalytic efficiency and higher membrane binding ability of mini-LOX. The truncation also yields in reduced substrate specificity, which aids in higher binding rates and hence product formation. The chromatogram indicates 48% higher yield with mini-LOX when compared to the catalysis using the native LOX.

[0053] The yield of catalysis of isoeugenol to vanillin by mini-LOX was analyzed by performing HPLC. The HPLC analysis includes a mobile phase comprising a mixture of methanol and 0.01% acetic acid at a ratio of 65:35, with flow rate of lml/min and at an absorbance of 280nm. The HPLC analysis further includes vanillin standard.

[0054] FIG 8 illustrates the chromatogram of vanillin and isoeugenol. HPLC analysis was performed for the obtained vanillin from catalysis of isoeugenol by mini-LOX. The yield of vanillin obtained by catalysis of isoeugenol by mini-LOX enzyme was 48%-50% higher when compared with native LOX enzyme. The peaks in the chromatogram indicated the presence of vanillin and isoeugenol. The retention time for vanillin was 4.0 minutes and the retention time for isoeugenol was 11.1 minutes.

[0055] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

Example 1: Catalysis of methyl isoeugenol to veratraldehyde by mini-LOX

[0056] The present invention relates to a biochemical method for production of vanillin and related phenylpropanoids and derivatives. Veratraldehyde is a phenylpropanoid commonly used as a flavoring agent and an odorant. Veratraldehyde is also used as an intermediate in various pharmaceutical substances. [0057] The catalysis of oxidative cleavage of methyl isoeugenol by mini-LOX results in the production of veratraldehyde. The substrate methyl isoeugenol dissolved in ethanol is taken at a concentration of I OmM and dispersed in buffer media. The enzyme mini-LOX is added to the substrate at a concentration of 10% to initiate the reaction. The reaction is carried out by agitating at 180 rpm at a temperature of 30°C for 72 hours. The concentration of veratraldehyde was examined at regular intervals.

[0058] FIG 9 illustrates the schematic representation of catalysis of methyl isoeugenol to veratraldehyde by mini-LOX. The catalysis of methyl isoeugenol to veratraldehyde by mini-LOX is achieved under reaction conditions of 20mM borate buffer, pH of 8.5-9.0, at oxygen saturation of 50-100%, at a temperature of 15-30°C for 36 to 92 hours.

[0059] The yield of catalysis of methyl isoeugenol to veratraldehyde by mini-LOX was analyzed by performing HPLC. The HPLC analysis includes a mobile phase comprising a mixture of methanol and 0.01% acetic acid at a ratio of 65:35, with flow rate of lml/min and at an absorbance of 280nm. The HPLC analysis included veratraldehyde standard.

[0060] FIG 10 illustrates the chromatogram of veratraldehyde and methyl isoeugenol. HPLC analysis was performed for the veratraldehyde obtained from methyl isoeugenol catalyzed by mini-LOX. The yield of veratraldehyde obtained by catalysis of methyl isoeugenol by mini-LOX enzyme was 90% higher when compared with native LOX enzyme. The peaks in the chromatogram indicated the presence of vanillin and isoeugenol. The retention time for veratraldehyde was 4.0 minutes and the retention time for methyl isoeugenol was 15.4 minutes.

[0061] The present invention relates to a biochemical method for the production of vanillin and related phenylpropanoids and derivatives. The biochemical method of the present invention utilizes naturally derived enzyme with desired conversion and broad substrate specificity for commercially viable production of high-value flavor phenylaldehyde compounds. The method is simple, cost effective and natural. The biotechnologically produced vanillin and related phenylpropanoids are widely utilized in food, beverages, pharmaceuticals, cosmetics, agricultural industries and so on. The biochemical method employed is commercially viable and industrially applicable.