Title of Invention

"A PROCESS FOR THE PRODUCTION OF VERBENOL"

Abstract

The present invention provides a process for the production of verbenol, which comprises the steps of: growing parent culture Aspergillus niger and Penicillium digitatum separately on potato dextrose broth and YMG broth under a shaking condition of 150-200 rpm; isolating protoplast separately from the grown biomass followed by filtration in order to obtain filtrates separately ; mixing the filtrates in a ratio of about 1:1 containing equal number of protoplasts; incubating the mixture in a fusion medium;making serial dilutions followed by plating on to a minimal medium II to facilitate the growth of fusants or heterokaryon ;transferring the grown fusants/heterokaryons to a known complete medium to obtain sporulation which are further grown on a potato dextrose broth ; filtering the biomass and adding it to buffer solution to which alpha pinene is added; and extracting the product in dichloromethane to get verbenol.

Full Text

The present invention relates to a process for production of verbenol. More in particular the process involves fusion of strains for the production of verbenol. Verbenol is a high valued flavour compound which has wide usage in food industries and used as a food flavour additive starting from alpha pinene a waste product of eucalyptus peel oil and /or pine bark. The perception of natural as better than artificial has led to an increased demand for natural flavours (Gatfield IL. Food Industrial perspective. In Parliament TH, Croteau R, editors-Biogeneration of Aroma. Washington, DC: American Chemical Society, 1986: 11-7, as this is the major concern, there has been strong interest in the Biotechnological production of natural flavour compounds during the past. (Wasserman BP, Montville TJ, Korwek EL, Food Biotechnology, Food Technol 1988;42: 133-46, Feron G, Bonnarme P, Durand A. Prospects for the microbial production of food flavours. Trends Food Sci. Technol 1996: 7: 285-93. Murray WD, DuffSJB, Lanthier PH, Armstrong DW, Weish FW, Williams RE. Development of Biotechnological process for the production of natural flavours and fragrances. In Charalaarnbous G, editor Frontiers of flavour Amsterdam: Elserier, 1988:1-18. Microbial fermentation is regarded as a potential means for producing natural flavour substances and the field has a great deal of interest today. Production of 4,6,6 trimethyl bicyclo 3.1.1 Hept -2en-2ol by this means is considered as GRAS food additive by the US Food and Drug Administration. (Aretander.S. Perfumes and flavour chemicals. NJ: Montdair Press 1969). It is widely used in the flavour industry, for preserving food grains and also as the direct precursor for the manufacture of taxol a natural anticancerous drug. Obtaining high valued flavouring compound is of immense importance as the starting material are low cost and are easily available. It is also economically feasible as a high valued compound is produced. The compound 4,6,6 trimethyl bicyclo (3.1.1) hept 2en-2ol is present in the oil of Verbena,Dili, Spearmint, Eucalyptus, blackberry and strawberry (Guenther E. The essential oils (1952) Vol.2, 444. Fritzche brothers, Inc.New York, Publisher: D.Van Nostrand Company inc. It is commonly known as verbenol. The drawing accompanying this specification figurel represents the general bioconversion reaction as follows: As a natural source mainly distillation of leaves is utilized, the yield of the oil is very low ranging from 0.072% to 0.19% which contains number of other compounds in the oil as well. It is not enantiospecific and is not pure. To separate and purify a single compound is again a very expensive method. By the present method the compound can be produced in larger quantities and in purer form which will be economically viable and would be a valuable adjunct to food. The current market price of 4,6,6 trimethyl bicyclo (3.1.1) hept 2en-2ol is Rs.1,20,000/- per kg, bycyclic monoterpene hydrocarbon as shown by Ramadevi, J. and Bhattacharyya.P.K. (1978). J.lnd.Chem.Soc. Vol.IV 1131-1137. The monoterpene alphapinene is a byproduct from the bark of Eucalyptus tree which is usually a waste product. It is also available from turpentine oil as a by product. The conversion of low cost, low valued alpha pinene as substrate to 4,6,6 trimethyl bicyclo (3.1.1) hept -2en-2ol, a high valued and high priced flavour compound is utilized by bakeries, confectionary, icecream and beverage industries. It is also advantageous in the preservation of food grains and is an antioxidant. In the present work protoplasts have been isolated from parent type Aspergillus niger and P.digitatum strain, a local isolate. The fungal isolates were isolated from soil which was collected from the area where the lime trees are grown in CFTRI campus, Mysore. These cultures have been deposited in CFTRI culture collection centre A.niger with CFR No.218 and P.digitatum with CFR No.219. The two protoplasts are fused to obtain a new strain which has a higher conversion efficiency towards formation of 4,6,6 trimethyl bicyclo (3.1.1) hept -2en-2ol from alpha pinene. Recently this subject has received a lot of attention. Number of scientists have worked on similar lines (Na Kayama, H, 1978; Cad Waller, 1989; Cheetam, 1995) Nakayama, H, Yamaguchi, Y. Maclida, Iwasaki, S, Komtsu.A and Shinoda.A. D.Lmenthol (1978). Japan.Kokai source:CA, 89, 74217K. Cadwallader.KR, Braddock, R.J.Parish, M.E. and Higgins, DP. Bioconversion of (+) limonene by Pseudomonas gladioli (1989). Journal of Food Science Vol.54, 1241-45. Cheetham Peter, S.J. Biotransformations, New routes to food ingredients (1995). Chemistry and industry 3 April. Although there are some patents on the production of 4,6,6 trimethyl bicyclo (3.1.1) hept -2en-2ol utilizing Yeast and bacterial cultures but the conversion yield is very low and also no one has tried to get a better strain which has better conversion efficiency through protoplast fusion. There is no work of similar kind being carried out showing higher conversion efficiency. Therefore, there is an urgent need to have this type of a process which will help in the utilization of monoterpene hydrocarbon alpha pinene, a by product from the waste of food industries. The main objective of the present invention is to provide a novel biotechnological process mediated by a selected intergeneric fusant strain between A.niger and P.digitatum for the production of verbenol. Another object is to obtain higher conversion efficiency of alpha pinene to verbenol. Still another object is to optimize conditions for protoplast isolation, regeneration and fusion conditions between protoplasts of two different genera of fungal cultures producing high yield of verbenol. Accordingly, A process for the production of verbenol which comprises, a. growing parent culture A as A.niger and B parent culture as P.digitatum separately on potato dextrose broth and YMG broth respectively for 10-18 hours, at pH (4.5-6.5) and at temperature (25-35°C) and at 150-200 rpm, b. isolating protoplast from the grown biomass for using lysing enzymes followed by filtration using known methods to obtain filtrate, c. mixing filterate at 1:1 ratio containing equal number of protoplasts, d. incubating the mixture is in a prewarmed (28-30°C) known fusion medium I for 10-20 minutes and serially diluting and plating out on a known minimal medium II for the fusants to grow which is further taken on a known complete medium for 48 hours at 30°C till sporulation, e. growing the spores on a potato dextrose broth at a pH (5.5-7.0) for 18-22 hours at 28-32°C, f. filtering the biomass and reacting the filtered biomass in the buffer solution which is further incubated with a substrate alpha pinene for 4-8, g. extracting the product in dichloromethane which is further concentrated by known method to obtain verbenol. In an embodiment of the present invention the conditions for protoplast isolation is in a buffer having 0.05M citrate phosphate.and at a pH 5.8 with KCI as osmotic stabilizer having strength 0.7 M. In an embodiment of the present invention the minimal medium containing sucrose for regeneration of fused colonies is in the range of 20-30g/100 ml. In an embodiment of the present invention the biomass is grown in Potato Dextrose Broth in a preferable range of pH 5.0-6.5, 25-30°C, 110-120 rpm for a period of 20-25 h. The novelty of the invention lies in the intergeneric fusant strain for maximising yield of the compound by protoplast fusion where the yield is much more than which produced by the plant sources or by any other natural source being used in hither to known processes. The invention is related to the use of an improved strain of intergeneric fusion between A.niger and P.digitatum to produce a product by a novel biotechnological process. The culture has been deposited in CFTRI culture collection centre and designated as APFusant No. CFR220. The reported literature does not deal on the issue of protoplast fusion for obtaining higher yield of verbenol by biotransformation. The prior art dealing with the conversion of substrate, alpha pinene to verbenol is very low with a conversion rate of 15-20% only. Moreover, these reports are for yeast and bacterial strains. The isolated intergeneric fusant strain of A. niger and P. digitatum have the following characteristics. The spores were grey is colour unlike of the parent cultures, grows well on potato dextrose agar at 32°C. The spore size of A.niger wild type is 3.7 µm, P. digitatum 5.6 µm and for fusant 3.9 µm and colony size for A.niger 1.45 cm YW, P.digitatum 1.74 cm YW and for fusant 0.9 cm L/w. The time taken for sporulation in A.niger is 48 h at 30°C in PDA, P.digitatum 52 h at 37°C in YMGA and 52 h at 32°C in PDA. Citrate phosphate buffer at 0.05M was found good to keep protoplasts of A.niger at 0.1 OM for P.digitatum and 0.05M for the fusant. The fusant is a haploid as observed with geimsa stain microscopically. Stable fusants were identified by non sectoral growth by benomyl treatment (20 µg/15 ml media) and periodic product formation through 5-6 sub cultures. The fusant strain has a desirable characteristic of high converison rate of the substrate with 59% conversion rate which is a synergistic effect. The colony size is smaller with 0.9 cm diameter. It has blackish grey, spores with 3.9 µm size and a sporulation time of 52 h and biomass of 2 g after 20 h of growth. The growth temperature of fusant was found optimum at 32°C. Hence, the present invention provides a novel biotechnological process mediated by a selected fusant strain of Aspergillus niger and Penicillium digitatum for the production of verbenol generally described as below: A biotechnological process for production of verbenol using intergeneric fusant. The process comprises of the followsing steps - 1. Isolation of a novel intergeneric fusant strain between Aspergillus niger and Penicillium digitatum. 2. The fusion of two protoplasts were with fusgen PEG 4000 (20%) and incubation of protoplasts for 10 min. at 30°C. 3. The culture is maintained on potato dextrose agar at 32°C. 4. The growth cell biomass was taken in phosphate buffer (0.05M, pH 7.0) at 30°C and incubated with alpha pinene for only 6 h at 200 rpm. 5. The end product was extracted in dichloromethane and analyzed on GC and GCMS. The invention is further illustrated by the following examples. EXAMPLE 1 Protoplasting was tried with different enzymes and enzyme combinations. A combination of cellulase, glucuronidase and lysing enzyme was found suitable in the concentration of 1:1:1 for/A.n/ger and 1:2:1 for P.digitatum when incubated for 3 h with 20 mg biomass which was initially grown for 14 h in its exponential phase as shown in Table 1. Higher or lower amounts of enzymes did not help in regeneration frequency. Intially maleic acid buffer (0.2 M, pH 5.8) was tried. Table 1: Effect of enzyme concentration and time of incubation on the (%) yield of protoplast and regeneration frequency (Table Removed) Buffer used Maleic acid (0.2M), osmotic stabilizers MgCI2, Biomass taken 20 mg/ml, 14 h growth. EXAMPLE 2 In order to test different buffer systems and to optimize the type of buffer required for best protoplasting three buffers were tried. It was found that citrate phosphate buffer (0.05 M, pH 5.8) with KCI as osmotic stabilizer (0.7M) was found to be the best for protoplast yield in A.niger and citrate phosphate buffer (0.1M, pH 5.8) with KCI as osmotic stabilizer (0.7 M) was found to be the best for protoplast yield in P.digitatum where as citrate phosphate buffer (0.05M, pH 5.8) with KCI as osmotic stabilizer (0.7M) was found good for keeping the two protoplasts of A.niger and P.digitatum for maximum fusion and regeneration (Table 2). Table 2: Effect of different buffer systems on protoplast formation (Table Removed) Enzymes used for protoplasting: Cellulase + Glucuronidase + Lysing enzyme 1:1:1 for A.niger 1:2:1 for P.digitatum EXAMPLE 3 As the buffer and its molarity were standardized for protoplasting, conditions were optimized for best osmotic stabilizer. In view of this, different stabilizers (0.7 M) were tried namely KCI, NaCI and sucrose both for A.niger and P.digitatum. In view of this, different stabilizers (0.7 M) were tried namely KCI, NaCI and sucrose. It was found that KCI (0.7 M) was the best osmotic stabilizer for protoplasting and for regeneration frequency in both the cases (Table 3). Table 3: Effect of different Osmotic stabilizers (Table Removed) Enzymes for protoplasting cellulase + Glucuronidase + lysing enzyme 1:1:1 for A.niger, 1:2:1 for P.digitatum, Citrate phosphate buffer used 0.2M, pH 5.8. EXAMPLE 4 As the conditions for protoplasting were optimized for A.niger and P.digitatum, the protoplasts were isolated from A.niger and P.digitatum separately and then conditions were standardized for fusion. Initially in the fusion medium (PEG) polyethylene glycol 4000 concentration was varied from (10-30%). It was found that 20% was optimum where the fusion frequency and also the regeneration frequency was highest. Increased concentration of PEG did not help in enhancing the fusion. Table 4: Effect of polyethylene glycol (PEG) concentration on fusion and regeneration frequency (Table Removed) Fusion frequency is the percentage of the number of fusants colonies obtained to the total number of protoplasts taken. Regeneration frequency is the percentage of the number of colonies formed on MM to the total number of fusants inoculated. EXAMPLE 5 As the quantity of fusogen was standardized it was very important to study the time of incubation of the two parent protoplasts in the fusion medium. The incubation time was varied from 5-30 min. It was found that 10 min. were best. Lesser time did not help in fusion and incubating for longer time results in cell lysis (Table 5). Table 5: Effect of incubation time with the fusogen on fusion and regeneration frequency (Table Removed) Fusion medium contains PEG 4000, 20%; Temp. 30°C CalCI2.2H2O 0.01 M, pH 7.4 EXAMPLE 6 As the PEG concentration and time of incubation were standarized for fusion of protoplasts of A.niger and P.digitatum conditions were optimized for best temperature at which the fusion reaction should take place. The temperature was varied from 25 to 40°C and it was found that 30°C was the best for fusion and also regeneration. At lower temperature the fusion between the two protoplasts was not noticed and at higher temperature the protoplasts shrunk and lysed (Table 6). Table 6: Effect of temperature of Fusion medium on the fusion and regeneration fequency (Table Removed) Fusion medium, PEG 4000, 20%, incubation time 10 min., CaCI2.2H2O, 0.01 M, pH 7.4 EXAMPLE 7 As the conditions for maximum fusion were standardized and the fusants were obtained these had to be studied for its regeneration on minimal medium. The amount of sucrose added to the minimal medium played a critical role in regeneration of recombinant fusant colonies. Therefore the concentration of sucrose was varied from 10-30% and regeneration frequency was studied. Optimum concentration was found to be 20% and lesser or higher concentration did not help in enhancing the regeneration (Table 7). Table 7: Effect of sucrose concentration on regeneration of fusant in minimal medium. (Table Removed) EXAMPLE 8 As the conditions were optimized for protoplasting of A.niger and P.digitatum the two protoplasts were mixed equally and centrifuged in 0.7M KCI. The pelleted protoplasts were suspended in fusion medium containing 20% (w/v) polyethylene glycol in 0.01 M CaCI2.2H2O at pH 7.5. The two protoplasts were incubated for 10 minutes at 30°C and after washing with 0.7 M KCI were serially diluted and plated on minimal medium for its regeneration frequency. The fusants were also counted on haemocytometer which gave a yield of 5.0% fusion frequency and 1.50% of regeneration frequency. The colonies grown on minimal medium were measured and the size was found bigger than the parent colonies (Table 8). The fusant colonies resembled P.digitatum in colour phenotype but microscopically were alike A.niger. It shows the transfer of only a portion of the genome from donor to recipient because it shows colour phenotype of one parent and microscopic structure of the other parent (Table 8) Table 8: Morphological characteristics and protoplast yield in parent and fusant cultures. (Table Removed) EXAMPLE 9 The protoplasts of A.niger and P.digitatum were isolated under optimized conditions and together in equal number and incubated in fusion medium contaning PEG 4000; 20% at 30°C for 10 min. The fusants obtained were regenerated in minimal medium containing 20% sucrose. In order to study the number of recombinents and heterokaryons the minimal medium was supplemented with benomyl, a haploiding agent (20 ug/15 ml) and the fusants were grown. A total number of 173 colonies were obtained out of which 166 colonies (95.9%) were recombinants and 7 colonies (4.04%) were heterokaryons (Table 9). Table 9: Effect of benomyl on fusant colony regenerated on minimal medium. (Table Removed) Benomyl 20 µg/15 ml on MM EXAMPLE 10 Parent cultures of A.niger (A) and P.digitatum (B) were grown by known methods in potato dextrose broth for 14 hours. 20 mg/ml of A and B were separately taken and mixed with cellulase, glucuronidase and lysing enzyme (1 mg each) for A.niger and via ratio of 1:2:1 for P.digitatum for 3 h at 30°C to obtain respective protoplasts. Protoplasts of A and B were fused in 1:1 ratio using fusion medium containing 20% polyethylene glucol (PEG 4000) and 0.01 M CaCI2 incubated for 10 minutes at 30°C. The fusant AB was regenerated on czepek's medium for 52 hours at 32°C. 12 grams of fusant biomass grown in potato dextrose broth (pH 6.5) for 20 hours (12 g) and then taken in phosphate buffer (0.05M; pH 7.0) with 20 mg alpha pinene, incubated for 6 h at 30°C produced 11.75 mg of verbenol. The fusant strain converts 59% of alpha pinene which is four and a half fold increase as compared to the parent cultures A and B. Culutre A of A.niger converts 13% of alpha pinene obtaining 4.4 mg of verbenol and culture B of P.digitatum converts 12.7% of alpha pinene producing 4.3 mg of verbenol. Table 10: Biotransformation efficiency of parent cultures and the fusant (Table Removed) Growing medium: PDB, 20 h, pH 6.5, 30°C Production medium: Phosphate buffer (0.2 M) pH 7.0, 6 h incubation with alpha pinene, 20 mg alpha pinene/12 g Biomass, 30°C. In the present invention it is high lighted that a novel high yielding strain a fusant strain between A.niger and P.digitatum has been obtained which gave 59% conversion of alpha pinene, a low cost substrate to a high valued flavour compound which has multiple advantages. This higher conversion rate is being reported for the first time (Table 11). Table 11: Characteristics of parent cultures of A.niger and P.digitatum and of their fusant (Table Removed) Prior art: The bioconversion rate reported is 15-20% in bacterial and yeast cultures The main advantages of the present invention are:- 1. A high yielding strain has been obtained which produces verbenol, through protoplasting and fusion of two different generic strains one A.niger and other P.digitatum. 2. An economical process to obtain verbenol from biological means. 3. The product compound obtained by this means is enantiospecific. 4. The product is classified as natural and comes under GRAS category. 5. The incubation time with the substrate has been reduced to 6 h as compared to 40-44 h as in earlier studies. We claim: 1. A process for the production of verbenol, which comprises the steps of: (a) growing parent culture Aspergillus niger and Penicillium digitatum separately on potato dextrose broth and YMG broth respectively for 10-18 hours, at pH in the range of 4.5-6.5 and at temperature in the range of 25-35 deg C under a shaking condition of 150-200 rpm; (b) isolating protoplast separately from the grown biomass by a known process followed by filtration in order to obtain filtrates separately ; (c) mixing the filtrates obtained in step (b) above in a ratio of about 1:1 containing equal number of protoplasts; (d) incubating the mixture obtained in step (c) above in a known fusion medium I having a temperature in the range of 28-30 deg C for a period of 10-20 minutes; (e) making serial dilutions followed by plating on to a known minimal medium II in order to facilitate the growth of fusants/heterokaryon ; (f) transferring the grown fusants/heterokaryons obtained in step (e) above to a known complete medium followed by incubation at a temperature of about 30 deg C for a period of about 48 hours to obtain sporulation; (g) growing the spores obtained in step (f) above on a potato dextrose broth at a pH in the range of 5.5-7.0 and at a temperature in the range of 28-32 deg C for a period of 18-22 hours. (h) filtering the biomass and adding it to buffer solution followed by incubation with a substraite alpha pinene for a period of 4-8 hours; and (i) extracting the product in dichloromethane followed by concentration by a known method in order to obtain the said verbenol. 2. A process as claimed in claim 1, wherein isolating protoplast used in step (b) is carried out in a pH of about 5.8 having 0.05 M citrate phosphate buffer and 0.7 M KC1.

Documents:

394-del-2001-abstract.pdf

394-del-2001-claims.pdf

394-del-2001-correspondence-others.pdf

394-del-2001-correspondence-po.pdf

394-del-2001-description (complete).pdf

394-del-2001-form-1.pdf

394-del-2001-form-18.pdf

394-del-2001-form-2.pdf

394-del-2001-form-3.pdf