Method for preparation of fungal mutant with high hydrolytic activity

Abstract

A method for preparing a hyper-cellulolytic catabolite derepressed mutants of ascomycetes fungus, especially variants of Penicillium funiculosum. Selection media used to isolate such variants include amorphous cellulose and a high concentration of glucose. Cellulase activities of mutant ID-10, in particular such as FPase and β-glucosidase were 1.5 times higher than Penicillium funiculosum MRJ-16 (parent). Furthermore, fungal mutant morphology was changed and no pH adjustment was required throughout the enzyme production process.

Claims

1. A method of preparing a mutant fungal strain comprising: (a) preparing a mutant fungal strain by contacting Penicillium funiculosum MRJ-16 with an aerobic culture medium followed by treatment with UV light or N-methyl-N′-nitro-Nnitrosoguanidine (NTG) or ethyl methanesulfonate (EMS) mutagens or in combination; (b) screening for the mutant fungal strain having higher celluloytic activity as compared to the Penicillium funiculosum MRJ-16 by aerobic fermentation in an aerobic culture medium comprising amorphous cellulose and glucose at concentration of 1-4% (w/w) under conditions suitable for the production of enzymes; (c) obtaining a mutant fungal strain Penicillium funiculosum-ID-10; and (d) screening the efficiency of the enzyme produced by the mutant fungal strain Penicillium funiculosum-ID-10 by hydrolyzing biomass.

2. The method in of claim 1, wherein the aerobic culture medium comprises 4% glucose.

3. The method in of claim 1, wherein the fermentation in step (b) is carried in an aerated stirred tank having a glass jacketed vessel of 2 L and working volume of 1.8 L.

4. The method in of claim 1, wherein the aerobic culture medium comprises ammonium sulphate 5 g/L, KH.sub.2PO.sub.4 6 g/L, MgSO.sub.4*7H.sub.2O 1 g/L, CaCO.sub.3 5 g/L, Glycerol 2.5 g/L, Corn steep solids 30 g/L, cellulose 30 g/L and Tween-80 2 ml/L.

5. The method of claim 1, wherein the method is carried out in a fermenter, which was sterilized at 120° C. for 20 minutes and cooled at 30° C. along with maintaining pH of 5.5.

6. The method of claim 1, wherein the enzyme produced in step (b) is a cellulase enzyme.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1. Penicillium funiculosum MRJ-16 (parent) and ID-10 mutant colony on screening media plate

DETAILED DESCRIPTION OF THE INVENTION

(2) The present invention discloses a mutant fungal strain derived from Penicillim funiculosum MRJ-16, which has an ability to produce higher titer of cellulases enzymes especially FPase and β-glucosidase activity than the parent Penicillim funiculosum MRJ-16. The present invention in particular discloses a mutant fungal strain Penicillim funiculosum ID-10 (acc no. MTCC 25224 deposited on Oct. 23, 2018, at the Institute of Microbial Technology WITCH), Sector 39A, Chandigarh-160036, India), which possesses different growth, morphology and cellulose hydrolyzing properties in the presence of glucose than the parent strain from which it was derived.

(3) Present invention also discloses a method of preparing a mutant fungal strain from MRJ-16 which comprises: subjecting Penicillium funiculosum MRJ-16 to aerobic culture media followed by treatment with UV light and/or NTG (N-methyl-N′-nitro-N-nitrosoguanidine) and/or EMS (Ethyl methanesulfonate) and screening colonies for higher cellulolytic activity followed by several more mutations.

(4) Another aspect of the present invention discloses a method of selection of mutual fungal strain by visually selection on basis of cream colored pigmentation and measuring the amorphous cellulose hydrolyzed zone.

(5) In accordance with the present invention, a mutant strain, Penicillium funiculosum ID-10, characterized by (A) higher cellulase activity than Penicillium funiculosum MRJ-16 (parent strain) and (B) much higher β-glucosidase activity obtained by subjecting Penicillium funiculosum MRJ-16 to aerobic culture media followed by treatment with UV light and/or NTG and or EMS and screening colonies for higher cellulolytic activity followed by several more mutations. The process of screening mutant was done using solid media comprises amorphous cellulose ranging from 0.1% to about 0.4% and a catabolite repressor molecule like glucose and or xylose. Mutants were selected by measuring the diameter of hydrolyzed zone. Enzyme production from Penicillium funiculosum ID-10 mutant strain was done using cellulose or pretreated lignocellulosic biomass (rice straw, wheat straw or baggase) as carbon source. The enzyme preparation produced has mixture of necessary enzyme such as FPase, CMCase, Cellobiase, β-glucosidase, α-L arabinofuranosidase, β-xylosidase, cellobiohydrase, oxidases or mixture thereof. A part of enzyme produced is harvested and used as it was without downstream processing ie enzyme along with fungal mycelia and another part of enzyme was separated from mycelia, concentrated and used for pretreated lignocellulosic biomass saccharification.

(6) Having described the basic aspects of the present invention, the following non-limiting examples illustrate specific embodiment thereof.

Example No. 1—Screening of an Efficient Mutant for Enzyme Production

(7) The parent strain Penicillium funiculosum MRJ-16 mutant strain was used to create mutants for enzyme production. Mutations were done using UV or NTG or EMS mutagens alone or all together. The mutant strains were obtained after repeated multistage mutagenesis process. Mutants were selected sequentially on specially designed media containing amorphous cellulose and glucose at different concentration from 1-4% (w/w) (screening media composition in table no. 1). Mutant strain that hydrolyzes the amorphous cellulose in the presence of 4% glucose was selected after visualizing and measuring the hydrolyzed zone. Stability of mutant stain for enzyme production capability was tested for multiple cycles and then used for further study.

(8) TABLE-US-00001 TABLE NO. 1 Screening media composition S. No. Chemical Components Quantity (g/L) 1 Ammonium sulphate 1.4 2 KH.sub.2PO.sub.4 2.0 3 MgSO.sub.4•7H.sub.2O 0.3 4 CaCl.sub.2•3H.sub.2O 0.3 5 Urea 0.3 6 Tween-80 0.1 7 Peptone 0.25 8 Yeast Extract 0.10 9 ZnSO.sub.4•7H.sub.2O 0.0014 10 FeSO.sub.4•7H.sub.2O 0.005 11 MnSO.sub.4•H.sub.2O 0.0016 12 CoCl.sub.2•6H.sub.2O 0.002 13 Glucose 30-40 14 Amorphous cellulose 1-4 15 Agar 20 16 pH 5.0

(9) The visual observation of the Penicillium funiculosum MRJ-16 (parent) and mutant (ID-10) cultured on screening media plate showed that in the presence of 4% glucose, ID-10 mycelia remains cream colored till 10-13 days of incubation, while MRJ-16 started turning yellow before the onset of spores as shown in FIG. 1. More wrinkled surface was observed in case of MRJ-16 than ID-10. ID-10 produces small colony with wide hydrolysis zone than MRJ-16 mutant with colony diameter and hydrolyzed zone diameter of 12 mm and 20 mm respectively.

Example No. 2—Enzyme Production Comparison of Mutant ID-10 and MRJ-16 Parent Strains

(10) Fermentation process was carried out in aerated stirred tank bioreactor of 2 L glass jacketed vessel, with 1.8 L working volume. The media components of fermentation media used were ammonium sulphate 3.5 g/L, KH.sub.2PO.sub.4 4 g/L, MgSO.sub.4.7H.sub.2O 0.5 g/L, CaCO.sub.3 2.5 g/L, Glycerol 2.5 g/L, Corn steep solids 20 g/L, cellulose 20 g/L and Tween-80 2 ml/L. The fermenter containing 1.5 L medium was sterilized at 120° C. for 20 min. After cooling, the temperature was kept at 30° C., pH adjusted to 5.5 and inoculated with 10% active liquid seed (seed media composition in table no.2) of Penicillium ID-10 mutant strain. After 96 h of fermentation, the enzyme broth was collected, centrifuged and analysis of clear enzyme broth was done.

(11) TABLE-US-00002 TABLE NO. 2 Seed/Inoculum Media composition S. No. Chemical Components Quantity (g/L) 1 Ammonium Sulphate 4 2 KH.sub.2PO.sub.4 3 3 MgSO.sub.4•7H.sub.2O 0.1 4 CaCO.sub.3 2 5 Sucrose 5 6 Corn Steep Liquor 15 7 Cellulose 10 8 Tween-80 2 9 pH 5.5

(12) The results obtained after 96 hr of incubation were; parent MRJ-16 produces 15 g/L of protein, 62 IU/ml of β-glucosidase and 6.4 FPU/ml of filter paper activity, while ID-10 produces 16 g/L of protein, 87 IU/ml of β-glucosidase and 9.3 FPU/ml of filter paper activity.

Example No. 3—Enzyme Production Comparison of Mutant ID-10 and MRJ-16 Parent Strains

(13) Fermentation process was carried out in aerated stirred tank bioreactor of 2 L glass jacketed vessel, with 1.8 L working volume. The media components of fermentation media used were ammonium sulphate 5 g/L, KH.sub.2PO.sub.4 6 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, CaCO.sub.3 5 g/L, Glycerol 2.5 g/L, Corn steep solids 30 g/L, cellulose 30 g/L and Tween-80 2 ml/L. The fermenter containing 1.5 L medium was sterilized at 120° C. for 20 min. After cooling, the temperature was kept at 30° C., pH adjusted to 5.5 and inoculated with 10% active liquid seed (seed media composition in table no. 3) of Penicillium ID-10 mutant strain. After 96 h of fermentation, the enzyme broth was collected, centrifuged and analysis of clear enzyme broth was done.

(14) TABLE-US-00003 TABLE NO. 3 Seed/Inoculum Media composition S. No. Chemical Components Quantity (g/L) 1 Ammonium Sulphate 5 2 KH.sub.2PO.sub.4 6 3 MgSO.sub.4•7H.sub.2O 1 4 CaCO.sub.3 2.5 5 Sucrose 10 6 Corn Steep Liquor 10 7 Cellulose 20 8 Tween-80 2 9 pH 5.5

(15) The results obtained after 96 hr of incubation were; parent MRJ-16 produces 16.2 g/L of protein, 64 IU/ml of β-glucosidase and 6.2 FPU/ml of filter paper activity, while ID-10 produces 16 g/L of protein, 98 IU/ml of β-glucosidase and 10.2 FPU/ml of filter paper activity.

Example No. 4—Enzyme Production in the Presence of Glucose

(16) In order to demonstrate the glucose repression on enzyme production, cellulose used as carbon source was replaced with glucose. Enzyme production from Penicillium funiculosum ID-10 mutant strain was carried out under the conditions and media composition as described in example no.2 and 3 except cellulose. Concentrated solution of glucose was autoclaved separately and added into media at 4% w/v concentration. Fermentation was lasted approximately for 120 h, enzyme harvested and analyses were done (Table 5).

(17) TABLE-US-00004 TABLE NO. 4 Enzyme production from MRJ-16 and parent strain in the presence of glucose Concentration BGL Mutants (% w/v) FPU/ml (IU/ml) Penicillium funiculosum MRJ-16 Cellulose 3% 6.47 62 mutant (Parent strain) Glucose 4% 0.12 ND Penicillium funiculosum ID-10 Cellulose 3% 10.3 97 Glucose 4% 3.21 29

Example No. 5—Diversity of Enzyme Secreted

(18) Cellulases enzymes production using Penicillium funiculosum ID-10 mutant strain was performed according to example no.2. The secretome analyses were done it is comprise enzyme activities of β-glucosidase 87 IU/ml, Filter paper activity 9.3 FPU/ml, endoglucanase 125 IU/ml, α-L arabinofuranosidase 0.05 IU/ml, β-xylosidase 1.2 IU/ml, xylanase 227 IU/ml, pectinase 97 IU/ml and oxidases 6.54 IU/ml analyzed using respective substrates.

Example No. 6—Diversity of Enzyme Secreted

(19) Cellulases enzymes production using Penicillium funiculosum ID-10 mutant strain was performed according to example no.3. The secretome analyses were done it is comprise enzyme activities of β-glucosidase 98 IU/ml, Filter paper activity 10.2 FPU/ml, endoglucanase 132 IU/ml, α-L arabinofuranosidase 0.05 IU/ml, β-xylosidase 1.2 IU/ml, xylanase 247 IU/ml, pectinase 95 IU/ml and oxidases 5.23 IU/ml analyzed using respective substrates.

Example No. 7—Hydrolysis of Pre-Treated Lignocellulosic Biomass Using Enzyme as Such

(20) The efficiency of enzyme produced was determined by its ability to hydrolyze lignocellulosic biomass such as acid pretreated rice straw and produce sugars. Enzyme broth produced in example no.6 was used as such without any downstream processing. Hydrolysis was performed at high substrate loading of biomass i.e. 20% at pH 4-5, 50 mM citrate buffer, temperature 50° C. at enzyme loadings of 6 FPU/g of dry biomass. Sugars released were determined at regular interval of time by HPLC. Enzyme cocktail worked efficiently and leads to 63% glucan conversion in 48 h.