Enzyme cocktail with hyperactive lipase through solid-state fermentation by the novel strain Penicillium sp. Y-21

Lipase is a kind of industrial enzyme preparation with various catalytic abilities and is widely used in food, energy, medicine and other fields. To increase lipase and enzyme cocktail activity through solid-state fermentation, the novel strain Penicillium sp. Y-21 was obtained through ethyl methanesulfonate (EMS) mutation from the novel strain Y, which was isolated from soils. Solid-state fermentation by strain Y-21 using agricultural byproducts was carried out in tray bioreactors. The optimum culture composition for enzyme cocktail fermentation was soybean meal 20 g, 3% (w/w) glucose, 1% (w/w) peptone, 5% (w/w) lard, 0.04% (w/w) CaCl2, 0.04% (w/w) FeCl3, 28 °C for 72 h. The enzyme cocktail produced by strain Y-21 is a kind of multienzyme complex, containing xylanase, glucanase, acidic protease, pectinase, cellulase and lipase, and their enzymatic activities (unit: U g−1) were 8000, 6000, 8000, 2000, 3000 and 120, respectively. During the fermentation process, the lipase coding genes pel, pha, and p12 were also studied and amplified from the RNA of Penicillium sp. Y-21 by RT-PCR. The results showed that the pel gene played an important role in enzyme production. Afterwards, an enzyme cocktail can be added to chicken feed as an additive, which improves animal growth and feed efficiency.


EMS mutagenesis.
The spores of Penicillium sp.Y were obtained by washing a 5-day incubation on plate agar.A certain amount of spore suspension and original EMS solution were used to prepare spore suspensionS containing 4% EMS (w/w) 19 .After mixing and shaking for 0-90 min, the control group received the same volume of saline as EMS.After mutagenesis, 2% Na 2 S 2 O 3 of the same volume as EMS was added, and after standing for 10 min, the mixture was centrifuged at 3,000 rpm for 5 min and 1 mL of treatment solution was left.The treatment solution was diluted 100 times with sterile water and coated.
Solid-state fermentation substrate composition.Wheat bran, soybean meal, rice bran, cottonseed meal and rapeseed meal were used as the main carbon and nitrogen sources.Then the total moisture content of the substrate was controlled at 60%.Optimization of fermentation considered substrate composition, additional carbon and nitrogen sources and their concentrations, different metal ions and concentrations, water content, inoculum and the effect of induced oil on enzyme cocktail production.The spores of Penicillium sp.Y-21 were inoculated into fermentation medium, and cultured at 28 °C for 72 h.

Tray bioreactor design and configuration.
The bioreactor (Fig. 1) was applied for the fermentation of cocktail lipase.The filtered air entered the chamber from the bottom, passed around the trays, and went out from the top.The bottom of the trays was perforated (2-3 mm) to allow the oxygen into the fermentation medium.The fermentation bed was approximately 1.5 cm deep to avoid any temperature gradient across the bed 20 .The temperature in the fermentation chamber was set at 28 °C, and the air moisture was approximately 70-80% to minimize water loss caused by evaporation through the bed.The trays were filled with 300 g of dry substrate, and samples were taken every 12 h for determination of lipase activity.The duration of the fermentation process was 72-96 h.Animal experimental procedures and measurements.A total of 9000 yellow broilers were collected and randomly divided into two group: control and treatment.Broiler chickens were randomly assigned into 2 experimental diets using a factorial arrangement with 2 enzyme cocktail supplemental levels (0 and 0.05%, w/w) in basal diets.Analytical methods.The enzyme activity of lipase was determined by means of GB/T 23535-2009.The unit of lipase activity is defined as hydrolyzing 1 μmol of titratable fatty acid within 1 min under a certain temperature and pH (U g −1 ).The protein content was determined by the Bradford method using bovine serum albumin (BSA) as the standard protein 24 .
Ethical approval.This article does not contain any research with human or animal subjects performed by any of the authors.

Results and discussion
Chemical mutation of strain 21.Based on the previous test results, 4% EMS solution was used for wildtype strain Y mutagenesis for 80 min to increase the lipase activity.Twenty three mutant strains were selected to determine the lipase activity (Table S1).Strain 21, with the highest lipase activity, was selected for further study, and the enzyme activity reached 73.33 U g −1 .Compared with 48.67 U g −1 of the wild-type strain, the enzyme activity increased by 50.67%.Strain 21 was named Penicillium.sp.Y-21 according to 18S rRNA sequence results.Different substrates, such as wheat bran, soybean meal, rice bran, cottonseed meal and rapeseed meal, were selected as carbon and nitrogen sources for enzyme cocktail fermentation.The results showed that the maximum enzyme activity occurred in the soybean meal substrate by the mutant Penicillium sp.Y-21.The activity of lipase reached 115.60 U g −1 after 72 h fermentation at 28 °C (Fig. 2).

Effect of inoculant and water content on lipase fermentation. The effects of inoculation amount
and moisture on enzyme production by strain 21 were investigated.The results are showed in Fig. 3. Strain 21 with 15% spore inoculant had the maximum enzyme fermentation and its activity reached 112.57U g −1 in the Less inoculant could lead to longer fermentation times, and higher inoculants also affect enzyme production 25 .Substrate moisture also affected the enzyme fermentation and activity (Fig. 3B).Lipase activity was 123.04 U g −1 under the condition of 50% water content.The excessive amount of water added is not conducive to the ventilation and heat dissipation of the solid medium, which leads to lower enzyme activity.
Effect of additional carbon/nitrogen source on solid state fermentation.Glucose, fructose, lactose and maltose were added to the fermentation medium as additional carbon sources, and the optimal carbon concentration was also studied.The results are shown in Fig. 4A,B.Additional glucose can improve enzyme production and activity.Lactose and maltose have an inhibitory effect on enzyme fermentation, and 4% glucose content is the optimal concentration for the enzyme production of the strain, and the enzyme activity reaches 115.67 U g −1 because glucose can be directly utilized as monosaccharide, which is beneficial to enzyme production 26,27 .Beef extract, peptone, ammonium chloride and potassium nitrate were added to the culture medium as external nitrogen sources.Figure 4C and D show that additional peptone is favorable for enzyme production.Ammonium chloride, potassium nitrate and other inorganic nitrogen sources can decrease enzyme activity; 1% peptone is appropriate for enzyme production, and the lipase activity can reach 119.17 U g −1 .Organic nitrogen peptone contains not only protein and amino acids but also a small amount of sugar and growth factors.Therefore, the growth effect of the strain was significantly improved.
Effect of additional metal ions on solid state fermentation.Mg 2+ , Ca 2+ , Fe 3+ , K + and Ba 2+ were added to the fermentation medium, and then optimum concentrations of metal ions of 0.02%, 0.04%, 0.06%, 0.08% and 0.1% were selected.The results showed that the addition of Fe 3+ greatly enhanced the enzyme-producing ability of the strain, as shown in Fig. 5A,B.

Effect of additional oil on solid-state fermentation.
Olive oil, coconut oil, soybean oil, grease and lard were added to the fermentation medium, and the results are shown in Fig. 6A.It can be seen from the figure that the strain is an oil-inducing strain, and the ability to produce lipase is greatly enhanced by adding oil.The lipase activity reached 120.33 U g −1 after adding lard.The reason may be that lard contains more polysaturated fatty acids, which can improve the enzyme production of the strain.
The qRT-PCR results showed that the peak value of the fusion curve was single, which indicated that the primers had high specificity and no nonspecific band amplification.According to the results of relative quantitative analysis in Fig. 6B, the gene expression of pel after adding induced oil was 4.30 times that without adding oil, and the gene expression of pha and p12 were 0.56 and 0.49 times that without adding oil, as shown in Fig. 5B.The results showed that high expression of the pel gene could effectively improve the lipase production of the strain, while the pha and p12 genes played a negative role in lipase production.

Cocktail lipase fermentation and purification.
Based on the optimum results shown above, a tray bioreactor was designed for lipase production.The ctivity of lipase, proteinase and xylanase were determined during fermentation and the results are shown in Table S3.Lipase and proteinase were generated simultaneously during the solid fermentation process, and their activity reached a maximum at 72 h of fermentation.Lipase and proteinase production were significantly higher in the tray bioreactor, and their production yields are 75 and 35% higher in the tray bioreactor than in the flask bioreactor.The results were in accordance with previous reports that filamentous fungi had better performance for enzyme production in static bed [28][29][30][31][32] .
The lipase and proteinase were purified 63.24 and 10.33 times, respectively, through ammonium sulfate precipitation and chromatography.The recovery rates were 13.67% and 14.39%, and the specific activities were 1366.67U mg −1 and 8540 U mg −1 , respectively, as shown in Table S4.First, the protein was precipitated with ammonium sulfate at 50% saturation, and the target protein was purified by ion chromatography.The SDS-PAGE of the purified protein is shown in Fig. 7.The purified lipase is a single band with a molecular weight of 36.2 kDa (lipase) and 64.7 kDa (proteinase).The results showed that the enzyme cocktail contained xylanase, glucanase, acidic protease, pectinase, cellulase and lipase, and their enzymatic activity units were 8000 ± 600 U g −1 , 6000 ± 500 U g −1 , 8000 ± 600 U g −1 , 2000 ± 150 U g −1 ,3000 ± 200 U g −1 and 120 ± 50 U g −1 , respectively.This enzyme cocktail includes relatively high xylanase, glucanase and protease activity but relatively low pectinase, cellulase and lipase activities, especially lipase activity, which was minimumal and only reached 120 U g −1 .
Dietary supplementation of animal diets with exogenous enzymes has been suggested as a strategy to increase nutrient digestibility and improve feed efficiency.Generally, 0.1% of exogenous enzyme cocktail (w/w) is usually added to chicken and pig feed as an additive (DB33/T 459-2003).Excessive enzyme cocktail loading is recommended by the association to increase the efficiency of substrate digestibility.To understand the enzymatic hydrolysis effect, 9000 yellow broilers were collected to verify the substrate digestibility efficiency after adding 0.05% of cocktail enzyme.Nine thousand yellow broilers were divided into two groups: control and treatment.The average daily gain (ADG, g day −1 ), average daily feed intake (ADFI, g day −1 ) and feed conversion rate (FCR) were monitored and analyzed as shown in Table S5.
As shown in Table S5, ADG was 17.12 g day −1 and 33.15 g day −1 after adding the cocktail enzyme in days 1-21 and 32.57 in days 1-50, respectively, and ADG increase by 0.58 g day −1 in 50 d.In addition, ADFI and FCR decreased by 1.39 g day −1 and 0.05 in 50 d, respectively.Especially during days 22-50, the ADFI decreased by 2.34 g day −1 , and the FCR decreased by 0.08, which showed that the addition of the cocktail to the chicken feed could significantly improve animal growth and increase feed efficiency in this study.The multienzyme complexes used in the experiments had combinations of 6 different enzymes and the enzymes xylanase, glucanase, acidic

Figure 3 .
Figure 3.Effect of inoculant (A) and substrate moisture (B) on enzyme fermentation.

Figure 4 .Figure 5 .
Figure 4. (A).Effect of additional carbon source and its content on enzyme production.(B).Effect of carbon source concentration on enzyme production by strains.(C).Effect of additional nitrogen source on enzyme production by strains.(D).Effect of nitrogen source concentration on enzyme production by strains.

Figure 6 .
Figure 6.Effect of induced oil on lipase production.