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Eur Food Res TechnolDOI 10.1007/s00217-014-2172-5

ORIgInal PaPER

Production optimization, purification, and characterization of a novel acid protease from a fusant by Aspergillus oryzae and Aspergillus niger

Caihong Li · Defeng Xu · Mouming Zhao · Lijun Sun · Yaling Wang

Received: 29 november 2013 / Revised: 8 January 2014 / accepted: 16 January 2014 © Springer-Verlag Berlin Heidelberg 2014

Introduction

acid proteases (E.C.3.4.2.3) are endopeptidases with molec-ular masses of 30–45 kDa and depend on aspartic acid resi-dues for their catalytic activity. acid proteases show maxi-mal activity at low pH and thus find extensive applications in the production of seasoning materials [1, 2]. Filamentous fungi are exploited for the production of industrial enzymes due to their ability of growing on solid substrates and pro-ducing a wide range of extracellular hydrolyzing enzymes. Aspergillus oryzae is an industrially important filamentous fungus that has been used for over a 1,000 years in tradi-tional oriental fermented food, such as sake, soy sauce, and miso (soybean paste) [3, 4]. The most significant advan-tage of A. oryzae is that it can produce copious amounts of various hydrolytic enzymes, and it is therefore used as an important source of the enzymes that degrade raw materi-als. Particularly, the protease produced by A. oryzae is one of the major reasons for its wide popularity in fermentation industry [5, 6]. In the case of soy sauce fermentation, acid protease (aspartic protease) is relatively deficient compared with neutral and alkaline proteases [7].

The most important protease in sake brewing, however, is the acid protease because of the acid condition of fer-mentation mash. acid protease is considered to be con-tributing to the efficient solubilization of steamed soybean in the acid mash [8]. Therefore, it is urgent to breed high-producing strains of acid protease for the degradation of raw proteins in soy sauce fermentation. Through genome recombination between A. oryzae and Aspergillus niger, we obtained an excellent fusant A. oryzae Fg76, which exhib-ited an enhancement by 82.19 % in acid protease activity compared with the parental strain of A. oryzae Hn3042, and the research has been published in the Journal of Indus-trial Microbiology and Biotechnology [9].

Abstract The production of a novel acid protease was enhanced by 44 % through statistical optimization. The cul-tural parameters, such as inoculum size, temperature, mois-ture content, and incubation time, were 8.59 × 105 g−1 dry koji, 31 °C, 57 %, and 86 h, respectively. This novel acid protease was purified by 17 folds with a recovery yield of 33.56 % and a specific activity of 4,105.49 U mg−1. Far-UV circular dichroic spectra revealed that this purified pro-tease contained 7.1 % α-helix, 64.1 % β-sheet, and 32 % aperiodic coil. This novel acid protease was active over the temperature range of 35–55 °C with optimum tempera-ture of 40 °C and was stable in the pH range of 2.5–6.5 with optimum pH of 3.5. Mn2+ enhanced its activity while Co2+ showed inhibitory effect. With casein as substrate, the kinetic parameters of Km, Vmax, energy of activation (Ea), and attenuation index of inactivation velocity by heat induc-ing (λ) were 0.96 mg ml−1, 135.14 μmol min−1 mg−1, 64.11 kJ mol−1, and 0.59, respectively.

Keywords Aspergillus · acid protease · Production · Purification · Characterization

C. li Institute of Biochemistry and Molecular Biology, guangdong Medical College, Dongguan 523808, China

D. Xu (*) · l. Sun · Y. Wang guangdong Provincial Key laboratory of aquatic Product Processing and Safety, Key laboratory of advanced Processing of aquatic Products of guangdong Higher Education Institution, College of Food Science and Technology, guangdong Ocean University, Zhanjiang 524088, Chinae-mail: stone_xdf@163.com

M. Zhao School of light Industry and Food Sciences, South China University of Technology, guangzhou 510640, China

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Protease production is highly regulated by the cultural parameters [10], and thus the cultural parameter optimization has become one of the main ways to enhance enzyme pro-duction. Conventionally, the fermentation process was opti-mized by using the one-factor-at-a-time strategy. This single-variable optimization strategy was relatively simple and did not require statistical analysis [11]. By one-factor-at-a-time strategy, the influence of variables on targets can be effec-tively judged and the approximate range of significant varia-bles can be determined consequently. This strategy, however, is time-consuming, requiring more experimental data sets and missing the interactions among variables [12]. There-fore, this approach must be complemented with the statistical methodologies, which are efficient experimental strategies to seek optimal conditions for multivariable system. Statistical methodology, such as central composite design (CCD), has been successfully employed for optimization in some bio-processes and showed satisfactory results [13, 14].

The excellent fusant A. oryzae Fg76, although, was obtained by genome recombination [9], the production and properties of the novel acid protease have not yet con-ducted. Therefore, the first objective of this study was to optimize the cultural parameters for the acid protease pro-duction by A. oryzae Fg76. One-factor-at-a-time method was first used to seek for the approximate range of signifi-cant variables. When the results of first stage are obtained, CCD was employed to determine the optimum levels for maximizing the acid protease production. Then, for clear understanding of the properties and to facilitate extensive applications, the purification and characterization of this novel acid protease were also carried out subsequently.

Materials and methods

Materials

Casein, tyrosine, acrylamide, bis-acrylamide, sodium dode-cyl sulfate (SDS), soybean isolation (SPI), bovine serum albumin (BSa), tetramethylethylenediamine (TEMED), silver salt dye, trypsin, and β-mercaptoethanol were pur-chased from Sigma; molecular weight marker kit (14.2–97.4 kDa) was from Takara; A. oryzae Hg76, a fusant with higher activity of acid protease, was obtained by genome recombination [9] and preserved in our lab. all other chem-icals used were analytical grade from guangzhou Chemical Reagent Factory of China.

Preparations of inoculum, seed culture medium, and cultivation

Aspergillus oryzae Hg76 was grown on the potato dextrose agar slant medium, cultured at 30 °C for 96 h, and then

stored at 4 °C until use. The conidia from a fully sporulated slant were dispersed in 10 ml of 0.1 % Tween-80 solution by dislodging them with a sterile loop under aseptic con-ditions. The conidia suspension was prepared and used as inoculums with the final concentration of 2.8 × 106 conidia ml−1.

The basal medium for A. oryzae Hg76 seed culture was composed of the following substances and fractions, i.e., wheat bran/flour/water = 4:1:3 (w:w:v). The mixtures were fully stirred, and 5.0 g of this substrate was added into a 100-ml conical flask as seed koji culture medium, followed by moist heat sterilization at 121 °C for 30 min. after inoculation with 1.0 ml of the inoculums, the conical flasks were placed in an incubator and cultivated at 30 °C for 96 h.

Extraction and assay of the acid protease

Extracellular acid protease was produced when A. oryzae Fg76 was cultured on the culture medium. Extracellular proteases were extracted from the mold bran with water containing 0.1 M sodium chloride (bran/solvent = 1:4) at 40 °C and vibration of 150 rpm for 1 h. The extract was centrifuged at 8,000×g and 4 °C for 10 min. Then the culture supernatant was filtrated through a 0.45 μm mem-brane (Millipore Inc., USa), and the filtrate was collected as the crude enzyme solution. acid protease activity was determined according to the procedure described by Tello-Solis [14] Protein concentration was determined by the method of Bradford [15] using bovine serum albumin as standard.

Experimental design and optimization

One‑factor‑at‑a‑time design

The effect of inoculum size on the enzyme production was determined by inoculating the conidia suspension at the size of 1.25, 2.5, 3.75, 5, 6.25, 7.5, 8.75, and 10 × 105 g−1 dry koji, respectively; the other parameters such as tem-perature, water content, and fermentation time were set at 30 °C, 50 %, and 90 h. The effect of incubation tempera-ture on enzyme activity was evaluated at 20, 25, 30, 35, and 40 °C, respectively. Then the influence of moisture content was examined by setting it at 20, 30, 40, 50, 60, and 70 %, respectively; the parameters of inoculum size and tempera-ture were set at values according to the results above. The effect of incubation time on enzyme production was subse-quently carried out by setting the incubation time at 40, 50, 60, 70, 80, 90, and 100 h, while the values of other parame-ters were assigned according to the above experiments. The enzyme production was evaluated by monitoring the activ-ity of acid protease in koji.

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Optimization design

Based on the results of the first stage, the optimization experiment was carried out by CCD. For statistical calcula-tion, the relation between the coded values and actual val-ues is described as follows:

where xi is the coded value of the independent variable, Xi is the actual value of independent variable, X0 is the actual value of the Xi at the center point, and ΔXi is the step change of independent variable. The title and levels of the selected variables are shown in Table 1. The enzyme activ-ity was taken as the dependent variable or response of the design experiments.

For predicting the optimal point, a second-order polyno-mial function was fitted to correlate relationship between independent variables and response. Quadratic equation for the independent variables was expressed as follows:

(1)xi =Xi − X0

DeltaXi

,

where Y stands for the predicted enzyme activity, a0 denotes the model intercept; χ1, χ2, χ3,…, χn are the codes of selected parameters based on one-factor-at-a-time exper-imental results; ai,…, aij are regression coefficients calcu-lated from the experimental data by multiple regression. all experimental designs were randomized and experiments were performed in triplicate. all statistical and mathemati-cal analysis of the results from CCD was done with Design Expert software (Version 7.4.1.0, Stat-Ease Inc., Min-neapolis, USa). Both linear and quadratic effects of the major variables on enzyme production were calculated, as well as their possible interactions. Their significance was evaluated by variance analysis (anOVa). The fitness of the model was evaluated by the determination of adjusted R2 coefficients. The validation of the model optimum value of selected variables was obtained by solving the regres-sion equation using Design Expert. The predicted optimum

(2)Y = α0 +

n∑

i=1

αiχi +

n∑

i=1

αiiχ2i +

n∑

i=1

∑

i<j

αijχiχj,

Table 1 Experimental design and results of CCD experiment

Factors Codes levels

−1 0 1

Inoculum (×105 g−1 dry koji) X1 6.25 7.5 8.25

Temperature (°C) X2 24 28 32

Moisture content (%) X3 40 50 60

Time (h) X4 70 80 90

Run X1 X2 X3 X4 Y (U mg−1 dry koji)

1 1 1 1 −1 1,914 ± 18

2 1 1 −1 −1 1,758 ± 22

3 1 −1 1 1 1,865 ± 17

4 −1 1 −1 1 1,539 ± 21

5 1 −1 −1 1 1,688 ± 25

6 −1 −1 1 −1 1,534 ± 19

7 −1 1 1 1 1,742 ± 16

8 −1 −1 −1 −1 1,469 ± 20

9 −1.682 0 0 0 1,658 ± 23

10 +1.682 0 0 0 1,954 ± 25

11 0 −1.682 0 0 1,559 ± 17

12 0 +1.682 0 0 1,792 ± 23

13 0 0 −1.682 0 1,424 ± 21

14 0 0 +1.682 0 1,715 ± 20

15 0 0 0 −1.682 1,689 ± 19

16 0 0 0 +1.682 1,855 ± 22

17 0 0 0 0 1,349 ± 18

18 0 0 0 0 1,328 ± 20

19 0 0 0 0 1,365 ± 21

20 0 0 0 0 1,346 ± 24

21 0 0 0 0 1,333 ± 19

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value was confirmed by the experiments with the selected optimum values of variables.

Purification and identification of acid protease

The purification of protease was performed accord-ing to the method of Wang et al. [16] with modifications. The crude enzyme extract obtained through extract-ing moldy bran with water containing 0.1 M naCl was precipitated with saturated ammonium sulfate solution. Precipitate was collected with 40–85 % saturation and centrifuged at 10,000×g for 30 min. The pellet was dis-solved in minimal volumes of 50 mM phosphate buffer (pH 6.0) and dialyzed against the same buffer to remove the trace amounts of ammonium sulfate. The dialysate was applied onto a DEaE-cellulose ion-exchange column (2.5 × 20 cm, 50 ml). Bound protein was eluted with a salt gradient of 0–1.0 M naCl. The active fractions were concentrated through ultrafiltration (a nominal molecu-lar weight cutoff of 10,000 Da, Millipore Inc., USa), fol-lowed by gel-filtration chromatography with Sephadex g-75 (1 × 50 cm, 100 ml). The active fractions were pooled and the homogeneity was confirmed by gel elec-trophoresis. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PagE) was carried out using 12 % acrylamide in gels as described by Vishwanatha [17]. Pro-tein bands were visualized by silver staining according to the procedure described by Sharam [18]. Phosphorylase b (92.7 kDa), BSa (66.4 kDa), ovalbumin (44.3 kDa), car-bonic anhydrase (29.0 kDa), SPI (20.1 kDa), and lysozyme (14.3 kDa) were used as molecular weight marker stand-ards. native PagE was performed in the absence of SDS and β-mercaptoethanol.

Identification of the purified protein was conducted with mass spectrometry according to the procedure described by Susnea with minor modifications [19]. The purified protein in gel was digested with trypsin, and the tryptic peptides were collected over a 300 μm ID, 15 cm C18 reversed phase column with an agilent 1100 HPlC pump. The lC-ESI-MS/MS analysis was recorded with Esquire 3000 ion trap (Bruker Daltonics, Bellerica, Ma, USa) as previously described by Declan Williams [20]. Digestion mixtures determined by lC-MS/MS were directly used for database search employing the MaSCOT peptide mass finger print-ing (PMF) search engine (http://www.matrixscience.com).

Spectral characterization of the acid protease

The UV absorption spectrum of acid protease was meas-ured using a Unico 2100 spectrophotometer (Shanghai, China). The relative fluorescence intensity of the purified acid protease was measured by using a Hitachi F-4500 spectrophotometer (Tokyo, Japan), and the protein solution

having absorbance of 0.05 at 280 nm in 50 mM phosphate buffer (pH 6.0) was scanned. The excitation spectrum was recorded in the range of 200–300 nm, fixing the emission wavelength at 330 nm. The excitation wavelength was fixed at 280 nm (maximum excitation for the protein) and emis-sion spectrum was recorded in the range of 300–400 nm. The spectra were recorded with 5 nm bandwidth for both excitation and emission monochromators.

Scanning of circular dichroic (CD) spectra was con-ducted according to the method described by Sharam [18] with slight modifications. CD spectra of the purified pro-tease was scanned using a Bio-logic MOS-450/aF-CD (Bio-logic, France) fitted with a xenon lamp. The far-UV CD spectrum was recorded from 190 to 250 nm using a 1 mm path length cell and the protein concentration of 0.1 mg ml−1 in 50 mM citric acid–sodium citrate buffer (pH 3.6). The near-UV CD spectrum was recorded in the range of 250–350 nm using a 10 mm path length cell and the protein concentration was 1 mg ml−1 in 50 mM cit-ric acid–sodium citrate buffer (pH 3.6). The secondary structure analysis of this acid protease in solution was per-formed using the CDSSTR method described by Sreerama [21] with reference database SP43.

Kinetics characterization of the acid protease

Effect of temperature and pH on the activity of acid protease

The effect of temperature and pH on activity was evaluated by Qiu’s method [22] with modifications. The optimum temperature for activity was determined in the range of 35–65 °C and casein was used as substrate. The maximum activity obtained within the range was taken as 100 %, and the residual activities were calculated accordingly. The thermal stability of acid protease was determined by incubating the purified acid protease in 50 mM citric acid–sodium citrate buffer (pH 3.6) within the temperature range of 35–65 °C for 1 h. The samples were immediately cooled in ice and the residual activity was assayed. The unincu-bated enzyme activity was taken as 100 %, and the residual activities were evaluated accordingly.

The optimum pH for the acid protease in activity was determined within the pH range of 2.0–6.5. Buffers used were 0.1 M glycine–HCl (pH 2.0–4.5) and 0.1 M acetate (pH 5.0–6.5). The activity was assayed at 40 °C. Maximum activity determined within the pH range was expressed as 100 %, and the residual activities were calculated accord-ingly. pH stability of the protease was evaluated by incu-bating the enzyme in relative pH buffer ranging from 2.0 to 6.5 for 1 h, and then the activity was determined at 40 °C. Maximum activity was expressed as 100 % and the residual activities were measured accordingly.

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Effect of metal ions and protease inhibitors on the activity of acid protease

The influence of various metal ions (Zn2+, K+, Fe2+, Cu2+, na+, Mg2+, Mn2+, Ca2+, and Co2+) and specific inhibitors on caseinolytic activity of the acid protease was determined according to the method described by nilantha-lakshman [23] with minor modifications. The enzyme was preincu-bated with corresponding metal ion solutions in appropri-ate concentrations for 1 h before adding substrate, and then the residual activity was measured according to the method described above. The inhibitory effect of specific inhibi-tors on the caseinolytic activity of purified acid protease was evaluated within the range of 0–100 mM. The inhibi-tors used were EDTa (metallo proteases), leupeptin (cys-tine proteases), PMSF (serine proteases), and pepstatin a (aspartate protease). after incubating the purified enzyme with specific inhibitors at 40 °C for 1 h, the residual activ-ity was determined under above conditions. The control enzymatic activity, without the inhibitor, was taken as 100 % and the residual activity was evaluated accordingly.

Determination of dynamic parameters of the acid protease

The determination of kinetic parameters was carried out by the method described by alam [24] with slight modifica-tions. The Km and Vmax of the purified acid protease were determined with casein in 50 mM citric acid–sodium cit-rate buffer (pH 3.6) as substrate. The enzyme was incu-bated with the substrate ranging from 0 to 20 mg ml−1 for 10 min at 40 °C. The relation between reaction velocity and substrate concentrations was fitted by the Eq. 3 and a dou-ble-reciprocal lineweaver–Burk plot was used for estimat-ing Km and Vmax

where V is the reaction velocity, μg ml−1 min−1; S is the substrate concentrations, mg ml−1; the Michaelis constant Km corresponds to substrate concentration at 1/2Vmax.

The energy of activation (Ea) of the purified enzyme was calculated according to the method described by Maran-goni [25]. The plot of lnK versus 1/T within the tempera-ture range of 20–45 °C yields a straight line with slope—Ea/R and the Eq. 4 can be used to obtain the Ea

where K is the reaction rate; A is the frequency factor, hav-ing the same dimensions as the rate constant K and relating to the frequency of collisions between reactant molecules; R is the ideal gas constant (8.314 J mol−1 K−1); T is the temperature in degree of Kelvin.

(3)1

V=

Km

Vmax·

1

S+

1

Vmax,

(4)ln K =−Ea

RT+ ln A,

The thermal stability of the purified acid protease was investigated by incubating the enzyme at 45 °C for a fixed time, and the residual activity was determined. The relation between incubation time and residual activity was fitted by the Eq. 5

where N is the residual activity and N0 denotes the initial activity of enzyme; λ is the attenuation index, indicative of the inactivation velocity by heat inducing; t is the incuba-tion time, h.

Results and discussion

Influence of single factor on the production of acid protease

The influences of inoculum size, incubation temperature, moisture content, and cultural time on acid protease produc-tion were evaluated individually, and the results were illus-trated in Fig. 1a–d. From Fig. 1a, the enzyme activity expe-rienced a continuous increase with the increase in inoculum size. The enzyme activity was observed only 310 U g−1 dry koji at the concentration of 1.25 × 105 ml−1, and the value reached 1,722 U g−1 dry koji when the concentra-tion of inoculum was 7.5 × 105 ml−1, which was increased over 5 times. after that, the enzyme activity was increased slightly. as for the temperature, Fig. 1b indicated that the enzyme activity reached a maximum at 30 °C and showed significant decrease when the temperature got higher or lower. Moisture content exerted a significant effect on enzyme production. as shown in Fig. 1c, the enzyme activ-ity is increased sharply from 356 to 1,799 U g−1 dry koji when the moisture content is increased from 20 to 50 %, after which the activity dropped slightly. For the parame-ter of incubation time, the tendency was observed similar to the moisture content. The maximum enzyme activity of 1,753 U g−1 dry koji was observed when the incuba-tion time reached 80 h, and since then the value declined slightly.

Optimization of cultural conditions for enzyme production

Based on the results of single-factor experiments, the parameters of inoculum size (X1), temperature (X2), mois-ture content (X3), and incubation time (X4) were further opti-mized with CCD. The variables, levels, design, and results were shown in Table 1. By applying model significance and multiple regression analysis on the experimental data in Table 1, a second-order polynomial equation was estab-lished to describe the correlation between the significant variables and enzyme production in terms of coded values

(5)N(t) = N0e−�t

,

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(6)

Y = 1, 744.44 + 88X1 + 69.27X2 + 109.84X3 + 49.35X4 + 21.58X21 − 24.56X2

2 − 62.03X23

+ 9.56X24 + 29.48X1X2 + 8.12X1X3 + 19.65X1X4 + 14.62X2X3 − 29.62X2X4 + 19.88X3X4.

Fig. 1 Influences of cultural conditions on the production of acid protease from the novel fusant A. oryzae Hg76. at the end of fermentation, moldy bran was extracted with water con-taining 0.1 M naCl for 60 min. activity was determined as described in the ‘‘Protease assay’’ section. a effect of inoculum size on the production of acid protease from A. oryzae Hg76; b effect of tempera-ture on the production of acid protease from A. oryzae Hg76; c effect of moisture content on the production of acid protease from A. oryzae Hg76; d effect of incubation time on the pro-duction of acid protease from A. oryzae Hg76

according to the anOVa for the above model (Table 2), the obtained model was significant (P < 0.0001) and the lack of fit was not significant (P = 0.4375 > 0.05). The value of adjusted R2 was 0.9890, suggesting that the experi-mental data were in good agreement with predicted values.

The quantitative analysis was conducted by compar-ing the coefficient values. as shown in Table 2, the P val-ues of X1, X2, X3, and X4 were lower than 0.01, and their coefficients were positive, demonstrating that they were the significant variables on enzyme activity. according to the coefficients in Table 2, moisture content has the most important influence on enzyme production, followed by inoculum size. Comparatively, the temperature and time were less important. This result was largely consistent with other reports, but still a little different from Vishwanatha et al. [26] investigated the effect of cultivation time, incu-bation pH, and temperature on acid protease production by a new isolated A. oryzae MTCC 5341, and they found fer-mentation time and temperature of incubation were the crit-ical factors for acid protease activity. generally, the similar enzymes from different species display different properties, and thus the difference in cultural conditions between our findings and other reports is probably due to the enzymatic structural difference.

Verification of the adjusted model

The verification of the adjusted model was conducted, and the correlation coefficient between experimental and pre-dicted values was 70.61 %, demonstrating that there was a highly fit degree between the observed values in experiment and the values predicted by Eq. 6. The observed enzyme activity of 1,936 U g−1koji was achieved with inoculum of 8.59 × 105 g−1 dry koji, temperature of 31 °C, moisture content of 57 %, and incubation time of 86 h. The enzyme production in optimized condition is increased by about 44 % compared to the production before optimization.

Purification of the acid protease

To purify the acid protease, the first step adopted the method of ammonium sulfate precipitation. The precipitate in 40–85 % saturation was loaded onto a DEaE-cellulose column and eluted with naCl in a linear gradient from 0 to 1.0 M. The eluted fractions were photometry measured at 280 nm, and the chromatogram was shown in Fig. 2a. Four peaks appeared in Fig. 2a (P1, P2, P3, and P4) with P2 being the main peak for acid protease activity at 0.3–0.35 M naCl. Through ion exchange, the enzyme activity

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was purified 3.26 folds with a recovery yield of 52.80 %. The active fractions of P2 were concentrated by ultrafiltra-tion (a nominal molecular weight cutoff of 10 kDa, Mil-lipore Inc., USa) and then fractionated on Sephadex g-75 column (Fig. 2b). as shown in Fig. 2b, the fraction of P2 was further separated into two parts (Pa and Pb), with the fraction Pb responsible for the majority of the activity. By these steps of purification, the extracellular acid protease was purified 17.09 folds with a yield of 33.56 % and spe-cific activity of 4,105.49 U mg−1 protein. The results of purification in each step were summarized in Table 3.

The SDS-PagE profiles of the acid protease after puri-fication procedure were illustrated in Fig. 2c. as shown in Fig. 2c, although the enzyme was purified as two peaks from gel-filtration chromatography (Pa and Pb), the major activity lied in Pb. Pb appeared nearly as a single band on SDS-PagE (line 5) and native PagE (Fig. 2d). These results indicated that this acid protease was a monomeric enzyme, and thus the possibility of homomultimer was ruled out. The molecular weight of this acid protease was approximately 50 kDa as estimated by SDS-PagE using protein markers of known molecular weight, which was a bit larger than the reported 47 kDa [26].

lC-MS/MS technique was employed to identify the purified protein band in Fig. 2d, and the results were illus-trated in Fig. 3 and Table 3. From the results searched directly with protein database (Table 3), this novel acid

protease had higher similarity to an alpha-amylase a from A. niger and a hypothetical protein from A. oryzae RIB40 than other proteins, indicating the fusant inherited both properties from the parental strain of A. oryzae and A. niger. Furthermore, the precise mass weight of this acid protease determined with lC-MS/MS was 52 kDa, basi-cally consistent with the value by SDS-PagE.

Spectral characterization of the purified acid protease

absorption of radiation in the near-UV by proteins depends on the Tyr and Trp content (and to a very small extent on the amount of Phe and disulfide bonds). The UV absorp-tion spectrum of the purified acid protease was shown in Fig. 4a. The enzyme has a maximum absorption at 280 nm. The fluorescence spectrum of the enzyme exhibited a maxi-mum excitation at 280 nm, and the maximum emission was determined to be 335 nm (Fig. 4b), indicating that the tryp-tophan residues were in a fairly hydrophobic environment. The conformation of acid protease in solution was subse-quently determined by CD spectra scanning in 50 mM cit-ric acid–sodium citrate buffer (pH 3.6). The far-UV CD spectrum was shown in Fig. 4c. The spectrum showed a minimum in the region of 213–217 nm, indicating a pre-dominant β structure. Two positive bands were seen at 198 and 232 nm when the spectrum was recorded at pH 3.6. The near-UV CD spectrum was shown in Fig. 4d. The enzyme

Table 2 Regression coefficients and variance analysis for the fitted quadratic polynomial model

* Significant at P < 0.05; ** significant at P < 0.01

Variable Coefficient F value Prob > F

Intercept 1,744.44 – –

X1 88 205.32 <0.0001**

X2 69.27 127.22 <0.0001**

X3 79.84 408.04 <0.0001**

X4 49.35 64.57 0.0002**

X12 21.58 32.62 0.0012**

X22 −24.56 42.24 0.0006**

X32 −62.03 269.53 <0.0001**

X42 9.56 6.4 0.0447*

X1X2 29.48 13.49 0.0104*

X1X3 8.12 2.48 0.1667

X1X4 19.65 5.99 0.0499

X2X3 14.62 8.02 0.0299*

X2X4 −29.62 13.63 0.0102*

X3X4 19.88 14.81 0.0085**

Source Sum of squares Degrees of freedom Mean square F value Prob > F

Model 3.88E+005 14 27,725.91 129.95 <0.0001**

lack of fit 433.39 2 216.69 1.02 0.4375

Pure error 846.80 4 211.70

Total 3.89E+005 20

R2 = 0.9967; R2Adj = 0.9890

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had peaks at 258, 264, 273, 280, 289, and 302 nm, and the minima were observed at 261, 269, 277, 285, and 294 nm, corresponding, respectively, to phenylalanine, tyrosine, and

tryptophan residues in the enzyme. The presence of these bands strongly suggested that the acid protease from A. oryzae Hg76 belonged to the family of aspartic proteases.

Fig. 2 Purification of the acid protease from fusant of A. oryzae Hg76. a Purification of acid protease was done by DEaE-cellulose; b Fraction P2 was further purified by molecular sieve chromatog-raphy; c Electrophoretogram of proteins during the purification procedures was visualized by SDS-PagE: lane 1 standard molecu-

lar weight markers; lane 2 crude extract; lane 3 ammonium sulfate precipitation (45–80 % saturation); lane 4 DEaE-cellulose column chromatography; lane 5 Sephadex g-75 column chromatography; d native-PagE pattern in gel electrophoresis for the purified acid pro-tease

Table 3 Purification and identification of the acid protease from A. oryzae Hg76

Purification step Total protein (mg) Total activity (U) Specific activity (U mg−1) Yield (%) Purification (fold)

Crude supernatant 72.3 ± 1.8 16,992 ± 22 235.02 ± 11 100 1

ammonium sulfate precipitation 38.4 ± 2.1 13,483 ± 34 351.12 ± 14 79.35 ± 3.2 1.49 ± 0.17

DEaE-cellulose 11.7 ± 1.3 8,972 ± 19 766.84 ± 22 52.80 ± 2.4 3.26 ± 0.22

Sephadex g-75 1.42 ± 0.4 5,702 ± 27 4,015.49 ± 25 33.56 ± 2.9 17.09 ± 0.54

Rank Protein name accession no. Protein MW Protein PI Protein score Protein score CI (%)

1 China a, orthorhombic crystal structure (space group P21212), of Aspergillus niger alpha-amylase a

gi|114794116 52,456.1 4.48 322 100

2 Hypothetical protein (Aspergillus oryzae RIB40) gi|169773935 54,775.3 4.48 320 100

3 Taka-amylase a (Taa-g1) precursor gi|166531 54,721.3 4.55 301 100

4 China a and possible catalytic residues of Taka-amylase a

gi|230754 52,368 4.4 289 100

5 alpha-amylase B precursor (1,4-alpha-d-glucan glucano-hydrolase B)

gi|1703301 54,886.4 4.52 221 100

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Fig. 3 lC-MS/MS mass spectrum of the purified acid protease (in Fig. 2d), indenti-fied after in-gel digestion with trypsin

Fig. 4 Spectral characterization of the acid protease from the fusant of A. oryzae Hg76. a UV absorption spectra of the acid protease; b protein fluorescence was excited at 280 nm and emission was monitored from 300 to 400 nm; c far-UV CD spectra of the acid protease, 0.1 mg ml−1 of protein in 50 mM citric acid–sodium cit-rate buffer (pH 3.6); d near-UV CD spectra of acid protease

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The results of CD studies of this enzyme were well in accordance with other studies of acid proteases from dif-ferent fungal origins [17, 21]. Subsequently, the CDSSTR method was used to estimate the secondary structure com-ponents, using SP43 as the reference database. The results showed this purified protease contained 64.1 % β structure, 7.1 % α-helix, and 32 % aperiodic coil. Compared with the acid protease from the parental strain A. oryzae Hn3042, the acid protease from A. oryzae Hg76 showed a slight decrease in the content of helix and a moderate increase in sheet and coil structure. In our study, the near-UV CD spec-tra of the purified protease showed similar spectra character to the aspartic proteases from A. oryzae MTCC5341 [17].

Kinetics characterization of the acid protease

Effect of temperature and pH on the activity of acid protease

The influence of temperature on the activity of acid pro-tease from A. oryzae Hg76 was investigated by varying

temperature from 35 to 65 °C. The plot of residual activ-ity versus temperature was shown in Fig. 5a. It can be seen that the enzyme was active over a wide temperature range of 35–55 °C with an optimum activity at 40 °C. The influ-ence of pH on the activity was investigated by varying pH from 2.0 to 6.5. The results showed that this acid protease was stable within the pH range of 3.0–6.5, and the opti-mum pH for activity was 3.5 (Fig. 5b). It has been reported that the acid proteases from A. oryzae commonly have the optimum pH in the range of 2.5–4.0 [26]. For most acid proteases, they are stable under moderate acid conditions (pH 4.0–6.0) and display maximal activity at low pH val-ues (about 3.0–5.0). The appropriate range for a specific enzyme must be determined empirically, and the purified enzyme was within this range.

The stability of enzymes remains a critical aspect in industrial applications [22]. The effect of temperature and pH on the protease activity was tested with different tem-perature and pH. The enzyme stability analysis at various temperatures showed that the enzyme was a mesophilic protease remaining active at temperatures ranging from 35

Fig. 5 Kinetics characteriza-tion of the acid protease from the fusant of A. oryzae Hg76. Effect of temperature (a) and pH (b) on the activity (filled cir‑cle) and stability (open circle) of the acid protease; c effect of metal ions on the enzyme activity

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to 55 °C with the optimal activity at temperature of 40 °C and the enzyme activity declined as temperature went above 55 °C (Fig. 5a), indicating the conformational insta-bility at high temperatures. The effect of pH on the enzyme activity was examined in the pH range from 2.0 to 6.5. The result showed the activity was stable within a broad pH range from 3.0 to 6.5, and the optimal activity was observed in pH 3.5 (Fig. 5b). The activity declined with further increasing pH. Compared with the acid protease from A. oryzae MTCC5341 [17], this purified protease exhibited slight decline in optimal temperature and pH. The change of activity at different temperature and pH might result from the slight change of enzyme conformation.

Effects of metal ions and protease inhibitors on the activity of acid protease

The effect of metal ions on activity was illustrated in Fig. 5c. as shown in Fig. 5c, the catalytic activity of the purified acid protease was improved by Mn2+ with an increase about 30 % but was inhibited by Co2+ with a decrease about 20 % compared to the control. The Mn2+-dependent activity improvement indicated that the enzyme required Mn2+ for its optimal activity, and this phenome-non might be attributed to Mn2+ involvement in stabiliza-tion of the enzyme molecular structure as reported in some

of the extracellular acid proteases derived from Rhizopus oryzae [2]. In fact, Mn2+ is known as inducers and stabiliz-ers of many enzymes protecting them from conformational changes [24].

Inhibitory effect of specific inhibitors on the enzyme activity was evaluated referring to the reported concentra-tions [26], and specific protease inhibitors were employed to identify the group at active site of this protease (Table 4). The enzyme retained above 90 % of relative activity in presence of leupeptin (10 μM), precluding the role of –SH group in enzyme activity. Similarly, PMSF (10 mM) did not affect the enzyme activity; hence, serine participation at the active site of the enzyme has also been ruled out. Inef-fectiveness of EDTa (10 mM) denied the possibility of the enzyme being a metalloenzyme. The strong inhibition by

Table 4 Effect of protease inhibitors on the activity of purified acid proteases

Inhibitors and its concentration Residual activity (%)

CK 100

PMSF (1 mM) 98.3 ± 1.2

EDTa (10 mM) 95.7 ± 2.5

leupeptin (10 μM) 93.6 ± 1.7

Pepstatin (10 μM) 2.1 ± 1.1

Fig. 6 Characterization of the dynamic parameters of the purified acid protease. a lineweaver–Burk double-recip-rocal plot of the acid protease. b arrhenius plot used in determi-nation of Ea. c Thermal stability of native enzyme activity from the initial value of N0 to differ-ent values of Nh

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93 % at 10 μM of pepstatin a pointed toward the presence of aspartate at active site. Therefore, this purified protease was proved to be aspartic protease, similar to the reports for acid proteases [26].

Determination of kinetics parameters of Km, Vmax, Ea, and λ of the acid protease

Based on the double-reciprocal lineweaver–Burk plot, the kinetic parameters of Km and Vmax were calculated and the results were shown in Fig. 6a. according to the fitted linear equation, Km and Vmax of the purified protease were deter-mined to be 0.96 mg ml−1 and 135.14 μmol min−1 mg−1, respectively, which was a remarkable decrease com-pared with that of parental strain A. oryzae Hn3042 (1.22 mg ml−1 and 181.82 μmol min−1 mg−1). The effect of temperature on the catalytic velocity was characterized with the parameter of Ea. In our study, the purified enzyme was incubated with casein for 10 min at the tempera-ture range from 20 to 45 °C and the relative activities of the enzyme at different temperatures were fitted with the temperatures (Fig. 6b). The linear-logarithmic equation of arrhenius plot was used to calculate the Ea of the puri-fied protease, and the value was 64.11 kJ mol−1, which is a closely similar to that of parental strain A. oryzae Hn3042 (64.61 kJ mol−1). In our research, this purified acid pro-tease was incubated throughout the time range at 45 °C, and the value of λ was determined to be 0.59 (Fig. 6c).

It is well known that the properties of enzyme are closely relative with the certain conformation of enzyme in solution, and the kinetic parameters of Km, Vmax, Ea, and λ have direct linkage with the enzyme properties, and they can be briefly used to explain the kinetic characteristics of enzymes [24]. Compared with the acid protease from parental strain A. oryzae Hn3042, this purified extracellular protease from A. oryzae Hg76 showed a slight decline in Km and Ea while a slight increase in decay index λ, which attributed to the slight change of dimensional structure.

Conclusions

Through statistical optimization, the production of a novel acid protease from A. oryzae Hg76 was enhanced by 44 %. and by continuous purification, this protease was purified 17 folds with a recovery yield of 33.56 % and a specific activity of 4,105.49 U mg−1. Characterizations in spectra and kinetics revealed that this novel acid protease pos-sessed some different properties from its parental strain A. oryzae Hn3042. Based on this investigation, the acid pro-tease’s production can be enhanced by regulating the cul-tural parameters and thus its potential applications in fer-mentation is predictable.

Acknowledgments The authors gratefully acknowledge the national natural Science Foundation of China (31201309) for the financial support.

Conflict of interest none.

Compliance with Ethics Requirements This article does not con-tain any studies with human or animal subjects.

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