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Neutral protease expression and optimized conditions for the degradation of blood cells using recombinant Pichia pastoris
International Biodeterioration & Biodegradation 93 (2014) 235e240
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Neutral protease expression and optimized conditions for the degradation of blood cells using recombinant Pichia pastoris Hao Zhang, Boru Zhang, Yanbin Zheng, Anshan Shan*, Baojing Cheng Laboratory of Biotechnology, Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 February 2014 Received in revised form 25 May 2014 Accepted 27 May 2014 Available online 5 July 2014
Significant amount of blood waste is generated by the livestock industry. However, the poor utilizability of blood cells limited the application of blood. In our experiment, to degrade blood cells effectively, a recombinant Pichia pastoris strain with the Aspergillus oryzae neutral protease gene was generated using genetic engineering methods. The recombinant strains were screened for high protease activity on agar plates and blood cells fermentation medium using blood cells as the nitrogen source. Single-factor experiments and response surface methodology (RSM) were applied to predicted the degree of hydrolysis (DH) of blood cells and the predicted maximal DH appeared at the region where the pH, time, and temperature were 8.1, 116.6 h, and 33.9 C, respectively. Under the proposed optimized conditions, the experimental DH value for recombinant pGAPZaA-NpI-GS115 was 49.05%. Therefore, the efficient degradation of blood cells by this recombinant stain may offer an environmental-friendly solution for the degradation of blood waste and other organic matter of similar molecular composition and more meaningfully may supply a high-quality and low-cost protein source for feed industry. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Neutral protease expression Optimized conditions Degradation Blood cells Recombinant Pichia pastoris
1. Introduction The livestock industry provided a significant amount of blood but is commonly not fully utilized or even discarded (Wang et al., 1997; Ockerman and Hansen, 2000). Blood is a potentially lowcost protein source, and recycling blood would reduce the oxygen burden created by the biodegradation of the pollution from s i Oliva, 2001). The protein slaughterhouse wastewaters (Pare content of blood cells (BCs) of the whole blood is 34e38% (Mandal et al., 1999). The BCs, which are produced by separating the adtevak from whole blood, mainly contain the ferrous hemoglobin in the form of heme-Fe with high bioavailability (Duarte et al., 1999; et al., 2000). The spray-dried BCs are highMartínez Gracia quality protein resources and have a favorable amino acids (AAs) profile (Williams, 1994). Therefore, the BCs have been widely used et al., 2011). However in feed for animals (Saguer et al., 2003; Fruge the low digestibility and metallic taste of hemoglobin (Hb) are not desirable when BCs are added to a variety of food and feed products et al., 2011). To solve these problems, various attempts (e.g., (Toldra microbial fermentation) have been made to process BCs, especially by breaking the blood cell membrane to improve the availability of
* Corresponding author. Tel.: þ86 451 55190685. E-mail address: [email protected] (A. Shan). http://dx.doi.org/10.1016/j.ibiod.2014.05.024 0964-8305/© 2014 Elsevier Ltd. All rights reserved.
rez-G BCs (Pe alvez et al., 2011; Zheng et al., 2012). After being processed, complex molecules, such as macromolecular proteins, are degraded into peptides of various molecular weights and free s i Oliva, 2001; Gaskell and Smith, 2007; amino acids (FAAs) (Pare Guo et al., 2007). Meanwhile, a large quantity of bacterial proteins are produced counterpoising the AAs contents of the BCs. Therefore, the biodegraded BCs have benefits on digestibility, s i Oliva, 2001; Chen palatability, and economic efficiency (Pare et al., 2013), and are sufficient for the nutritional requirements of animals (Yan, 1995; He, 2008). Additionally the biodegraded BCs have exerted positive effect on weaned and finishing pigs (King'ori et al., 1998; M'ncene et al., 1999; Chen et al., 2013). Being a kind of incision enzyme, neutral proteases can be used for the hydrolysis of various proteins. They can break down the macromolecular proteins into amino acids, with the appropriate pH value from 4.0 to 11.0 (Xiao, 2006). Additionally, they have been widely used in food, medicine and feed industry for their effective enzymatic hydrolysis reaction rate, low industrial pollution and mild reaction condition. Neutral proteases are the earliest protein enzymes used in industrial production, and the representative one is neutral protease I produced by Aspergillus oryzae (Zhou 2010). The Pichia pastoris expression system offers economy, ease of manipulation, the ability to perform complex post-translational modifications, and high expression levels. However, little references have been found that using P. pastoris to degrade BCs before.
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In this experiment, we developed a recombinant P. pastoris strain, named pGAPZaA-NpI-GS115, for the high-level production of neutral protease. Two statistical methods including single-factor experiments and BoxeBehnken design (BBD; three factors and three levels) of RSM were employed to optimize the BCs fermentation conditions. 2. Materials and methods 2.1. Strains, vectors, enzymes, reagents, and growth medium All of the strains, vectors and enzymes used in this experiment are included in Table 1. A. oryzae 3.042 (CICC 2339) purchased from the China Center of Industrial Culture Collection (CICC) is an widely used industrial strain which was screened by UV mutagenesis for high-yield of protease. All of the culture medium and methods used for Pichia are described in the Pichia expression manuals (Cregg et al., 2009). All of the chemical reagents were of analyticalreagent grade. 2.2. DNA cloning of neutral protease I (NpI) from A. oryzae 2.2.1. RNA extraction For separation of RNA, A. oryzae 3.042 was cultured in potato extract glucose medium. 100 ml of culture medium was loaded into a 250-ml conical flask and autoclaved at 121 C for 20 min. Each flask was inoculated with 1.0 ml of the spore solution and shaken at 30 C, 120 rpm for 72 h. Through the above-described process, the fungal hyphae of A. oryzae, which was used for extracting RNA, turned to lots of small balls with a diameter of 3e8 mm (Guo and Ma, 2008). The fungal mycelia were collected and rapidly frozen in liquid nitrogen. The total RNA was extracted using the Fungal RNA Kit (OMEGA, USA). 2.2.2. One-step RT-PCR Reagents from the one-step RT-PCR kit (TaKaRa, Dalian, China) were used to prepare the master mix solutions according to the describe of Yang et al. (2013). Both RT and DNA polymerization were conducted in the same tube without the subsequent addition of enzymes or buffer. The PCR program was 50 C for 30 min, 94 C for 2 min, 30 cycles of 94 C for 30 s and 55 C for 30 s, and a final extension at 72 C for 2 min using two complementary deoxyoligonucleotides (oligos): A (forward), 50 -CTCGAGATGCGGGGTCTTCTACTAGCTG-30 ; B (reverse), 50 -GCGGCCGCGAATTCGAAGCGGCGGAC-30 (the restriction sites XhoI (CTCGAG) and NotI (GCGGCCGC) are shown in italics in the oligos). The PCR products were detected by electrophoresing through 1% agarose gels in 1 TAE buffer (40 mM Triseacetate (pH 8.0) and 1 mM ethylenediaminetetraacetic acid). The purified PCR product was ligated into the cloning vector pMD-18T. After transformation of the vector into Escherichia coli DH5a competent cells, the transformants (pMD-18T-NpI) were screened on LB plates (1% tryptone, 1% NaCl, 0.5% yeast extract, and 1.5% agar, pH 7.0) containing 50 mg/ml ampicillin. The pMD-18T-NpI plasmid was identified through restriction enzyme analysis and then sequencing by Sangon (Shanghai, China). Table 1 Strains, vectors and enzymes used in this experiment. Strains, vectors and enzymes
Source
Aspergillus oryzae strain 3.042 Escherichia coli strain DH5a P. pastoris strain GS115 The expression vector pMD-18T The expression vector pGAPZaA Enzymes
Preserved in our laboratory Purchased from Takara (Dalian, China) Purchased from Invitrogen Purchased from Takara (Dalian, China) Purchased from Invitrogen Purchased from Takara (Dalian, China)
2.3. Construction of the expression vectors The pMD-18T cloning vector and the pGAPZaA plasmid were digested with the restriction enzymes XhoI and NotI and then ligated with T4 DNA ligase. The recombinant plasmids were transformed into E. coli DH5a, and Zeocin™-resistant transformants were screened on low-salt Luria-Bertani (LLB) agar plates containing 25 mg/ml Zeocin™. The DNA plasmid was prepared, and the insert fragments were identified by restriction enzyme digestion (XhoI and Not I) and further confirmed by sequencing (Sangon Biotech, Shanghai, China). 2.4. Transformation and screening of P. pastoris expression strains The pGAPZaA-NpI recombinant plasmid and pGAPZaA plasmid (negative control) were linearized using the SacI restriction enzyme. The linearized plasmid and GS115 competent cells were mixed and transferred to an ice-cold 0.2 cm electroporation cuvette. The mixture was incubated on ice for 5 min and pulsed with a MicroPulser (Bio-Rad, Hercules, CA, USA; conditions: 2.5 kV, 200 U, and 25 mF). 1 ml of ice-cold 1 M sorbitol was added immediately to the cuvette. The cuvette contents were transferred to a sterile 15-ml tube and incubated without shaking at 30 C for 2 h. The cells were plated on YPDZ agar plates (1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar, and 100 mg/ml Zeocin™) for 3e4 days at 30 C until colonies formed. 2.5. Expression of NpI gene in P. pastoris The positive P. pastoris transformants (named pGAPZaA-NpIGS115) were cultured at 200 rpm for 12e16 h in a shaking flask containing 10 ml YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) at 30 C and then expanded into 100 ml of YPD medium cultured at 200 rpm for 96 h in a shaking flask at 30 C. For screening of the neutral protease productivity (a vector without any neutral protease gene insert was used as a control), the neutral protease in the culture supernatants was monitored by measuring the expressed recombinant neutral protease by SDSePAGE. 2.6. Screening of pGAPZaA-NpI-GS115 based on the neutral protease activity The neutral protease activity was assayed by the casein FolineCiocalteu method described by Oda and Murao (1974). 1 ml of the enzyme solution was added to 1 ml of 1.14% casein solution in a 0.1 M Na2HPO4eNaH2PO4 buffer with a pH of 7.5. After incubation at 40 C for 10 min, the reaction was stopped by the addition of 2.0 ml of 0.44 M trichloroacetic acid followed by centrifugation at 12,000 g for 10 min. The 1 ml supernatant was mixed with 5 ml of 0.4 M sodium carbonate and 1 ml of the FolineCiocalteu reagent. After incubation at 40 C for 20 min, the optical density was measured at 680 nm. One unit of enzyme activity was defined as the enzyme quantity that liberates 1 mg of tyrosine per ml of the reaction mixture per min. The positive transformant that expressed the highest neutral protease level was selected and used for the time-course studies. 2.7. Optimized conditions for fermenting blood cells using pGAPZaA-NpI-GS115 2.7.1. Degradation of BCs Liquid-state fermentation (LSF) was conducted in a 250-ml flask that contained 3 g of wheat bran, 3 g of blood cells, and 100 ml of buffered solution. The pH of the medium was adjusted to 7.5. The prepared medium was used as the basic substrate for the pGAPZaANpI-GS115.
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The inoculated medium was incubated at 30 C for 120 h and then incubated in boiling water for 15 min to inactivate the enzyme. The mixtures in the flasks were then removed by filtration using filter paper. The filtrate was diluted to 100 ml with distilled water to determine the degree of hydrolysis (DH). 2.7.2. Single-factor experiments The purpose of single-factor experiments was to determine the fermentation conditions individually, such as temperature, time, and pH, to measure their contribution toward the DH. 2.7.3. Response surface methodology A three-level, three-variable BBD (Design-Expert v. 8.0) was applied to determine the best combination of fermentation variables to maximize the DH of the BCs. Based on the single-factor experiments, the initial conditions were a temperature of 34 C, a time duration of 108 h, and a pH of 8.0. Table 2 lists the BBD matrix and the response values that were used to develop the model. The response value obtained from each trial is an average of triplicates. 2.7.4. Degree of hydrolysis The degree of hydrolysis was presented as the percentage of cleaved peptide bonds:
DH ð%Þ ¼
h 100; htot
(1)
where h (hydrolysis equivalents) is the number of hydrolyzed bonds, which is expressed as millimolar equivalents per gram of protein (mmol/g of protein), and htot is the total amount of peptide bonds per protein equivalent, which can be determined from the amino acid composition and is 8.62 mmol/g for BCs. The amino nitrogen content was determined using a modified ninhydrin colorimetric method (Zheng et al., 2014). 2.8. Statistical analysis The data obtained from the BBD design were fitted with a second-order polynomial equation as follows:
Y ¼ b0 þ
2 X
bi Xi þ
i¼1
2 X
bii Xi2 þ
i¼1
X X
bij Xi Xj ;
(2)
i¼1 j¼iþ1
where Y is the predicted response, b0 is a constant, bi is a linear coefficient, bii is a quadratic coefficient, bij is an interaction coefficient, and Xi and Xj are independent variables. The statistical significance of the model and the model variables were determined at probability values (P) of 0.05 and 0.01. 3. Results and discussion 3.1. Cloning of the NpI gene from A. oryzae, construction of expression plasmid, and screening of recombinant transformants The NpI gene which encoding the neutral protease of A. oryzae strain 3.042 was amplified by RT-PCR using the reverse transcription product as the template and cloned into the pMD-18T vector. Table 2 Factor levels for pGAPZaA-NpI-GS115 that were used to optimize the DH. Code
pH (A)
Time (B, h)
Temperature (C, C)
1 0 1
7 8 9
96 108 120
31 34 37
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The pMD-18T-NpI recombinant plasmid was identified by restriction enzyme analysis and DNA sequencing. The sequence data were identical to the sequence published in GenBank (Accession No. AF099904.1). The correct DNA fragment was used to construct the expression vector. Fig. 1 explains how the expression vector was constructed. The correct DNA fragment was inserted into the pGAPZaA plasmid to construct the recombinant vector. The recombinant vector was identified by XhoI and NotI digestion, and the corresponding fragment (1905 bp) was obtained (Fig. 2). The target fragment was confirmed by gene sequencing to be the same as the original sequence of the NpI gene in GenBank, and the recombinant vector was denoted pGAPZaA-NpI. P. pastoris strain GS115 was transformed with the pGAPZaA-NpI linearized plasmid. After incubation on YPD plates with Zeocin™ (100 mg/ml) for three days at 30 C, approximately 80 colonies were formed. The pGAPZaA vector without any insert was also transformed into P. pastoris GS115 and used as a control. 3.2. Screening of pGAPZaA-NpI-GS115 based on the neutral protease activity All of the colonies were cultured in YPD medium to assay the neutral protease activity, and the clone with the highest expression level of neutral protease was selected for the time-course studies. The NpI gene was expressed in P. pastoris and had the expected size of 46 kDa, as verified by SDSePAGE (Fig. 3). 3.3. Effects of pH on the DH of BCs As shown in Fig. 4(a) (fixed levels: blood cells ¼ 3 g, temperature ¼ 34 C, time ¼ 120 h), the peak DH of pGAPZaA-NpIGS115 appeared at the pH value of 8.0. The pH value can play an important role in controlling the expression of many genes of fungi (Penalva et al., 2008). In this study, the DH increased gradually to the optimal level and then exhibited a minor decrease due to enzyme production, which is similar to the results reported by Gautam et al. (2011) and Bansal et al. (2012). 3.4. Effects of time on the DH of BCs As shown in Fig. 4(b) (fixed levels: blood cells ¼ 3 g, temperature ¼ 34 C, initial pH ¼ 7.5), the peak DH values of pGAPZaA-NpI-GS115 appeared at 108 h. The evaluation of the time to peak is of prime importance for enzyme biosynthesis by fungi (Kuhad and Singh, 1993). Upon further incubation, the enzyme yields declined gradually due to the release of protease and a decrease in the pH of the medium (Yao et al., 2012), which may be due to the depletion of nutrients and the accumulation of other byproducts, such as protease, in the fermentation medium (Romero et al., 1998). 3.5. Effects of temperature on the DH of BCs As shown in Fig. 4(c) (fixed levels: blood cells ¼ 3 g, time ¼ 120 h, initial pH ¼ 7.5), the peak DH of pGAPZaA-NpI-GS115 appeared at a temperature of 34 C. As reported, the optimum growth temperature for P. pastoris GS115 is 28e30 C, and higher temperatures (above 40 C) altered the cell membrane composition and simulated protein catabolism, causing cell death (Cregg et al. 2009). A study conducted by Yang and Qiu (2006) investigated a temperature range from 30 to 60 C and found that 55 C resulted in the highest neutral protease activity. The optimal temperature for DH was between the optimal growth temperature for P. pastoris GS115 and the optimal temperature for neutral protease activity,
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Fig. 1. Map of construction of the expression vector pGAPZaA-NpI.
Fig. 2. Enzyme digestion of the pGAPZaA-NpI recombinant plasmid. Lane M1: DL5000 DNA marker; Lane 1: pGAPZaA-NpI recombinant plasmid; Lane 2: product from the digestion of pGAPZaA-NpI with XhoI; Lane 3: product from the digestion of pGAPZaANpI with NotI; Lane 4: product from the digestion of pGAPZaA-NpI with XhoI and NotI; Lane M2: DL10000 DNA marker.
Fig. 3. SDSePAGE analyses of yeast cultures expressing secreted NpI. Lane M: protein molecular weight marker; Lane 1: sample from the negative control P. pastoris; Lanes 2e7: samples from NpI-expressing P. pastoris.
H. Zhang et al. / International Biodeterioration & Biodegradation 93 (2014) 235e240
Fig. 4. Effects of pH (a), time (b), and temperature (c) on the DH.
and this finding can be attributed to the combined effect of both the growth temperature for P. pastoris and the optimal temperature for neutral protease activity.
239
of the model. The P value for model was significant (P < 0.01) and the lack of fit was not significant (P > 0.05), confirming the validity of the model. The model was found to be adequate for predictions within the range of the experimental variables. The values of the coefficients of Eq. (3) were calculated and their significance tested using the Design-Expert v. 8.0 software; these are listed in Table 4. Each P value was used as a tool to determine the significance of each coefficient, which, in turn, may indicate the pattern of the interactions between the variables. As observed in Table 4, the quadratic term coefficients (X2) was significant with P values at the 0.01 level, the linear coefficient (X1 * X3) was significant with a P value at the 0.05 level, and the quadratic term coefficient (X1, X3 and X2) were significant with a P value at the 0.01 and 0.05 level respectively. The other coefficients (X1, X3, X1 * X2, and X2 * X3) were not significant (P > 0.05). The cleavage of the peptide bonds of the protein substrate has been found to be markedly influenced by the conditions under which the substrate is hydrolyzed with bacterial proteases (Box et al., 1978; Wang and Xu, 2007; Zhu and Row, 2011; Ovissipour et al., 2012). In this study, after the proteases were produced from the recombinant pGAPZaA-NpI-GS115, the cleavage of the peptide bonds of the BCs was influenced by the fermentation conditions. The adjusted determination coefficient ðR2Adj Þ was used as the correlation measure to test the goodness of fit of the regression equation. The value of R2Adj (0.9269) for Eq. (3) was reasonably close to 1. These results indicate a high degree of correlation between the experimental and predicted values. Good correlations between the experimental results and the results predicted by RSM models of proteolytic reactions have been rerez-Ga lvez et al., ported by several researchers (Chen et al., 2010; Pe 2011; Zheng et al., 2012). The very low value of the coefficient of variation (C.V.) (1.84% for pGAPZaA-NpI-GS115) clearly indicates the very high degree of precision and reliability of the experimental values. The optimal conditions indicated by RSM are pH ¼ 8.1, time ¼ 116.6 h, and temperature ¼ 33.9 C with a desirability DH of 49.46%. Under the abovementioned conditions, the experimental DH of fermented blood cells with pGAPZaA-NpI-GS115 is 49.05%, which is much higher than 35.13% and 32.91% that reported by Zheng et al. (2012, 2014), respectively. Table 3 The DH of pGAPZaA-NpI-GS115 at various pH values, temperatures, and times.
3.6. Optimization of the fermentation conditions for DH
Run
The influence of the factors on the DH of pGAPZaA-NpI-GS115 was determined using the factorial design described in the previous section. The best explanatory model equations for the DH values for pGAPZaA-NpI-GS115 obtained from the uncoded fermentation data are described in Eq. (3):
DH ¼ 740:03728 þ 75:57892 pH þ 1:55667 Time þ 23:12006 Temperature þ 0:022292 pH Time þ 0:33667 pH Temperature þ 3:05556E 003 Time Temperature 4:11725 pH2 7:89757E 003 Time2 0:30553 Temperature2 : (3) The experimental and predicted values for the DH of pGAPZaANpI-GS115 under various combinations of the independent variables are presented in Table 3. The results indicate that the DH for pGAPZaA-NpI-GS115 ranged from 40.76% to 49.40% depending on the experimental conditions. The statistical testing of the model was performed using ANOVA, which was required to test the significance and adequacy
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 a
Code values
DHd
Real values
X1
X2
X3
X1a
X2b
X3c
Experimental
0 0 1 1 0 1 1 1 1 1 1 0 0 0 0 0 0
1 1 0 1 1 1 1 1 0 0 0 1 0 0 0 0 0
1 1 1 0 1 0 0 0 1 1 1 1 0 0 0 0 0
8 8 9 7 8 9 9 7 7 7 9 8 8 8 8 8 8
120 96 108 120 120 120 96 96 108 108 108 96 108 108 108 108 108
37 31 31 34 31 34 34 34 37 31 37 37 34 34 34 34 34
46.91 43.32 43.26 43.55 46.44 46.77 43.19 41.04 42.81 41.27 40.76 43.35 48.50 48.33 49.01 49.40 49.22
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.58 0.53 0.49 0.56 0.25 0.33 0.46 0.29 0.12 0.58 0.35 0.44 0.17 0.34 0.27 0.22 0.51
Predicted 46.97 43.26 43.53 43.76 46.23 46.71 42.98 41.10 41.65 41.27 41.65 43.56 48.89 48.89 48.89 48.89 48.89
X1 ¼ pH. X2 ¼ Time. c X3 ¼ Temperature. d DH represents the average degree of hydrolysis of triplicate experiments. The DH was calculated using the equation Y ¼ 0.98A570 0.01 (R2 ¼ 0.997), which was derived from the standard curve of completely hydrolyzed BCs (absorbance at 570 nm versus the concentration of the hydrolyzate). b
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Table 4 ANOVA of the response surface quadratic polynomial model of pGAPZaA-NpI-GS115. Source
Sum of squares df
Model 146.65 X1epH 3.52 X2eTime 20.38 X3eTemperature 0.026 0.29 X1X2 X1X3 4.08 X2X3 0.048 X12 71.38 X22 5.45 X32 31.84 Residual 4.84 Lack of fit 4.00 Pure error 0.85 Cor total 151.49 Std. dev. Mean C.V. % PRESS
0.83 45.13 1.84 65.26
9 1 1 1 1 1 1 1 1 1 7 3 4 16
Mean square
F-value P-value
16.29 3.52 20.38 0.026 0.29 4.08 0.048 71.38 5.45 31.84 0.69 1.33 0.21 e
23.54 5.09 29.45 0.038 0.41 5.90 0.07 103.13 7.87 46.00 e 6.27 e e
R-squared Adj R-squared Pred R-squared Adeq precision
0.0002 0.0586 <0.0010 0.8506 0.5407 0.0455 0.7991 <0.0001 0.0263 0.0003 e 0.0541 e e
***a ***a
**b ***a **b ***a
0.9680 0.9269 0.5693 13.295
df, degrees of freedom. a Significance at the 0.01 level. b Significance at the 0.05 level.
4. Conclusions In this work, a recombinant P. pastoris pGAPZaA-NpI-GS115 was successfully constructed, and under the indicated conditions, the experimental DH of fermented blood cells with this recombinant is 49.05%. The blood cells degradation result indicates that future application of pGAPZaA-NpI-GS115 in the conversion of waste blood generated by the livestock industry into protein hydrolyzates is very promising; the resulting protein hydrolyzates could be utilized as animal feed or nitrogen fertilizers. Such technology represents a valuable approach with two intrinsic advantages, namely, the recycling of agro-industrial residues and the concomitant aggregation of value to these inexpensive raw materials. Conflict of interest The authors declare that they have no conflicts of interest related to this work. Acknowledgments This project was financially supported by the National Basic Research Program (Grant No. 2012CB124703), the National Key Technology R&D Program (2013BAD10B03), the China Agriculture Research System (CARS-36), and the Program for Innovative Research Team of Universities in Heilongjiang Province (2012TD003). References Bansal, N., Tewari, R., Soni, R., Soni, S.K., 2012. Production of cellulases from Aspergillus niger NS-2 in solid state fermentation on agricultural and kitchen waste residues. Waste Manag. 32, 1341e1346. Box, G.E.P., Hunter, J.S., Hunter, W.G., 1978. Statistics for Experimenters: an Introduction to Design, Data Analysis, and Model Building. Wiley, New York, pp. 245e255. Chen, H.C., Ju, H.Y., Wu, T.T., Liu, Y.C., Lee, C.C., Chang, C., Chung, Y.L., Shieh, C.J., 2010. Continuous production of lipase-catalyzed biodiesel in a packed-bed reactor: optimization and enzyme reuse study. BioMed Res. Int. 2011. Chen, Q., Zhang, H., Zheng, Y., Shan, A., Bi, Z., 2013. Effects of enzymatically hydrolyzed blood cells on growth performance and intestinal characteristics of newly weaned piglets. Livestock Sci. 157, 514e519. Cregg, J.M., Tolstorukov, I., Kusari, A., Sunga, J., Madden, K., Chappell, T., 2009. Expression in the yeast Pichia pastoris. Meth. Enzymol. 463, 169e189.
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Caracterizatzio i revaloracio de la fraccio plasm Pare atica de la sang de porc procedent d'escorxadors industrials (Doctoral thesis). University of Girona, Spain. Penalva, M.A., Tilburn, J., Bignell, E., Arst Jr., H.N., 2008. Ambient pH gene regulation in fungi: making connections. Trends Microbiol. 16, 291e300. Romero, F., Garacia, L.A., Diaz, M., 1998. Protease production from whey at high concentration by Serratia marcescens. Resour. Environ. Biotechnol. 2, 93e115. Saguer, E., Altarriba, S., Lorca, C., Pares, D., Toldra, M., Carretero, C., 2003. Colour stabilization of spray-dried porcine red blood cells using nicotinic acid and nicotinamide. Food Sci. Technol. Int. 9, 301e307. s, D., Saguer, E., Carretero, C., 2011. Hemoglobin hydrolysates from Toldr a, M., Pare porcine blood obtained through enzymatic hydrolysis assisted by high hydrostatic pressure processing. Innov. Food Sci. Emerg. Technol. 12, 435e442. Wang, L., Flores, R.A., Johnson, L.A., 1997. Processing feed ingredients from blends of soybean meal, whole blood, and red blood cells. Trans. ASAE 40, 691e697. Wang, W.D., Xu, S.Y., 2007. Degradation kinetics of anthocyanins in blackberry juice and concentrate. J. Food Eng. 82, 271e275. Williams, A.P., 1994. Amino acid requirements of the veal calf and beef steer. Amino Acids Farm Anim. Nutr., 329e349. Xiao, H.Q., 2006. Study on Screening of High-yield Proteinase-producing Strain and Enzyme Characterizations. Hunan Agricultural University, China. Yan, G.L., 1995. Study on the microbial production of blood meal by the use of vector. J. Zhangjiakou Agric. College 11, 10e14. Yang, K., Li, Y., Duan, Z., Guo, R., Liu, Z., Zhou, D., Yuan, F., Tian, Y., 2013. A one-step RT-PCR assay to detect and discriminate porcine reproductive and respiratory syndrome viruses in clinical specimens. Gene 531, 199e204. Yang, S.P., Qiu, D.Q., 2006. Study on characters of neutral protease from Aspergillus oryzae. J. Zhanjiang Ocean Univ. 26, 22e25. 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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Neutral protease expression and optimized conditions for the degradation of blood cells using recombinant Pichia pastoris Hao Zhang, Boru Zhang, Yanbin Zheng, Anshan Shan*, Baojing Cheng Laboratory of Biotechnology, Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 February 2014 Received in revised form 25 May 2014 Accepted 27 May 2014 Available online 5 July 2014
Significant amount of blood waste is generated by the livestock industry. However, the poor utilizability of blood cells limited the application of blood. In our experiment, to degrade blood cells effectively, a recombinant Pichia pastoris strain with the Aspergillus oryzae neutral protease gene was generated using genetic engineering methods. The recombinant strains were screened for high protease activity on agar plates and blood cells fermentation medium using blood cells as the nitrogen source. Single-factor experiments and response surface methodology (RSM) were applied to predicted the degree of hydrolysis (DH) of blood cells and the predicted maximal DH appeared at the region where the pH, time, and temperature were 8.1, 116.6 h, and 33.9 C, respectively. Under the proposed optimized conditions, the experimental DH value for recombinant pGAPZaA-NpI-GS115 was 49.05%. Therefore, the efficient degradation of blood cells by this recombinant stain may offer an environmental-friendly solution for the degradation of blood waste and other organic matter of similar molecular composition and more meaningfully may supply a high-quality and low-cost protein source for feed industry. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Neutral protease expression Optimized conditions Degradation Blood cells Recombinant Pichia pastoris
1. Introduction The livestock industry provided a significant amount of blood but is commonly not fully utilized or even discarded (Wang et al., 1997; Ockerman and Hansen, 2000). Blood is a potentially lowcost protein source, and recycling blood would reduce the oxygen burden created by the biodegradation of the pollution from s i Oliva, 2001). The protein slaughterhouse wastewaters (Pare content of blood cells (BCs) of the whole blood is 34e38% (Mandal et al., 1999). The BCs, which are produced by separating the adtevak from whole blood, mainly contain the ferrous hemoglobin in the form of heme-Fe with high bioavailability (Duarte et al., 1999; et al., 2000). The spray-dried BCs are highMartínez Gracia quality protein resources and have a favorable amino acids (AAs) profile (Williams, 1994). Therefore, the BCs have been widely used et al., 2011). However in feed for animals (Saguer et al., 2003; Fruge the low digestibility and metallic taste of hemoglobin (Hb) are not desirable when BCs are added to a variety of food and feed products et al., 2011). To solve these problems, various attempts (e.g., (Toldra microbial fermentation) have been made to process BCs, especially by breaking the blood cell membrane to improve the availability of
* Corresponding author. Tel.: þ86 451 55190685. E-mail address: [email protected] (A. Shan). http://dx.doi.org/10.1016/j.ibiod.2014.05.024 0964-8305/© 2014 Elsevier Ltd. All rights reserved.
rez-G BCs (Pe alvez et al., 2011; Zheng et al., 2012). After being processed, complex molecules, such as macromolecular proteins, are degraded into peptides of various molecular weights and free s i Oliva, 2001; Gaskell and Smith, 2007; amino acids (FAAs) (Pare Guo et al., 2007). Meanwhile, a large quantity of bacterial proteins are produced counterpoising the AAs contents of the BCs. Therefore, the biodegraded BCs have benefits on digestibility, s i Oliva, 2001; Chen palatability, and economic efficiency (Pare et al., 2013), and are sufficient for the nutritional requirements of animals (Yan, 1995; He, 2008). Additionally the biodegraded BCs have exerted positive effect on weaned and finishing pigs (King'ori et al., 1998; M'ncene et al., 1999; Chen et al., 2013). Being a kind of incision enzyme, neutral proteases can be used for the hydrolysis of various proteins. They can break down the macromolecular proteins into amino acids, with the appropriate pH value from 4.0 to 11.0 (Xiao, 2006). Additionally, they have been widely used in food, medicine and feed industry for their effective enzymatic hydrolysis reaction rate, low industrial pollution and mild reaction condition. Neutral proteases are the earliest protein enzymes used in industrial production, and the representative one is neutral protease I produced by Aspergillus oryzae (Zhou 2010). The Pichia pastoris expression system offers economy, ease of manipulation, the ability to perform complex post-translational modifications, and high expression levels. However, little references have been found that using P. pastoris to degrade BCs before.
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In this experiment, we developed a recombinant P. pastoris strain, named pGAPZaA-NpI-GS115, for the high-level production of neutral protease. Two statistical methods including single-factor experiments and BoxeBehnken design (BBD; three factors and three levels) of RSM were employed to optimize the BCs fermentation conditions. 2. Materials and methods 2.1. Strains, vectors, enzymes, reagents, and growth medium All of the strains, vectors and enzymes used in this experiment are included in Table 1. A. oryzae 3.042 (CICC 2339) purchased from the China Center of Industrial Culture Collection (CICC) is an widely used industrial strain which was screened by UV mutagenesis for high-yield of protease. All of the culture medium and methods used for Pichia are described in the Pichia expression manuals (Cregg et al., 2009). All of the chemical reagents were of analyticalreagent grade. 2.2. DNA cloning of neutral protease I (NpI) from A. oryzae 2.2.1. RNA extraction For separation of RNA, A. oryzae 3.042 was cultured in potato extract glucose medium. 100 ml of culture medium was loaded into a 250-ml conical flask and autoclaved at 121 C for 20 min. Each flask was inoculated with 1.0 ml of the spore solution and shaken at 30 C, 120 rpm for 72 h. Through the above-described process, the fungal hyphae of A. oryzae, which was used for extracting RNA, turned to lots of small balls with a diameter of 3e8 mm (Guo and Ma, 2008). The fungal mycelia were collected and rapidly frozen in liquid nitrogen. The total RNA was extracted using the Fungal RNA Kit (OMEGA, USA). 2.2.2. One-step RT-PCR Reagents from the one-step RT-PCR kit (TaKaRa, Dalian, China) were used to prepare the master mix solutions according to the describe of Yang et al. (2013). Both RT and DNA polymerization were conducted in the same tube without the subsequent addition of enzymes or buffer. The PCR program was 50 C for 30 min, 94 C for 2 min, 30 cycles of 94 C for 30 s and 55 C for 30 s, and a final extension at 72 C for 2 min using two complementary deoxyoligonucleotides (oligos): A (forward), 50 -CTCGAGATGCGGGGTCTTCTACTAGCTG-30 ; B (reverse), 50 -GCGGCCGCGAATTCGAAGCGGCGGAC-30 (the restriction sites XhoI (CTCGAG) and NotI (GCGGCCGC) are shown in italics in the oligos). The PCR products were detected by electrophoresing through 1% agarose gels in 1 TAE buffer (40 mM Triseacetate (pH 8.0) and 1 mM ethylenediaminetetraacetic acid). The purified PCR product was ligated into the cloning vector pMD-18T. After transformation of the vector into Escherichia coli DH5a competent cells, the transformants (pMD-18T-NpI) were screened on LB plates (1% tryptone, 1% NaCl, 0.5% yeast extract, and 1.5% agar, pH 7.0) containing 50 mg/ml ampicillin. The pMD-18T-NpI plasmid was identified through restriction enzyme analysis and then sequencing by Sangon (Shanghai, China). Table 1 Strains, vectors and enzymes used in this experiment. Strains, vectors and enzymes
Source
Aspergillus oryzae strain 3.042 Escherichia coli strain DH5a P. pastoris strain GS115 The expression vector pMD-18T The expression vector pGAPZaA Enzymes
Preserved in our laboratory Purchased from Takara (Dalian, China) Purchased from Invitrogen Purchased from Takara (Dalian, China) Purchased from Invitrogen Purchased from Takara (Dalian, China)
2.3. Construction of the expression vectors The pMD-18T cloning vector and the pGAPZaA plasmid were digested with the restriction enzymes XhoI and NotI and then ligated with T4 DNA ligase. The recombinant plasmids were transformed into E. coli DH5a, and Zeocin™-resistant transformants were screened on low-salt Luria-Bertani (LLB) agar plates containing 25 mg/ml Zeocin™. The DNA plasmid was prepared, and the insert fragments were identified by restriction enzyme digestion (XhoI and Not I) and further confirmed by sequencing (Sangon Biotech, Shanghai, China). 2.4. Transformation and screening of P. pastoris expression strains The pGAPZaA-NpI recombinant plasmid and pGAPZaA plasmid (negative control) were linearized using the SacI restriction enzyme. The linearized plasmid and GS115 competent cells were mixed and transferred to an ice-cold 0.2 cm electroporation cuvette. The mixture was incubated on ice for 5 min and pulsed with a MicroPulser (Bio-Rad, Hercules, CA, USA; conditions: 2.5 kV, 200 U, and 25 mF). 1 ml of ice-cold 1 M sorbitol was added immediately to the cuvette. The cuvette contents were transferred to a sterile 15-ml tube and incubated without shaking at 30 C for 2 h. The cells were plated on YPDZ agar plates (1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar, and 100 mg/ml Zeocin™) for 3e4 days at 30 C until colonies formed. 2.5. Expression of NpI gene in P. pastoris The positive P. pastoris transformants (named pGAPZaA-NpIGS115) were cultured at 200 rpm for 12e16 h in a shaking flask containing 10 ml YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) at 30 C and then expanded into 100 ml of YPD medium cultured at 200 rpm for 96 h in a shaking flask at 30 C. For screening of the neutral protease productivity (a vector without any neutral protease gene insert was used as a control), the neutral protease in the culture supernatants was monitored by measuring the expressed recombinant neutral protease by SDSePAGE. 2.6. Screening of pGAPZaA-NpI-GS115 based on the neutral protease activity The neutral protease activity was assayed by the casein FolineCiocalteu method described by Oda and Murao (1974). 1 ml of the enzyme solution was added to 1 ml of 1.14% casein solution in a 0.1 M Na2HPO4eNaH2PO4 buffer with a pH of 7.5. After incubation at 40 C for 10 min, the reaction was stopped by the addition of 2.0 ml of 0.44 M trichloroacetic acid followed by centrifugation at 12,000 g for 10 min. The 1 ml supernatant was mixed with 5 ml of 0.4 M sodium carbonate and 1 ml of the FolineCiocalteu reagent. After incubation at 40 C for 20 min, the optical density was measured at 680 nm. One unit of enzyme activity was defined as the enzyme quantity that liberates 1 mg of tyrosine per ml of the reaction mixture per min. The positive transformant that expressed the highest neutral protease level was selected and used for the time-course studies. 2.7. Optimized conditions for fermenting blood cells using pGAPZaA-NpI-GS115 2.7.1. Degradation of BCs Liquid-state fermentation (LSF) was conducted in a 250-ml flask that contained 3 g of wheat bran, 3 g of blood cells, and 100 ml of buffered solution. The pH of the medium was adjusted to 7.5. The prepared medium was used as the basic substrate for the pGAPZaANpI-GS115.
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The inoculated medium was incubated at 30 C for 120 h and then incubated in boiling water for 15 min to inactivate the enzyme. The mixtures in the flasks were then removed by filtration using filter paper. The filtrate was diluted to 100 ml with distilled water to determine the degree of hydrolysis (DH). 2.7.2. Single-factor experiments The purpose of single-factor experiments was to determine the fermentation conditions individually, such as temperature, time, and pH, to measure their contribution toward the DH. 2.7.3. Response surface methodology A three-level, three-variable BBD (Design-Expert v. 8.0) was applied to determine the best combination of fermentation variables to maximize the DH of the BCs. Based on the single-factor experiments, the initial conditions were a temperature of 34 C, a time duration of 108 h, and a pH of 8.0. Table 2 lists the BBD matrix and the response values that were used to develop the model. The response value obtained from each trial is an average of triplicates. 2.7.4. Degree of hydrolysis The degree of hydrolysis was presented as the percentage of cleaved peptide bonds:
DH ð%Þ ¼
h 100; htot
(1)
where h (hydrolysis equivalents) is the number of hydrolyzed bonds, which is expressed as millimolar equivalents per gram of protein (mmol/g of protein), and htot is the total amount of peptide bonds per protein equivalent, which can be determined from the amino acid composition and is 8.62 mmol/g for BCs. The amino nitrogen content was determined using a modified ninhydrin colorimetric method (Zheng et al., 2014). 2.8. Statistical analysis The data obtained from the BBD design were fitted with a second-order polynomial equation as follows:
Y ¼ b0 þ
2 X
bi Xi þ
i¼1
2 X
bii Xi2 þ
i¼1
X X
bij Xi Xj ;
(2)
i¼1 j¼iþ1
where Y is the predicted response, b0 is a constant, bi is a linear coefficient, bii is a quadratic coefficient, bij is an interaction coefficient, and Xi and Xj are independent variables. The statistical significance of the model and the model variables were determined at probability values (P) of 0.05 and 0.01. 3. Results and discussion 3.1. Cloning of the NpI gene from A. oryzae, construction of expression plasmid, and screening of recombinant transformants The NpI gene which encoding the neutral protease of A. oryzae strain 3.042 was amplified by RT-PCR using the reverse transcription product as the template and cloned into the pMD-18T vector. Table 2 Factor levels for pGAPZaA-NpI-GS115 that were used to optimize the DH. Code
pH (A)
Time (B, h)
Temperature (C, C)
1 0 1
7 8 9
96 108 120
31 34 37
237
The pMD-18T-NpI recombinant plasmid was identified by restriction enzyme analysis and DNA sequencing. The sequence data were identical to the sequence published in GenBank (Accession No. AF099904.1). The correct DNA fragment was used to construct the expression vector. Fig. 1 explains how the expression vector was constructed. The correct DNA fragment was inserted into the pGAPZaA plasmid to construct the recombinant vector. The recombinant vector was identified by XhoI and NotI digestion, and the corresponding fragment (1905 bp) was obtained (Fig. 2). The target fragment was confirmed by gene sequencing to be the same as the original sequence of the NpI gene in GenBank, and the recombinant vector was denoted pGAPZaA-NpI. P. pastoris strain GS115 was transformed with the pGAPZaA-NpI linearized plasmid. After incubation on YPD plates with Zeocin™ (100 mg/ml) for three days at 30 C, approximately 80 colonies were formed. The pGAPZaA vector without any insert was also transformed into P. pastoris GS115 and used as a control. 3.2. Screening of pGAPZaA-NpI-GS115 based on the neutral protease activity All of the colonies were cultured in YPD medium to assay the neutral protease activity, and the clone with the highest expression level of neutral protease was selected for the time-course studies. The NpI gene was expressed in P. pastoris and had the expected size of 46 kDa, as verified by SDSePAGE (Fig. 3). 3.3. Effects of pH on the DH of BCs As shown in Fig. 4(a) (fixed levels: blood cells ¼ 3 g, temperature ¼ 34 C, time ¼ 120 h), the peak DH of pGAPZaA-NpIGS115 appeared at the pH value of 8.0. The pH value can play an important role in controlling the expression of many genes of fungi (Penalva et al., 2008). In this study, the DH increased gradually to the optimal level and then exhibited a minor decrease due to enzyme production, which is similar to the results reported by Gautam et al. (2011) and Bansal et al. (2012). 3.4. Effects of time on the DH of BCs As shown in Fig. 4(b) (fixed levels: blood cells ¼ 3 g, temperature ¼ 34 C, initial pH ¼ 7.5), the peak DH values of pGAPZaA-NpI-GS115 appeared at 108 h. The evaluation of the time to peak is of prime importance for enzyme biosynthesis by fungi (Kuhad and Singh, 1993). Upon further incubation, the enzyme yields declined gradually due to the release of protease and a decrease in the pH of the medium (Yao et al., 2012), which may be due to the depletion of nutrients and the accumulation of other byproducts, such as protease, in the fermentation medium (Romero et al., 1998). 3.5. Effects of temperature on the DH of BCs As shown in Fig. 4(c) (fixed levels: blood cells ¼ 3 g, time ¼ 120 h, initial pH ¼ 7.5), the peak DH of pGAPZaA-NpI-GS115 appeared at a temperature of 34 C. As reported, the optimum growth temperature for P. pastoris GS115 is 28e30 C, and higher temperatures (above 40 C) altered the cell membrane composition and simulated protein catabolism, causing cell death (Cregg et al. 2009). A study conducted by Yang and Qiu (2006) investigated a temperature range from 30 to 60 C and found that 55 C resulted in the highest neutral protease activity. The optimal temperature for DH was between the optimal growth temperature for P. pastoris GS115 and the optimal temperature for neutral protease activity,
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Fig. 1. Map of construction of the expression vector pGAPZaA-NpI.
Fig. 2. Enzyme digestion of the pGAPZaA-NpI recombinant plasmid. Lane M1: DL5000 DNA marker; Lane 1: pGAPZaA-NpI recombinant plasmid; Lane 2: product from the digestion of pGAPZaA-NpI with XhoI; Lane 3: product from the digestion of pGAPZaANpI with NotI; Lane 4: product from the digestion of pGAPZaA-NpI with XhoI and NotI; Lane M2: DL10000 DNA marker.
Fig. 3. SDSePAGE analyses of yeast cultures expressing secreted NpI. Lane M: protein molecular weight marker; Lane 1: sample from the negative control P. pastoris; Lanes 2e7: samples from NpI-expressing P. pastoris.
H. Zhang et al. / International Biodeterioration & Biodegradation 93 (2014) 235e240
Fig. 4. Effects of pH (a), time (b), and temperature (c) on the DH.
and this finding can be attributed to the combined effect of both the growth temperature for P. pastoris and the optimal temperature for neutral protease activity.
239
of the model. The P value for model was significant (P < 0.01) and the lack of fit was not significant (P > 0.05), confirming the validity of the model. The model was found to be adequate for predictions within the range of the experimental variables. The values of the coefficients of Eq. (3) were calculated and their significance tested using the Design-Expert v. 8.0 software; these are listed in Table 4. Each P value was used as a tool to determine the significance of each coefficient, which, in turn, may indicate the pattern of the interactions between the variables. As observed in Table 4, the quadratic term coefficients (X2) was significant with P values at the 0.01 level, the linear coefficient (X1 * X3) was significant with a P value at the 0.05 level, and the quadratic term coefficient (X1, X3 and X2) were significant with a P value at the 0.01 and 0.05 level respectively. The other coefficients (X1, X3, X1 * X2, and X2 * X3) were not significant (P > 0.05). The cleavage of the peptide bonds of the protein substrate has been found to be markedly influenced by the conditions under which the substrate is hydrolyzed with bacterial proteases (Box et al., 1978; Wang and Xu, 2007; Zhu and Row, 2011; Ovissipour et al., 2012). In this study, after the proteases were produced from the recombinant pGAPZaA-NpI-GS115, the cleavage of the peptide bonds of the BCs was influenced by the fermentation conditions. The adjusted determination coefficient ðR2Adj Þ was used as the correlation measure to test the goodness of fit of the regression equation. The value of R2Adj (0.9269) for Eq. (3) was reasonably close to 1. These results indicate a high degree of correlation between the experimental and predicted values. Good correlations between the experimental results and the results predicted by RSM models of proteolytic reactions have been rerez-Ga lvez et al., ported by several researchers (Chen et al., 2010; Pe 2011; Zheng et al., 2012). The very low value of the coefficient of variation (C.V.) (1.84% for pGAPZaA-NpI-GS115) clearly indicates the very high degree of precision and reliability of the experimental values. The optimal conditions indicated by RSM are pH ¼ 8.1, time ¼ 116.6 h, and temperature ¼ 33.9 C with a desirability DH of 49.46%. Under the abovementioned conditions, the experimental DH of fermented blood cells with pGAPZaA-NpI-GS115 is 49.05%, which is much higher than 35.13% and 32.91% that reported by Zheng et al. (2012, 2014), respectively. Table 3 The DH of pGAPZaA-NpI-GS115 at various pH values, temperatures, and times.
3.6. Optimization of the fermentation conditions for DH
Run
The influence of the factors on the DH of pGAPZaA-NpI-GS115 was determined using the factorial design described in the previous section. The best explanatory model equations for the DH values for pGAPZaA-NpI-GS115 obtained from the uncoded fermentation data are described in Eq. (3):
DH ¼ 740:03728 þ 75:57892 pH þ 1:55667 Time þ 23:12006 Temperature þ 0:022292 pH Time þ 0:33667 pH Temperature þ 3:05556E 003 Time Temperature 4:11725 pH2 7:89757E 003 Time2 0:30553 Temperature2 : (3) The experimental and predicted values for the DH of pGAPZaANpI-GS115 under various combinations of the independent variables are presented in Table 3. The results indicate that the DH for pGAPZaA-NpI-GS115 ranged from 40.76% to 49.40% depending on the experimental conditions. The statistical testing of the model was performed using ANOVA, which was required to test the significance and adequacy
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 a
Code values
DHd
Real values
X1
X2
X3
X1a
X2b
X3c
Experimental
0 0 1 1 0 1 1 1 1 1 1 0 0 0 0 0 0
1 1 0 1 1 1 1 1 0 0 0 1 0 0 0 0 0
1 1 1 0 1 0 0 0 1 1 1 1 0 0 0 0 0
8 8 9 7 8 9 9 7 7 7 9 8 8 8 8 8 8
120 96 108 120 120 120 96 96 108 108 108 96 108 108 108 108 108
37 31 31 34 31 34 34 34 37 31 37 37 34 34 34 34 34
46.91 43.32 43.26 43.55 46.44 46.77 43.19 41.04 42.81 41.27 40.76 43.35 48.50 48.33 49.01 49.40 49.22
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.58 0.53 0.49 0.56 0.25 0.33 0.46 0.29 0.12 0.58 0.35 0.44 0.17 0.34 0.27 0.22 0.51
Predicted 46.97 43.26 43.53 43.76 46.23 46.71 42.98 41.10 41.65 41.27 41.65 43.56 48.89 48.89 48.89 48.89 48.89
X1 ¼ pH. X2 ¼ Time. c X3 ¼ Temperature. d DH represents the average degree of hydrolysis of triplicate experiments. The DH was calculated using the equation Y ¼ 0.98A570 0.01 (R2 ¼ 0.997), which was derived from the standard curve of completely hydrolyzed BCs (absorbance at 570 nm versus the concentration of the hydrolyzate). b
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Table 4 ANOVA of the response surface quadratic polynomial model of pGAPZaA-NpI-GS115. Source
Sum of squares df
Model 146.65 X1epH 3.52 X2eTime 20.38 X3eTemperature 0.026 0.29 X1X2 X1X3 4.08 X2X3 0.048 X12 71.38 X22 5.45 X32 31.84 Residual 4.84 Lack of fit 4.00 Pure error 0.85 Cor total 151.49 Std. dev. Mean C.V. % PRESS
0.83 45.13 1.84 65.26
9 1 1 1 1 1 1 1 1 1 7 3 4 16
Mean square
F-value P-value
16.29 3.52 20.38 0.026 0.29 4.08 0.048 71.38 5.45 31.84 0.69 1.33 0.21 e
23.54 5.09 29.45 0.038 0.41 5.90 0.07 103.13 7.87 46.00 e 6.27 e e
R-squared Adj R-squared Pred R-squared Adeq precision
0.0002 0.0586 <0.0010 0.8506 0.5407 0.0455 0.7991 <0.0001 0.0263 0.0003 e 0.0541 e e
***a ***a
**b ***a **b ***a
0.9680 0.9269 0.5693 13.295
df, degrees of freedom. a Significance at the 0.01 level. b Significance at the 0.05 level.
4. Conclusions In this work, a recombinant P. pastoris pGAPZaA-NpI-GS115 was successfully constructed, and under the indicated conditions, the experimental DH of fermented blood cells with this recombinant is 49.05%. The blood cells degradation result indicates that future application of pGAPZaA-NpI-GS115 in the conversion of waste blood generated by the livestock industry into protein hydrolyzates is very promising; the resulting protein hydrolyzates could be utilized as animal feed or nitrogen fertilizers. Such technology represents a valuable approach with two intrinsic advantages, namely, the recycling of agro-industrial residues and the concomitant aggregation of value to these inexpensive raw materials. Conflict of interest The authors declare that they have no conflicts of interest related to this work. Acknowledgments This project was financially supported by the National Basic Research Program (Grant No. 2012CB124703), the National Key Technology R&D Program (2013BAD10B03), the China Agriculture Research System (CARS-36), and the Program for Innovative Research Team of Universities in Heilongjiang Province (2012TD003). References Bansal, N., Tewari, R., Soni, R., Soni, S.K., 2012. Production of cellulases from Aspergillus niger NS-2 in solid state fermentation on agricultural and kitchen waste residues. Waste Manag. 32, 1341e1346. Box, G.E.P., Hunter, J.S., Hunter, W.G., 1978. Statistics for Experimenters: an Introduction to Design, Data Analysis, and Model Building. Wiley, New York, pp. 245e255. Chen, H.C., Ju, H.Y., Wu, T.T., Liu, Y.C., Lee, C.C., Chang, C., Chung, Y.L., Shieh, C.J., 2010. Continuous production of lipase-catalyzed biodiesel in a packed-bed reactor: optimization and enzyme reuse study. BioMed Res. Int. 2011. Chen, Q., Zhang, H., Zheng, Y., Shan, A., Bi, Z., 2013. Effects of enzymatically hydrolyzed blood cells on growth performance and intestinal characteristics of newly weaned piglets. Livestock Sci. 157, 514e519. Cregg, J.M., Tolstorukov, I., Kusari, A., Sunga, J., Madden, K., Chappell, T., 2009. Expression in the yeast Pichia pastoris. Meth. Enzymol. 463, 169e189.
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