1 s2.0 S2589014X19301586 Mainext
1 s2.0 S2589014X19301586 Mainext
1 s2.0 S2589014X19301586 Mainext
A R T I C LE I N FO A B S T R A C T
Keywords: Engineered C. utilis strains were constructed to express three exogenous proteases, carboxypeptidase pepF,
Fermented mash of food waste Proteases aminopeptidase YPDF and endopeptidase pepA. These three proteases could be constitutively expressed and
Candida utilis secreted by the recombinant strains. The enzyme activities and the synergistic effect of three proteases were
Synergy investigated in hydrolysis of food waste fermentation mash. Synergism was apparently observed between en-
Enzymatic hydrolysis
dopeptidases and exopeptidases. The synergistic effect between two exopeptidases was less strong. The highest
amino acid yield was obtained by using P2A11, the combination of 15.5% pepF and 84.5% pepA. At 60 h, the
yield of amino acid by using P2A11 reached 23.9 mg/mL, and the protein hydrolysis rate was 51.2%. It was
73.8% of that by using 0.2% g/g commercial Novozymes Flavorzyme. This study is first focused on the fer-
mented mash of food waste, which was hydrolyzed into amino acid fertilizer by biological means, so as to
achieve the secondary utilization of waste.
1. Introduction compounds and nutrients in fermented mash of food waste (Tong et al.,
2018). It would also cause environmental pollution, as it is easily, can
Food waste refers to food waste produced in people's daily life and be destroyed by the native microflora acting as decomposers. Therefore,
food processing, which contains high organic and moisture content. The how to utilize the fermented mash of food waste and reduce environ-
amount of food waste is increasing rapidly as the population is growing mental pollution as far as possible is also a key problem. The fermen-
quickly and the Economy is developing fast (Li and Jin, 2015). The tation mash of kitchen waste contains a large number of nitrogen
major chemical components of kitchen waste are starch, protein, fat, compounds, such as protein, which can be explained to obtain amino
cellulose, and others; it has the characteristics of high water content, acids, the latter is pharmaceutical and biological feed raw materials,
high proportion of organic matter and high salt content. Therefore, has a certain economic value (Bahari et al., 2013; Ghasemi et al., 2014)
kitchen waste needs to be treated correctly and effectively to avoid .
environmental pollution (Wan et al., 1997; Kelley and Walker, 1999). Protein can be broken down into amino acid by acid, alkaline and
Bio-treatment is an ideal method to dispose organic waste or organic enzyme. Enzymatic hydrolysis does not produce racemization. The
pollution (Li et al., 2017). For example, anaerobic fermentation is a proteases are divided into two types: endopeptidase and exopeptidase
process of decomposing organic matter into methane and carbon di- according to their cleavage site. Endopeptidase act in the middle of the
oxide by anaerobic microorganism (Khan et al., 2018). In recent years, high molecular weight polypeptide chain and form a small molecular
there were more and more studies on the bio-production of high-value- weight peptide chain. Exopeptidase can be divided into carbox-
added product from food waste, such as biodiesel (Priyadarshi and ypeptidase and aminopeptidase, which cleave the amino acid residue
Paul, 2018), ethanol, volatile fatty acids, lactic acid, Welan gum, xan- from the free carboxyl end and the free amino end of the polypeptide
than gum, etc. (Li et al., 2017; Shen et al., 2017; Li et al., 2016; Darwin respectively. Usually, the deep hydrolysis of protein need the co-
et al., 2018). After the fermentation of carbohydrates in food waste, operation of several kinds of proteases (Zhang et al., 2013). Candida
there are still a considerable number of undegradable organic utilis has been commercially used in the production of food additives
⁎
Corresponding author at: Research Center for Molecular Biology, Institutes of Life and Health Engineering, College of Life Science and Technology, Jinan
University, Guangzhou 510632, PR China.
E-mail address: [email protected] (Z. Liu).
https://doi.org/10.1016/j.biteb.2019.100268
Received 22 May 2019; Received in revised form 21 June 2019; Accepted 22 June 2019
Available online 22 June 2019
2589-014X/ © 2019 Elsevier Ltd. All rights reserved.
T. Sun, et al. Bioresource Technology Reports 7 (2019) 100268
and nutritional feed supplements for > 70 years (Bzducha-Wrobel was carried out. The rDNA fragment was obtained from the genome of
et al., 2018). It not only has the advantages of acid resistance, protease C. utilis and was inserted into the plasmid pcGAPGA. The promoter
resistance, fast propagation speed, low culture conditions, but also has ScTEF1 and ScHXT7 fragments were inserted into the promoter region
the advantage of recognized safe. In this study, several different ami- of pcGAPGA respectively, then the pepF and YPDF fragments were in-
nopeptidases, carboxypeptidases and endoproteases were selected to serted into plasmids to form plasmids pcTEF1GA-rDNA-pepF,
express in Candida utilis, and the promoters of proteases were optimized pcHXT7GA-rDNA-pepF, pcTEF1GA-rDNA-YPDF, pcHXT7GA-rDNA-
to achieve the best expression efficiency. YPDF respectively.
Synergy means that different types of enzymes combine or act on The fragments of endoprotease gene were inserted into the vector of
different sites, and ultimately achieve the best results through mutual pcGAPGA and pcPGKGA-GFP respectively. The vector pcGAPGA-pepA
cooperation, and improve the production of products (Kostylev and and pcPGKZαA-pepA were obtained.
Wilson, 2014). But as the amount of enzyme increases, it is bound to YPDF expression cassette was inserted into the vector pcGAPGA-
increase the product. In this case, the yield of hydrolysis increases with pepA to obtain the two-gene co-expression vector GAP-pepA-HXT7-
the amount of enzyme protein. For a supplementary system, it is YPDF. Using ClonExpress II One Step Cloning Kit, pepF expression
meaningless. Synergistic effects usually refer to the efficiency of en- cassette was inserted into it to obtain three-gene co-expression vector
zyme release products at the same amount of mixture (Li et al., 2014). GAP-pepA-HXT7-YPDF-TEF1-pepF.
Therefore, synergy is often used to study the effectiveness of additional The expression plasmid containing promoter CuGAP was linearized
coenzyme. with SacI, plasmid containing promoter CuPGK was linearized with
This study focused on the fermented mash of food waste, which was NheI, plasmid containing promoter CuMAL was linearized with PstI, and
hydrolyzed into amino acid fertilizer by biological means, so as to plasmid containing promoter ScTEF1 and ScHXT7 were linearized with
achieve the secondary utilization of waste. It not only protects the en- ScaI. The linear DNA fragments were introduced into C. utilis to obtain
vironment, but also improves economic benefits. In the present work, the corresponding engineering strains using the lithium acetate and
by means of metabolic engineering and using the expression system of dithiothreitol method (Thompson et al., 1998).
Candida utilis, we constructed the engineering strains which can express
aminopeptidase, carboxypeptidase, and endoprotease separately, and 2.3. Enzymatic hydrolysis of food waste fermented mash
the strain which can co-express the three enzymes. After that, we in-
vestigated the synergistic effect among these three proteases and ob- In this study, the fermented mash of food waste was obtained from
tained the best ratio of the enzymes in hydrolysis of food waste fer- Guangdong Recyclean Low-Carbon Technology Co. Ltd. The fermented
mentation mash. The fermentation mash of food waste was degraded mash was a mixture of food waste after oil removal and ethanol fer-
into amino acid in depth, and it can be turned into amino acid rich mentation by waste yeast, the main components are shown in Table 1.
fertilizer. This study would help to achieve the goal of zero discharge of The fermented mash was adjusted to pH 6 with Na2HPO4 and separated
food waste utilization. into 50 g per bottle to sterilize. After cooling, the Novozyme flavorzyme
was loaded into the substrate at 0.1%, 0.2%, 0.4%, 0.6% and 0.8% g/g.
2. Methods The mixture was hydrolyzed at 45 °C for 72 h. Samples were collected
from the reaction mixture. Each sample from the hydrolysate was
2.1. Strains, plasmids and culture conditions centrifuged for 5 min at 12000 rpm. The supernatant was analyzed to
determine the amino acid yield using HPLC. According to the optimal
Yeast were cultured and fermented at 30 °C with 200 rpm in YPD amount of Novozyme flavorzyme, for synergistic experiment, the pro-
medium. Plasmids based on pcGAPGA were constructed in E. coli tein loading was also a constant value of 0.2% g/g substrate. The ratio
DH5α, which was cultured in LB medium at 37 °C with 200 rpm. A. of aminopeptidase, carboxypeptidase and endopeptidase was summar-
niger and B. coagulis were cultured in YPD medium. 50 μg/mL kana- ized and shown in Table 2, and enzyme hydrolysis was carried out at
mycin (Kan) were added to E.coli culture. The recombinant strains with 40 °C, 200 rpm. Samples (1 mL) were taken from the reaction mixture at
pcGAPGA plasmid were inoculated into yeast medium with 300 μg/ specific times. Each sample from the hydrolysate was heated in boiling
mL G418. water for 10 min to deactivate the enzymes and then cooled to room
temperature and subsequently centrifuged for 10 min at 12000 rpm.
2.2. Plasmid construction and yeast transformation The supernatant was removal of residual proteins and derivatized to
analysis amino acid output.
DNA manipulations were conducted following the standard proto-
cols (Green, 2012). In this study, nine protease genes were selected and The protein hydrolysis rate(%)
expressed in yeast strains. Among them, one carboxypeptidase gene, = amino acid content/Total protein content
named pepF (van den Hombergh et al., 1994), was cloned from As-
pergillus Niger genome by overlapping extension PCR. Two other car-
boxypeptidase genes, named Bc-S1 and Bc-S2, were obtained from the 2.4. Detection of amino acids
genome of Bacillus coagulans. Five aminopeptidase genes, named YPDF,
AP1, AP2, APC and APS, were cloned from the genome of Bacillus Samples were removed soluble protein by sulfonic salicylic acid
coagulans. The DNA fragment of endoprotease gene pepA (Archer, method. Amino acids were derivatized by PITC before they can be
1992) which has been modified in the previously study was from As-
pergillus niger. All cloned DNA fragments were cloned into a shuttle Table 1
plasmid pcGAPGA to construct the expression plasmids pcGAPGA-X Characteristics of fermented mash of food waste used in the experi-
respectively. (X represents the name of the inserted DNA fragment). ments.
In order to improve the expression efficiency of proteases, several Parameters Contenta (w/w, %)
effective promoters were used. Promoter PGK and MAL fragments were
inserted into pcGAPGA respectively, Then the pepF and YPDF frag- pH 3–4
Moisture 91.7% ± 1.8
ments were linked into plasmids by ClonExpress II One Step Cloning Kit Residual protein content 64.6% ± 0.5
to obtain plasmids pcPGKGA-pepF, pcMALGA-pepF, pcPGKGA-YPDF,
and pcMALGA-YPDF (Miyano et al., 2018). All values are based on wet mass. Values are given as means ±
The construction of a plasmid containing TEF1 or HXT7 promoter standard deviation (n = 3).
2
T. Sun, et al. Bioresource Technology Reports 7 (2019) 100268
In order to improving the expression efficiency and the activity of 3.4. Hydrolysis rate of protein
protease, several strong promoters in previously studies were used to
match with different target genes. The recombinant expression vectors When the hydrolysis time was 60 h, comparing with the single
3
T. Sun, et al. Bioresource Technology Reports 7 (2019) 100268
protease groups, protein hydrolysis rate of the synergistic groups were 4. Discussion
all increased, except Co-expression. Among them, the amino acid yield
of P2A11 was the highest. The protein hydrolysis rate of 0.2% g/g Fla. The composition of food waste is complex, and it is difficult to se-
was 71.5%. The hydrolysis rate of P2A11 was 51.2% and was 81.5% of parate the components. So far, fermenting food waste and producing
that by 0.2% Flavorzyme, The hydrolysis rates of Y10A3, P10Y3 and X9Y4 high value-added output is one of the effective ways to utilize it.
were similar. The hydrolysis rates of Co-expression were lower than However, the output of fermentation mash is large, and it contains a lot
YPDF, and need further improvement (Fig. 5.). of protein. How to utilize it and reduce environmental pollution as far
as possible is also a key problem to be solved. In this study, engineering
yeasts were constructed to degrade the protein in fermentation mash
3.5. Fermented mash of food waste hydrolysis curve and produce amino acid fertilizer. Therefore, the fermentation mash of
food waste could be fully utilized, and the purpose of graded utilization
The time course of amino acids production from fermented mash of of food waste could be achieved.
food waste by using P2A11 and 0.2% Flavorzyme were conducted. It can In this study, C. utilis was used to express exogenous protease genes
be seen that the content of amino acid increased with the increase of for its better acid and protease resistance. Aspergillus can secrete a
hydrolysis time from 12 to 60 h. At 72 h, the content of amino acids variety of proteases with high enzymatic activity. Bacillus coagulans,
produced by using P2A11 and 0.2% Flavorzyme all tended to be stable. which can grow under high temperature and acidic conditions, also
Compared with 0.2% fla protease, the hydrolysis rate of P2A11 was secretes various proteases. Therefore, this study transferred the pro-
lower. At 60 h, the amino acid content of P2A11 was 21.4 mg/mL and tease genes from Aspergillus and Bacillus coagulans into C. utilis and
reached 73.8% of that by using 0.2% Flavorzyme, which was 29 mg/mL constructed the engineering strains which can express three proteases
(Fig. 6.). separately and the strain which can co-express the three enzymes.
Enzyme activity analysis confirmed that all genes were successfully
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T. Sun, et al. Bioresource Technology Reports 7 (2019) 100268
integrated into the genome of C. utilis and could be stably expressed and
secreted into culture supernatants. The enzyme activity of recombined
YPDF was lower than that of pepF and pepA. Compared with single
gene expression strains, the enzyme activity of three gene Co-expression
strains was slightly increased, but was not significant. It would be that
the introduction of three exogenous genes made the strain overload.
The effects of pH and temperature on the enzyme activities of pepF,
YPDA and pepA were analyzed. All enzymes maintained high activities
at pH 3–5, a condition in same with the characteristics of fermented
mash of food waste (pH 3–4). Then the proteases will work more effi-
ciently.
The residual proteins in fermentation mash of food waste can be
Fig. 4. Synergistic effect of pepF and pepA (A and B), YPDF and pepA (C and
effectively hydrolyzed by Novozymes Flavorzyme. Under the action of
D), pepF and YPDF (E and F), X and YPDF (G and H) on fermentation mash of
0.2% g/g Flavorzyme, the protein hydrolysis rate reaches 71.5%. food waste. (X representative P2A11), The corresponding hydrolysis time for (A,
However, when the concentration of Flavorzyme was increased to 0.8% C, E, G), (B, D, F, H) is 12 h, and 60 h respectively. Values with different letters
g/g, the amino acid yield did not increase significantly. The reason may within the figures indicate significant differences (p < 0.05). n = 2.
be that with the increase of enzyme amount, the prolongation of en-
zymatic hydrolysis time or the decrease of acidic polypeptide, the
et al., 2012).
phenomena of resistance to flavorzyme protease will appear, similar
In this study, the synergistic effect of three proteases in hydrolyzing
phenomena occurred in the hydrolysis of soybean by flavorzyme pro-
the fermentation mash of food waste was investigated. For synergistic
tease (Kanu et al., 2009) and soybean meal by alkaline protease (Zhao
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T. Sun, et al. Bioresource Technology Reports 7 (2019) 100268
Fig. 4. (continued)
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T. Sun, et al. Bioresource Technology Reports 7 (2019) 100268
The time course of amino acids yields was investigated. The amino enzyme molecules, and indirectly affect the hydrolysis efficiency
acids content by using P2A11 was lower than that by using 0.2% g/g (Kamnerdpetch et al., 2007). Furthermore, heat treatment can trans-
Flavorzyme all the time. After hydrolyse for 60 h, the amino acids yield form proteins into open polymers and make proteases bind with sub-
of P2A11was 23.85 mg/mL, and was 73.8% of that by using 0.2% g/g strate more easily, it will help to promote the hydrolysis of protein
Flavorzyme. With the prolongation of hydrolysis time, the increases of (Agboola and Dalgleish, 1996; Turgeon et al., 1992). In addition, ul-
amino acid content were all slow down. It would be that with the in- trasound was an effect-assist (Ma et al., 2011). Therefore, more assis-
crease of content of short peptide in reaction system, some hydrophobic tant treatment should be investigated to promote the hydrolysis effi-
amino acids were exposed, and the emulsifying ability of solution in- ciency in the future.
creased, then the stereoscopic repulsion produces stability that affects
the interaction between enzymes and substrate molecules (Michael 5. Conclusion
Fountoulakis, 1998).
In further study, the mass fraction of the substrate should be opti- In this study, the metabolic engineering method was used to con-
mized. Because the viscosity of substrate will affect the diffusion of struct engineering C. utilis strains which can produce proteases for
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T. Sun, et al. Bioresource Technology Reports 7 (2019) 100268
degradation of fermentation mash of food waste. Synergism was ob- pulp protein hydrolyzation process by the combination of protease enzyme systems.
served among endopeptidase, carboxypeptidase and aminopeptidase. Enzym. Microb. Technol. 40 (4), 508–514.
Kanu, P.J., Kanu, J.B., Sandy, H., E, B.A., Kande, J., Mornya, P.M.P., Huiming, Z., 2009.
Comparing with the single protease, the synergistic of pepF and pepA Optimization of enzymatic hydrolysis of defatted sesame flour by different proteases
increased the yield of amino acid significantly. The hydrolysis rate of and their effect on the functional properties of the resulting protein hydrolysate. Am.
P2A11 was 51.2% at 60 h when using the fermentation mash of food J. Food Technol. 4 (6), 226–240.
Kelley, T.R., Walker, P.M., 1999. Bacterial concentration reduction of food waste
waste as substrate. amended animal feed using a single-screw dry-extrusion process. Bioresour. Technol.
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substrate. Carbohydr. Polym. 151, 684–691.
Supplementary data to this article can be found online at https:// Li, P., Xie, Y., Zeng, Y., Hu, W., Kang, Y., Li, X., Wang, Y., Xie, T., Zhang, Y., 2017.
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