i An update to this article is included at the end

Bioresource Technology Reports 7 (2019) 100268

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Bioresource Technology Reports

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Producing amino acid fertilizer by hydrolysis of the fermented mash of food T

waste with the synergy of three proteases expressed by engineered Candida
utilis
⁎
Tao Suna, Wenjuan Xiaoa, Cuifeng Jianga, Jue Wanga, Zehuan Liua,b,
a
Research Center for Molecular Biology, Institutes of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, PR China
b
Guangdong Recyclean Low-Carbon Technology Co. Ltd., PR China

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
Food waste refers to food waste produced in people's daily life and
food processing, which contains high organic and moisture content. The
amount of food waste is increasing rapidly as the population is growing
quickly and the Economy is developing fast (Li and Jin, 2015). The
major chemical components of kitchen waste are starch, protein, fat,
cellulose, and others; it has the characteristics of high water content,
high proportion of organic matter and high salt content. Therefore,
kitchen waste needs to be treated correctly and effectively to avoid
environmental pollution (Wan et al., 1997; Kelley and Walker, 1999).
Bio-treatment is an ideal method to dispose organic waste or organic
pollution (Li et al., 2017). For example, anaerobic fermentation is a
process of decomposing organic matter into methane and carbon di-
oxide by anaerobic microorganism (Khan et al., 2018). In recent years,
there were more and more studies on the bio-production of high-value-
added product from food waste, such as biodiesel (Priyadarshi and
Paul, 2018), ethanol, volatile fatty acids, lactic acid, Welan gum, xan-
than gum, etc. (Li et al., 2017; Shen et al., 2017; Li et al., 2016; Darwin
et al., 2018). After the fermentation of carbohydrates in food waste,
there are still a considerable number of undegradable organic
compounds and nutrients in fermented mash of food waste (Tong et al.,
2018). It would also cause environmental pollution, as it is easily, can
be destroyed by the native microflora acting as decomposers. Therefore,
how to utilize the fermented mash of food waste and reduce environ-
mental pollution as far as possible is also a key problem. The fermen-
tation mash of kitchen waste contains a large number of nitrogen
compounds, such as protein, which can be explained to obtain amino
acids, the latter is pharmaceutical and biological feed raw materials,
has a certain economic value (Bahari et al., 2013; Ghasemi et al., 2014)
.
Protein can be broken down into amino acid by acid, alkaline and
enzyme. Enzymatic hydrolysis does not produce racemization. The
proteases are divided into two types: endopeptidase and exopeptidase
according to their cleavage site. Endopeptidase act in the middle of the
high molecular weight polypeptide chain and form a small molecular
weight peptide chain. Exopeptidase can be divided into carbox-
ypeptidase and aminopeptidase, which cleave the amino acid residue
from the free carboxyl end and the free amino end of the polypeptide
respectively. Usually, the deep hydrolysis of protein need the co-
operation of several kinds of proteases (Zhang et al., 2013). Candida
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: zhliu@jnu.edu.cn (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.
and nutritional feed supplements for > 70 years (Bzducha-Wrobel
et al., 2018). It not only has the advantages of acid resistance, protease
resistance, fast propagation speed, low culture conditions, but also has
the advantage of recognized safe. In this study, several different ami-
nopeptidases, carboxypeptidases and endoproteases were selected to
express in Candida utilis, and the promoters of proteases were optimized
to achieve the best expression efficiency.
Synergy means that different types of enzymes combine or act on
different sites, and ultimately achieve the best results through mutual
cooperation, and improve the production of products (Kostylev and
Wilson, 2014). But as the amount of enzyme increases, it is bound to
increase the product. In this case, the yield of hydrolysis increases with
the amount of enzyme protein. For a supplementary system, it is
meaningless. Synergistic effects usually refer to the efficiency of en-
zyme release products at the same amount of mixture (Li et al., 2014).
Therefore, synergy is often used to study the effectiveness of additional
coenzyme.
This study focused on the fermented mash of food waste, which was
hydrolyzed into amino acid fertilizer by biological means, so as to
achieve the secondary utilization of waste. It not only protects the en-
vironment, but also improves economic benefits. In the present work,
by means of metabolic engineering and using the expression system of
Candida utilis, we constructed the engineering strains which can express
aminopeptidase, carboxypeptidase, and endoprotease separately, and
the strain which can co-express the three enzymes. After that, we in-
vestigated the synergistic effect among these three proteases and ob-
tained the best ratio of the enzymes in hydrolysis of food waste fer-
mentation mash. The fermentation mash of food waste was degraded
into amino acid in depth, and it can be turned into amino acid rich
fertilizer. This study would help to achieve the goal of zero discharge of
food waste utilization.
2. Methods
2.1. Strains, plasmids and culture conditions
Yeast were cultured and fermented at 30 °C with 200 rpm in YPD
medium. Plasmids based on pcGAPGA were constructed in E. coli
DH5α, which was cultured in LB medium at 37 °C with 200 rpm. A.
niger and B. coagulis were cultured in YPD medium. 50 μg/mL kana-
mycin (Kan) were added to E.coli culture. The recombinant strains with
pcGAPGA plasmid were inoculated into yeast medium with 300 μg/
mL G418.
2.2. Plasmid construction and yeast transformation
DNA manipulations were conducted following the standard proto-
cols (Green, 2012). In this study, nine protease genes were selected and
expressed in yeast strains. Among them, one carboxypeptidase gene,
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
genome of Bacillus coagulans. Five aminopeptidase genes, named YPDF,
AP1, AP2, APC and APS, were cloned from the genome of Bacillus
coagulans. The DNA fragment of endoprotease gene pepA (Archer,
1992) which has been modified in the previously study was from As-
pergillus niger. All cloned DNA fragments were cloned into a shuttle
plasmid pcGAPGA to construct the expression plasmids pcGAPGA-X
respectively. (X represents the name of the inserted DNA fragment).
In order to improve the expression efficiency of proteases, several
effective promoters were used. Promoter PGK and MAL fragments were
inserted into pcGAPGA respectively, Then the pepF and YPDF frag-
ments were linked into plasmids by ClonExpress II One Step Cloning Kit
to obtain plasmids pcPGKGA-pepF, pcMALGA-pepF, pcPGKGA-YPDF,
and pcMALGA-YPDF (Miyano et al., 2018).
The construction of a plasmid containing TEF1 or HXT7 promoter
2
Bioresource Technology Reports 7 (2019) 100268
was carried out. The rDNA fragment was obtained from the genome of
C. utilis and was inserted into the plasmid pcGAPGA. The promoter
ScTEF1 and ScHXT7 fragments were inserted into the promoter region
of pcGAPGA respectively, then the pepF and YPDF fragments were in-
serted into plasmids to form plasmids pcTEF1GA-rDNA-pepF,
pcHXT7GA-rDNA-pepF, pcTEF1GA-rDNA-YPDF, pcHXT7GA-rDNA-
YPDF respectively.
The fragments of endoprotease gene were inserted into the vector of
pcGAPGA and pcPGKGA-GFP respectively. The vector pcGAPGA-pepA
and pcPGKZαA-pepA were obtained.
YPDF expression cassette was inserted into the vector pcGAPGA-
pepA to obtain the two-gene co-expression vector GAP-pepA-HXT7-
YPDF. Using ClonExpress II One Step Cloning Kit, pepF expression
cassette was inserted into it to obtain three-gene co-expression vector
GAP-pepA-HXT7-YPDF-TEF1-pepF.
The expression plasmid containing promoter CuGAP was linearized
with SacI, plasmid containing promoter CuPGK was linearized with
NheI, plasmid containing promoter CuMAL was linearized with PstI, and
plasmid containing promoter ScTEF1 and ScHXT7 were linearized with
ScaI. The linear DNA fragments were introduced into C. utilis to obtain
the corresponding engineering strains using the lithium acetate and
dithiothreitol method (Thompson et al., 1998).
2.3. Enzymatic hydrolysis of food waste fermented mash
In this study, the fermented mash of food waste was obtained from
Guangdong Recyclean Low-Carbon Technology Co. Ltd. The fermented
mash was a mixture of food waste after oil removal and ethanol fer-
mentation by waste yeast, the main components are shown in Table 1.
The fermented mash was adjusted to pH 6 with Na2HPO4 and separated
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.
The mixture was hydrolyzed at 45 °C for 72 h. Samples were collected
from the reaction mixture. Each sample from the hydrolysate was
centrifuged for 5 min at 12000 rpm. The supernatant was analyzed to
determine the amino acid yield using HPLC. According to the optimal
amount of Novozyme flavorzyme, for synergistic experiment, the pro-
tein loading was also a constant value of 0.2% g/g substrate. The ratio
of aminopeptidase, carboxypeptidase and endopeptidase was summar-
ized and shown in Table 2, and enzyme hydrolysis was carried out at
40 °C, 200 rpm. Samples (1 mL) were taken from the reaction mixture at
specific times. Each sample from the hydrolysate was heated in boiling
water for 10 min to deactivate the enzymes and then cooled to room
temperature and subsequently centrifuged for 10 min at 12000 rpm.
The supernatant was removal of residual proteins and derivatized to
analysis amino acid output.
The protein hydrolysis rate(%)
= amino acid content/Total protein content
2.4. Detection of amino acids
Samples were removed soluble protein by sulfonic salicylic acid
method. Amino acids were derivatized by PITC before they can be
Table 1
Characteristics of fermented mash of food waste used in the experi-
ments.
Parameters Contenta (w/w, %)
pH 3–4
Moisture 91.7% ± 1.8
Residual protein content 64.6% ± 0.5
All values are based on wet mass. Values are given as means ±
standard deviation (n = 3).
T. Sun, et al. Bioresource Technology Reports 7 (2019) 100268

Table 2 with different promoters were constructed and transformed into C.

Synergistic action of enzymes and experimental design. utilis. The activities of proteases under different promoters were in-
Total pepF YPDF pepA(mg/g) Total pepF YPDF pepA(mg/g) vestigated. Among them, the activity of aminopeptidase YPDF with
enzyme (mg/ (mg/ enzyme (mg/ (mg/ promoter scHXT7 was the highest, the activity of carboxypeptidase
g) g) g) g) pepF with promoter scTEF1 was the highest, and the activity of en-
dopeptidase pepA with promoter cuGAP was higher than that with
pepF 2 NS NS P1Y12 0.15 1.85 NS
YPDF NS 2 NS P2Y11 0.31 1.69 NS
promoter cuPGK. Three proteases with their optimal promoter combi-
pepA NS NS 2 P3Y10 0.46 1.54 NS nations were selected to construct three gene co-expression vectors and
P1A12 0.15 NS 1.85 P4Y9 0.62 1.38 NS transformed into C. utilis. Compared with scHXT7-YPDF and scTEF1-
P2A11 0.31 NS 1.69 P5Y8 0.77 1.23 NS pepF, the enzymatic activity of Cu-coexpression was improved, but it
P3A10 0.46 NS 1.54 P6Y7 0.92 1.08 NS
was lower than that of cuGAP-pepA (Fig. 2.).
P4A9 0.62 NS 1.38 P7Y6 1.08 0.92 NS
P5A8 0.77 NS 1.23 P8Y5 1.23 0.77 NS
P6A7 0.92 NS 1.08 P9Y4 1.38 0.62 NS 3.3. Synergism of different proteases
P7A6 1.08 NS 0.92 P10Y3 1.54 0.46 NS
P8A5 1.23 NS 0.77 P11Y2 1.69 0.31 NS
The enzymatic hydrolysis was carried out at a substrate loading of
P9A4 1.38 NS 0.62 P12Y1 1.85 0.15 NS
P10A3 1.54 NS 0.46 X1Y12 0.02 1.85 0.13
50 g with different dosages of Novozymes Flavorzyme set, as shown in
P11A2 1.69 NS 0.31 X2Y11 0.05 1.69 0.26 Fig. 5. At the beginning of 12 h, the yield of amino acids increased with
P12A1 1.85 NS 0.15 X3Y10 0.07 1.54 0.39 the increase of enzyme concentration. Amino acid yield was the highest
Y1A12 NS 0.15 1.85 X4Y9 0.1 1.38 0.52 when the enzyme content was 0.8%(Fig. 3A.). However, the amino acid
Y2A11 NS 0.31 1.69 X5Y8 0.12 1.23 0.65
yields by using 0.2%, 0.4%, 0.6% and 0.8% g/g after hydrolyzing for
Y3A10 NS 0.46 1.54 X6Y7 0.14 1.08 0.78
Y4A9 NS 0.62 1.38 X7Y6 0.17 0.92 0.91 60 h were approximately the same (p > 0.05) (Fig. 3B.). Considering
Y5A8 NS 0.77 1.23 X8Y5 0.19 0.77 1.04 the cost of industrial production, 0.2% g/g of Novozymes Flavorzyme
Y6A7 NS 0.92 1.08 X9Y4 0.21 0.62 1.17 was used in hydrolyzing fermentation mash of food waste.
Y7A6 NS 1.08 0.92 X10Y3 0.24 0.46 1.3 Fermentation mash of food waste was hydrolyzed by different
Y8A5 NS 1.23 0.77 X11Y2 0.26 0.31 1.43
Y9A4 NS 1.38 0.62 X12Y1 0.28 0.15 1.57
combinations of proteases. The results were shown in Fig. 4 (A-H).
Y10A3 NS 1.54 0.46 There was obvious synergism between pepF and pepA (Fig. 4 A and B).
Y11A2 NS 1.69 0.31 The amino acids yields of P2A11 and P10A3 reached 12.5 mg/mL and
Y12A1 NS 1.85 0.15 12.4 mg/mL respectively after 12 h of hydrolysis and were significantly
higher than that of pepF or pepA. At 60 h, the amino acids yield of all
combination groups were higher than that of the single protease groups.
detected (Fa-mei, 1999). Twenty kinds of natural amino acid standard
The best combination of enzymes was P2A11. The amino acids yield of
kit were purchased from Solarbio. Amino acid was determined by HPLC
P2A11 reached 23.2 mg/mL. They are 2.7 times as much as pepA and 2.1
with a Kromasil C18 column, column temperature box 38 °C, detection
times as much as pepF, respectively. P1A12 was the second-highest
wavelength 254 nm, injection volume 2 μL. The mobile phase A was
production of amino acids. It was 17.9 mg/mL and they are 2.0 times as
0.1 mol/L sodium acetate-acetonitrile (volume ratio 97:3) and the
much as pepA and 1.6 times as much as pepF, respectively. Comparing
mobile phase B was acetonitrile-water (volume ratio 4:1) solution.
among all synergistic groups, it was found that the synergism between
pepA and pepF was more significantly when pepA or pepF was in
2.5. Calculations and statistical analysis greater proportion. In contrast, the synergism was less apparently when
the proportions of pepA and pepF were close.
All experiments were conducted in duplicate. The data are pre- Synergism was also observed between YPDF and pepA in producing
sented as mean values ± standard deviation (SD). Statistical analysis amino acids (Fig. 4 C and D). At 12 h, the amino acids yields of Y1A12,
was carried out by the IBM SPSS statistics 22 using oneway ANOVA and Y2A11 and Y10A3 were 10.45 mg/mL, 10.53 mg/mL and 10.35 mg/mL.
Duncan's multiple range tests. Results were considered statistically They were all higher than that of the single YPDF or single pepA sig-
significant at 95% confidence interval (p < 0.05). nificantly. At 60 h, the synergism was more apparently. The amino acid
content of Y10A3 increased to 14.5 mg/mL. Y1A12 was the second-
3. Result highest production of amino acids. Similarly with the synergism be-
tween the pepA and pepF, the synergism between YPDF and pepA was
3.1. Selection of aminopeptidase and carboxypeptidase more significantly when one of the protease was in greater proportion,
and it was less apparently or disappeared when the proportions of
In this study, five aminopeptidases were selected from Bacillus proteases were close.
coagulans, two carboxypeptidases were selected from Bacillus coagulans In the study of the synergistic effects between two exopeptidase
and one from Aspergillus niger. The genes were obtained by PCR and (Fig. 4 D and E), the amino acids yields by using P10Y3, P11Y2, P8Y5 and
linked to pcGAPGA vector respectively. The recombinant vectors were P9Y4 were significantly higher than those by using YPDF and pepF at
transformed into C. utilis respectively. The activities of aminopeptidase 12 h. At 60 h, the amino acids yields by using P10Y3 reached to
and carboxypeptidase were determined (Fig. 1). Among the five ami- 13.28 mg/mL.
nopeptidases, the activity of YPDF was the highest and was 3.36 times In the study of the synergistic effects of three enzymes (Fig. 4 G and
higher than that of APC. Among the three carboxypeptidases, the ac- H). Compared with P2A11, the synergistic production of three proteases
tivity of pepF was 1.8 times higher than that of Bc-S1 and 1.4 times of (including synergistic effect of three enzymes mixtures and Co-expres-
Bc-S2. Therefore, YPDF and pepF were chosen for subsequent experi- sion) was significantly lower at 12 h and 60 h. With the increase of
ments. hydrolysis time, P2A11 increased significantly, but the synergistic effect
of three proteases did not. Compared with Co-expression, the sy-
3.2. Promoter optimization of protease nergistic of the other three enzymes were lower except X9Y4 and X10Y3.

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.
Fig. 1. Enzymatic activities of different aminopeptidases and carboxypeptidases.
Fig. 2. Activities of pepA, pepF and YPDF with different promoters.
protease groups, protein hydrolysis rate of the synergistic groups were
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.
was 71.5%. The hydrolysis rate of P2A11 was 51.2% and was 81.5% of
that by 0.2% Flavorzyme, The hydrolysis rates of Y10A3, P10Y3 and X9Y4
were similar. The hydrolysis rates of Co-expression were lower than
YPDF, and need further improvement (Fig. 5.).
3.5. Fermented mash of food waste hydrolysis curve
The time course of amino acids production from fermented mash of
food waste by using P2A11 and 0.2% Flavorzyme were conducted. It can
be seen that the content of amino acid increased with the increase of
hydrolysis time from 12 to 60 h. At 72 h, the content of amino acids
produced by using P2A11 and 0.2% Flavorzyme all tended to be stable.
Compared with 0.2% fla protease, the hydrolysis rate of P2A11 was
lower. At 60 h, the amino acid content of P2A11 was 21.4 mg/mL and
reached 73.8% of that by using 0.2% Flavorzyme, which was 29 mg/mL
(Fig. 6.).
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Bioresource Technology Reports 7 (2019) 100268
4. Discussion
The composition of food waste is complex, and it is difficult to se-
parate the components. So far, fermenting food waste and producing
high value-added output is one of the effective ways to utilize it.
However, the output of fermentation mash is large, and it contains a lot
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
and produce amino acid fertilizer. Therefore, the fermentation mash of
food waste could be fully utilized, and the purpose of graded utilization
of food waste could be achieved.
In this study, C. utilis was used to express exogenous protease genes
for its better acid and protease resistance. Aspergillus can secrete a
variety of proteases with high enzymatic activity. Bacillus coagulans,
which can grow under high temperature and acidic conditions, also
secretes various proteases. Therefore, this study transferred the pro-
tease genes from Aspergillus and Bacillus coagulans into C. utilis and
constructed the engineering strains which can express three proteases
separately and the strain which can co-express the three enzymes.
Enzyme activity analysis confirmed that all genes were successfully
T. Sun, et al. Bioresource Technology Reports 7 (2019) 100268

Fig. 3. (A) Amino acids yield of Flavorzyme at different concentrations at 12 h.

(B) Amino acids yield of Flavorzyme at different concentrations at 60 h.

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.
Fig. 4. (continued)
experiment, the protein loading was also a constant value of 0.2 g/g
substrate. It was found that endopeptidases and exopeptidases could
cooperate with each other and achieved better synergistic effect, and
the synergistic effect of pepA (endoproteolysis) with pepF (carbox-
ypeptidase) was better than that with YPDF (aminopeptidase). It was
also found that the synergism between endopeptidase and exopeptidase
was more significantly when endopeptidase or exopeptidase was in
dominant proportion. In contrast, the synergism was less apparently
when the proportions of endopeptidase and exopeptidase were close.
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Bioresource Technology Reports 7 (2019) 100268
Fig. 4. (continued)
The synergistic effect between two exopeptidases pepF and YPDF
was not obvious. At the first 12 h, the hydrolysis rate of the two exo-
peptidases was faster, but slower after 12 h. It was found that the initial
amino acid concentration increased rapidly when two exoproteins were
synergistic, but the amino acid yield rate was slow in the subsequent
hydrolysis process and the group lacked the ability of continuous hy-
drolysis.
Compared with the synergistic effect of pepF and pepA, the sy-
nergistic effect of the three enzymes was not obvious. For X3Y10, X9Y4
and X10Y3, the amino acid content was lower at 60 h than that at 12 h.
the possible reason is that the mixed enzyme acts as a catalyst, it can be
in full contact with the substrate in initial reaction stage; over time, the
hydrolysates increase continuously, which would inhibit the decom-
position of protein and even lead to the polymerization of hydrolysates
(Eijsink et al., 2004). In addition, in the presence of a variety of pro-
teases, mixed enzymes can catalyze at different positions of polypeptide
chains. When the mixed proteases are too much in reaction system, they
will hydrolyze themselves because they are proteins too. All these
would affect the experimental results ultimately (Bickerstaff, 1993;
Mildner et al., 1994).
In this study, the highest amino acids yield was obtained by using
P2A11, which was 23.85 mg/mL after 60 h hydrolysis. The protein hy-
drolysis rate of P2A11 reached 51.2% at 60 h, which was much higher
than that of Kamnerdpetch's study. The protein hydrolysis rate was 44%
at 60 h in their work (Kamnerdpetch et al., 2007).
T. Sun, et al. Bioresource Technology Reports 7 (2019) 100268

Fig. 5. Protein hydrolysis rate.

The time course of amino acids yields was investigated. The amino
acids content by using P2A11 was lower than that by using 0.2% g/g
Flavorzyme all the time. After hydrolyse for 60 h, the amino acids yield
of P2A11was 23.85 mg/mL, and was 73.8% of that by using 0.2% g/g
Flavorzyme. With the prolongation of hydrolysis time, the increases of
amino acid content were all slow down. It would be that with the in-
crease of content of short peptide in reaction system, some hydrophobic
amino acids were exposed, and the emulsifying ability of solution in-
creased, then the stereoscopic repulsion produces stability that affects
the interaction between enzymes and substrate molecules (Michael
Fountoulakis, 1998).
In further study, the mass fraction of the substrate should be opti-
mized. Because the viscosity of substrate will affect the diffusion of
enzyme molecules, and indirectly affect the hydrolysis efficiency
(Kamnerdpetch et al., 2007). Furthermore, heat treatment can trans-
form proteins into open polymers and make proteases bind with sub-
strate more easily, it will help to promote the hydrolysis of protein
(Agboola and Dalgleish, 1996; Turgeon et al., 1992). In addition, ul-
trasound was an effect-assist (Ma et al., 2011). Therefore, more assis-
tant treatment should be investigated to promote the hydrolysis effi-
ciency in the future.
5. Conclusion
In this study, the metabolic engineering method was used to con-
struct engineering C. utilis strains which can produce proteases for

Fig. 6. Amino acids production from fermented mash of food waste.

7
T. Sun, et al.
degradation of fermentation mash of food waste. Synergism was ob-
served among endopeptidase, carboxypeptidase and aminopeptidase.
Comparing with the single protease, the synergistic of pepF and pepA
increased the yield of amino acid significantly. The hydrolysis rate of
P2A11 was 51.2% at 60 h when using the fermentation mash of food
waste as substrate.
Acknowledgements
This research was supported by the Program for New Century
Excellent Talents in University (NCET-05-0745), the Science and
Technology Planning Project of Guangdong Province
(2012B020311005), and the Scientific Research Foundation for the
Returned Overseas Chinese Scholars, State Education Ministry China.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biteb.2019.100268.
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 Update
Bioresource Technology Reports
Volume 11, Issue , September 2020, Page

DOI: https://doi.org/10.1016/j.biteb.2020.100540
 Bioresource Technology Reports 11 (2020) 100540
Contents lists available at ScienceDirect
Bioresource Technology Reports
journal homepage: www.journals.elsevier.com/bioresource-technology-reports
Erratum regarding previous published articles
Owing to a publisher error declaration/conflict of interest state­
ments were not included in the published versions of the following
articles, that appeared in previous issues of Bioresource Technology
Reports.
The appropriate declaration/conflict of interest statements, pro­
vided by the authors, are included below.
1. “Linking the nitrous oxide production and mitigation with the mi­
crobial community in wastewater treatment: A review” (Bioresource
Technology Reports, 2019; 7: 100191)
https://doi.org/10.1016/j.biteb.2019.100191
Conflict of Interest: The authors declare that they have no com­
peting interests.
2. “Intensifying soluble dietary fiber production and properties of
soybean curd residue via autoclaving treatment” (Bioresource
Technology Reports, 2019; 7: 100203)
https://doi.org/10.1016/j.biteb.2019.100203
Conflict of Interest: The Authors declare that we have no financial
and personal relationships with other people or organizations that
can inappropriately influence our work, there is no professional or
other personal interest of any nature or kind in any product, service
and/or company that could be construed as influencing the position
presented in our manuscript.
3. “Rapid granulation of aerobic granular sludge: A mini review on
operation strategies and comparative analysis” (Bioresource
Technology Reports, 2019; 7: 100206)
https://doi.org/10.1016/j.biteb.2019.100206
4. “Bio-butanol production from rice straw — Recent trends, possibi­
lities, and challenges” (Bioresource Technology Reports, 2019; 7:
100224)
https://doi.org/10.1016/j.biteb.2019.100224
Conflict of Interest: The Authors wish to confirm that there are no
known conflicts of interest associated with this publication and
DOI of original article: https://doi.org/10.1016/j.biteb.2019.100224
https://doi.org/10.1016/j.biteb.2020.100540
Available online 08 September 2020
2589-014X/ © 2020 Elsevier Ltd. All rights reserved.
T
there has been no significant financial support for this work that
could have influenced its outcome.
5. “Combined electrocoagulation and electrooxidation process in
electro membrane bioreactor to improve membrane filtration ef­
fectiveness” (Bioresource Technology Reports, 2019; 7: 100237)
https://doi.org/10.1016/j.biteb.2019.100237
Conflict of Interest: The authors certify that they have no affiliations
with or involvement in any organization or entity with any financial
interest (such as honoraria; educational grants; participation in
speakers' bureaus; membership, employment, consultancies, stock
ownership, or other equity interest; and expert testimony or patent-
licensing arrangements), or non-financial interest (such as personal
or professional relationships, affiliations, knowledge or beliefs) in
the subject matter or materials discussed in this manuscript.
6. “Membrane filtration of alkali-depolymerised kraft lignin for bio­
logical conversion” (Bioresource Technology Reports, 2019; 7:
100250)
https://doi.org/10.1016/j.biteb.2019.100250
Conflict of Interest: The authors declare that there are no conflicts of
interest associated with the work.
7. “Producing amino acid fertilizer by hydrolysis of the fermented
mash of food waste with the synergy of three proteases expressed by
engineered Candida utilis” (Bioresource Technology Reports, 2019;
7: 100268)
https://doi.org/10.1016/j.biteb.2019.100268
Conflict of Interest: The authors declare that they have no conflict of
interest
8. “Optimization of photo fermentation in corn stalk through phos­
phate additive” (Bioresource Technology Reports, 2019; 7: 100278)
https://doi.org/10.1016/j.biteb.2019.100278
Conflict of Interest: The Authors have no conflict of interest in this
manuscript.