Overexpression of Escherichia coli Phytase in Pichia pastoris and Its

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Over-expression of E. coli Phytase in Pichia pastoris and Its Biochemical Properties Hsueh-Ming Tai, Li-Jung Yin, Wei-Chuan Chen, and Shann-Tzong Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf401853b • Publication Date (Web): 05 Jun 2013 Downloaded from http://pubs.acs.org on June 15, 2013

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Journal of Agricultural and Food Chemistry

Over-expression of E. coli Phytase in Pichia pastoris and Its Biochemical

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Properties

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Hsueh-Ming Tai1,2, Li-Jung Yin3, Wei-Chuan Chen4 and Shann-Tzong Jiang 1,4

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3

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Keelung 20224, Taiwan * To whom correspondence should be addressed. Tel: +886-4-26328001 Ext. 15375; Fax: +886-943207241; E-Mail: [email protected]

Department of Food and Nutrition, Providence University, No. 200, Sec. 7, Taiwan Boulevard, Salu, Taichung 43301, Taiwan 2 Nugen Bioscience (Taiwan) Co, Ltd., No. 29, 40th Rd., Taichung Industry Park, Taichung, Taiwan. Department of Seafood Science, National Kaohsiung Marine University, No.142 Hai-Chuan Rd. Nan-Tzu, Kaohsiung 81143, Taiwan Department of Food Science, National Taiwan Ocean University, No.2 Beining Rd.,

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Running title: Over-expression of E. coli phytase in Pichia pastoris

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ACS Paragon Plus Environment


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Abstract

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To obtain Pichia pastoris mutant with E. coli phytase gene, which was

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synthesized according to P. pastoris codon preference, a mature phytase cDNA of E.

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coli being altered according to the codons usage preference of Pichia pastoris was

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artificially synthesized and cloned into expression vector of pGAPZαC. The final

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extracellular phytase activity was 112.5 U/mL after 72 h cultivation. The phytase,

25

with a molecular mass of 46 kDa, was purified to electrophoretical homogeneity after

26

Ni Sepharose 6 Fast Flow chromatography. The yield, purification fold, and specific

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activity were 63.97%, 26.17 and 1.57 kU/mg, respectively. It had an optimal pH and

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temperature of 4.0-6.0 and 50 oC, respectively, and was stable at pH 3.0-8.0 and 25-40

29

o

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Cu2+, Zn2+, Hg2+, Fe2+, Fe3+, phenylmethyl sulfonylfluoride (PMSF) and

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N-tosyl-L-lysine chloromethyl ketone (TLCK), but activated by Mg2+, Ca2+, Sr2+,

32

Ba2+,

33

N-ethylmaleimide (NEM). It revealed higher affinity to calcium phytate than to other

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phosphate conjugates.

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Keywords: phytase, E. coli, Pichia pastoris, cloning, expression.

C. The purified recombinant phytase was resistant to trypsin and highly inhibited by

glutathione

(GSH),

ethylene

diamine

tetraacetic

36 37

2


acid

(EDTA)

and

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38 Introduction

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Phytic acid, a major component of plant seeds, is about 1-3% in many cereals

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and oilseeds and amounts to 60-90% of total phosphorus.1 Although phytates are very

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important in many physiological functions, especially in seed germination, they are

43

considered solely as anti-nutrients due to the binding with starch and protein and their

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strong chelating ability with divalent minerals such as Ca2+, Mg2+, Zn2+ and Fe2+.2

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Conversely, as a strong chelator of iron and zinc, phytate in plant foods actually can

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serve as an antioxidant to reduce free radical formation mediated by these metals.2

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The formation of insoluble mineral-phytate complexes at physiological pHs is

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considered to be the major reason for poor mineral bioavailability, since these

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complexes are hardly absorbed in human gastrointestinal tract.3 Since many minerals

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are involved in the activation of intra- and extracellular enzymes, a deficiency of any

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one of these essential minerals might result in severe metabolic disorders and

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compromise the health of the organism. Some minerals deficiencies are common in

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developing countries, but mineral sub-deficiencies are also found in developed

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countries.4

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Effective reduction of phytic acid can be obtained via the action of both enzymatic and nonenzymatic degradation.3

Enzymatic degradation can be done by

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the addition of either isolated form of wild type or recombinant exogenous phytic acid

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degrading enzymes from various sources of fungi and bacteria. Phytase can reduce the

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anti-nutritional factor of phytic acid and also eutrophication, caused by the excretion

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of undigested phytic acid by monogastrics due to the lack of adequate levels of

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phytase in their digestive tracts.3 Phytases from several plants and microbial species

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have been purified and characterized.5

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are from the secretion by gastrointestinal tract, production by microorganisms inside

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tracts, and from foods or feeds with the addition of microbial phytases.

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microbial phytases are usually sensitive to pH and heat, which consequently greatly

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lowered their activities during processing or passing through the gastrointestinal

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tracts.6

Most of phytases in animal digestive tracts

However,

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Nonenzymatic hydrolysis during food processing or by physical removal of

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phytate-rich parts from plants seed are also frequently employed to reduce the phytate

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levels in the final foods. Phytases (myo-inositol hexakisphosphate 3- or

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6-phosphohydrolases; EC 3.1.3.8 or EC 3.1.3.26) are phosphatases that can catalyze

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the sequential hydrolysis of myo-inositol--hexakisphosphate or phytic acid (InsP6) to

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lower the formation of phosphorylated myo-inositol derivatives and inorganic

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phosphate.5 Biotechnological engineering to optimize phytases catalytic features is

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considered as a promising strategy to efficient reduction of phytate. Enhancement of

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thermal tolerance and increase in specific activity are two important issues not only

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for animal feed, but also for food processing applications of phytases. Various

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strategies have been used to obtain the phytases with higher thermal stability such as

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shifting in temperature optimum of the Escherichia coli phytase from 55 °C to 65 °C

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achieved by expression in Pichia pastoris after introducing 3 glycosylation sites into

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the amino acid sequence by site-directed mutagenesis.7

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Nevertheless, there are still limited sources of phytase that is suitable for all food

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applications. Thus, screening for ideal phytase producing bacteria with more

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favorable properties and biotechnological engineering to optimize phytase’s catalytic

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and stability features are still attracting researchers. Some of the criteria of ideal

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phytase is capable of remain highly active during food processing or preparation.

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Moreover, it is a favorable property of phytase to have high phytate-degrading

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capability even at room temperature, withstanding of acceptable temperatures such as

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in digestive tract of animals or human and a high activity over a broad pH range.3

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Monogastric animals such as pig and poultry, fish and also humans lack phytase

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to hydrolyze phytate.8 Supplemental dietary phytase is needed for them to utilize

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phosphorus and other minerals bound in phytates. Although currently phytases are

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used mainly as animal feed additives in diets of monogastric animals, there is a great

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potential for using in the processing and manufacturing of food for human

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consumption.9 Phytases used in animal feed can reduce/avoid the supplementation of

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inorganic phosphate,8 which consequently lead to lower the feed costs as well as the

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pollution caused by fecal phosphorous excretion of animals.10

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Phytases activity, thermal stability, pH stability, degradation of different phytate

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forms and production yields need to be improved to make these phytases possible for

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industrial application.8 However, there is no phytase that can fulfill all of the

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mentioned requirements.8 Site-directed mutagenesis has been used to improve the

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specific activity or thermal stability of phytases from Aspergillus fumigatus

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coli,7 and to change the pH profile of Aspergillus niger phytase.12 Fungal and E. coli

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phytases are acidic histidine phytases, which can degrade phytate at low pH in

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stomach where phytate exists in metal free form and results in myo-inositol

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monophosphate.13 It has been reported that the E. coli phytase was more resistant to

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pepsin activity than fungal phytases.14 Two types of phytases are known as 3-phytase

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(EC 3.1.3.8) and 6-phytase (EC 3.1.3.26).5 The phytase from E. coli belongs to

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6-phytase type.15 Augspurger et al. 8 reported that a bacterial phytase derived from E.

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coli liberated more P in broilers than two recombinant fungal phytases, based on

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increases in tibia ash relative to inorganic P supplementation.

11

and E.

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Chen et al.16 modified appropriately the medium composition and fermentation

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strategy of Escherichia coli phytase gene appA expressed in P. pastoris KM71-61

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strain to enhance phytase activity level from 118 to 204 U/ml at the flask scale.

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This study was to obtain Pichia pastoris mutant with E. coli phytase gene, which

116

was synthesized without altering the amino acid sequence according to P. pastoris

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codon preference. It was over-expressed and the biochemical properties of the

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purified recombinant E. coli phytase were determined.

119 120

Materials and Methods Microorganisms and plasmids

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Escherichia coli BL21 (DE3) (Stratagene, La Jolla, Calif., USA) was used as the

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donor of DNA. Escherichia coli TOP10F′ (Invitrogen, Carlsbad, Calif., USA) was

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used for the construction of E. coli genomic library and for DNA manipulations. The

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plasmid pUC57 cloning vector (GenScript, Piscataway, New Jersey, USA) was used

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for DNA fragment cloning. The DNA encoding phytase with His-tag were ligated into

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pGAPZαC (Invitrogen) expression vector. Pichia pastoris SMD1168H (Invitrogen)

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were used for expression host of phytase.

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Design and Synthesis of codon optimized phytase gene

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The usage biases of codon in E. coli and P. pastoris are significantly different. To

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further improve the expression level of recombinant phytase in P. pastoris, the

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optimized phytase gene was designed in favor of P. pastoris expression by artificially

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gene synthesized.17-18 The synthesized 1218-bp phytase gene showed 74% homology

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with the wild type phytase gene (Figure 1). This synthetic phytase sequence, in which

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319 nucleotides were changed, was designed to be preferential for P. pastoris by the

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online program DNAWorks. However, the encoding amino acid sequences of both

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wild type and synthetic genes were exactly the same. The GC content decreased from

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54.5% to 49.4%, closer to the actual GC content of other high expression genes in P.

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pastoris.19 Moreover, to avoid premature termination, the AT-rich codons were

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eliminated and changed according to the favor codons usage of P. pastoris expression.

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PCR synthesis and construction of phytase expression pGAPZαC Plasmid

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The phytase gene of Escherichia coli BL21(DE3) (GenBank Accession No.

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CP001509.3), with an additional His-tag sequence at 3′-end, was optimized using

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codon usage preference 17 and synthesized by polymerase chain reaction method. The

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standard techniques of recombinant DNA protocols were performed according to

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Ausubel et al.20 and Sambrook et al..21 A 1.29 kb DNA fragment was amplified. The

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PCR product was first cloned into pUC57 vector (GenScript) and then transformed

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into Escherichia coli Top 10 F′ competent cells. The nucleotide sequence of resulted

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products was confirmed using primers by sequencing. After midi-preparation, the

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correct plasmid was digested with Xho I and Xba I and finally ligated into pGAPZαC

150

vector (Novagen).

151

Transformation and selection in Pichia pastoris Host

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The pGAPZαC plasmid, ligated with phytase, was digested with BspHI in the

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GAP promoter region to linearize the vector and then transformed into Pichia pastoris

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SMD1168H. The colonies were selected by plating on YPDS agar plates (BD

155

Biosciences, Clontech) containing 1500 µg/mL Zeocin. The yeast colony integrated

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by recombinant pGAPZαC- phytase DNA with correct in-frame coding sequence into

157

the Pichia genome was used for the protein expression.

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Cultivation of Pichia pastoris SMD1168H and production of phytase

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The Pichia pastoris SMD1168H strain integrated with recombinant pGAPZαC

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-phytase DNA was cultivated in YPDS broth (BD Biosciences, Clontech) at 30 °C

161

with shaking at 250 rpm overnight. The resulted culture was inoculated into a medium

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containing 1% yeast extract, 2% peptone, 2% dextrose and 100 µg/mL Zeocin and

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incubated at 30 °C. The phytase activity, pH and viable cell counts (CFU/mL) were

164

monitored during 5 days incubation. After incubation, it was centrifuged at 13,000 ×g,

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4 °C for 30 min. The supernatant was used for the further purification.

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Determination of enzyme activity

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Phytase activity was determined according to Zou et al.22

After 30 min

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centrifugation at 13,000 ×g, 4 °C and being filtered through a 0.45 µm sterilized

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membrane (Gelman Sciences, Ann Arbor, MI) to remove cells, phytase sample (0.25

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mL) or collected phytase samples during purification process were incubated with 1

9



171

mL of 2 mM sodium phytate (dissolved in 100 mmol/L sodium acetate, pH 5.5). After

172

the hydrolysis, 1 mL of 15% (w/v) trichloroacetic acid (TCA) was added to stop the

173

reaction. The release of inorganic phosphate from sodium phytate was determined

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colorimetrically by adding ammonium molybdate, sulfuric acid and ferrous sulfate

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solution. After 10 min reaction, the absorbance at 700 nm was measured. One unit

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activity (U) was defined as the amount of enzyme that can liberate 1 µmol of

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inorganic phosphorus within 1 min reaction at 37 °C.22

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Purification of phytase

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The broth was centrifuged at 13,000 ×g, 4 °C for 30 min and then filtered

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through a 0.45 µm sterilized membrane (Gelman Sciences, Ann Arbor, MI) to remove

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cells. After being concentrated by Amicon ultrafiltration (cutoff: 10 kDa, Amicon

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Div., W. R. Grace and Co., Beverly, MA, USA). The concentrated sample was loaded

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onto a Ni Sepharose 6 Fast Flow column (2.6 × 4.7 cm) pre-equilibrated with a buffer

184

containing 20 mM sodium acetate and 10 mM imidazole (pH 5.5). After washed with

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the same buffer, the bound proteins were eluted with an elution buffer containing 20

186

mM sodium acetate and 200 mM imidazole (pH 5.5). The absorbance of eluents at

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280 nm was measured and the phytase activity in each fractions was determined

188

according to Zou et al.22

189

Determination of protein concentration

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190

Protein concentrations were determined by dye binding method

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serum albumin (BSA) as standard.

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De-glycosylation

23

using bovine

193

De-glycosylation was performed by using Enzymatic Protein De-glycosylation

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Kit (Sigma-aldrich, St. Louis, Missouri, USA) according to the protocols of the

195

manufacturer. Briefly, 100 µg of recombinant phytase were dissolved in 30 µL

196

distilled water. Ten mL of reaction buffer and 2.5 mL denaturation solution were

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added to the phytase solution and mixed gently. The mixture was heated at 100 °C for

198

5 min and then cooled to room temperature. About 2.5 mL TRITON X-100 solution

199

was added to the resulted sample. The phytase solution was then de-glycosylated by

200

mixing with 1 mL each of the PNGase F, O-glycosidase, and α-(2, 3, 6, 8,

201

9)-neuraminidase solutions and incubated at 37 °C for 3 h. De-glycosylated protein

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was analyzed by SDS–PAGE.

203

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

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SDS-PAGE analysis was performed with a 10% polyacrylamide gel according to 24

205

Laemmli.

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R-250 and de-stained according to Neuhoff et al..25 The low molecular weight

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calibration kit (GE Healthcare Bio-Sciences Corp., MA, USA) was used as markers

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[Phosphorylase β subunit (97.0 kDa), bovine serum albumin (66.0 kDa), ovalbumin

After electrophoretic running, gels were stained with Coomassie blue

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209

(45.0 kDa), carbonic anhydrase (30.0 kDa), soybean inhibitor (20.1 kDa),

210

α-lactalbumin (14.0 kDa)].

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Optimal pH and temperature of the purified phytase

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The optimum pH was determined by measuring the activity at various pH (100

213

mM glycine-HCl buffer 1.0, 2.0, 3.0, 4.0; 100 mM sodium acetate buffer 5.0, 6.0 and

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100 mM tris-HCl buffer 7.0, 8.0, 9.0) at 37 oC, while the optimal temperature was

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measured by incubating the reaction mixture at different temperatures (25o-90 °C) for

216

30 min using 1.5 mM sodium phytate as substrate.22 The maximal phytase activities

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at optimal pH and temperature were defined as 100%.

218

purified recombinant phytase were used in the determination of optimal pH and

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temperature.

220

pH and thermal stability of the purified phytase

About 0.225 nmol of the

221

The pH stability was determined by measuring the residual phytase activity after

222

30 min incubation at 37 oC, different pH values (100 mM glycine-HCl: pH 1.0–4.0),

223

100 mM sodium acetate: pH 4.0–6.0, 100 mM Tris-HCl: pH 6.0–9.0) according to

224

Zou et al. 22

225

The thermal stability was determined by measuring the residual phytase activity

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after 5 and 30 min incubation in 20 mM sodium acetate buffer (pH 5.5) at

227

temperatures from 25o to 95 oC according to Zou et al. 22

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Effects of metal ions, inhibitors or reducing agent

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To investigate the effect of metal ions, purified recombinant phytase in 20 mM

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sodium acetate buffer (pH 5.5) with 5 and 10 mM of Ag+, K+, Li+, Na+, Ba2+, Ca2+,

231

Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Mg2+, Mn2+, Ni2+, Zn2+ and Fe3+ were incubated at 37 oC.

232

After 30 min incubation, the residual phytase activity was measured according to Zou

233

et al.22

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To investigate different inhibitors effects, the purified phytase in 20 mM sodium

235

acetate buffer (pH 5.5) with 1 to 5 mM ethylene diamine tetraacetic acid (EDTA),

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ethylene

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p-chloromercuribenzoate (pCMB) and phenylmethyl sulfonyl fluoride (PMSF) or

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reducing agents such as dithiothreitol (DTT), glutathione (GSH), β-mercaptoethanol

239

(β-Me) was incubated at 37 oC for 30 min. The residual enzyme activity was

240

measured according to Zou et al.22

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Substrate specificity and kinetic analysis

glycol

tetraacetic

acid

(EGTA),

N-ethylmaleimide

(NEM),

242

To determine the substrate specificity, purified phytase in 20 mM sodium acetate

243

(pH 5.5) was incubated with 1.5 mM p-nitrophenyl phosphate (pNPP),

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sodium-phytate (with 9 sodium, Sigma-Aldrich Inc.), calcium-phytate (with 6 sodium,

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Sigma-Aldrich Inc.), fructose 1,6-bisphosphate (F-1,6-PP) and glucose 6-phosphate

246

(G-6-P) at 37 oC for 30 min. The activity was measured according to Zou et al.22 The

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kinetic parameters of KM and Vmax were determined by incubating the phytase with

248

various substrate concentrations (0.1-20 mM) in 20 mM sodium acetate buffer (pH

249

5.5) at 37 oC and calculated by Michaelis–Menten equation. The kcat was determined

250

with the equation: Vmax=kcat [E], where [E] is the enzyme concentration. The ratio of

251

kcat/KM was used to evaluate the catalytic efficiency of recombinant phytase.

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Pepsin and trypsin tolerance tests

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Pepsin in 80 mM glycine-HCl (pH 2.0) and trypsin in 80 mM NH4H2CO3 (pH

254

7.5) were used to evaluate the proteolytic effects on phytase. Purified recombinant

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phytase was incubated with various amounts of pepsin or trypsin at ratios

256

(protease/phytase) ranging from 0.002, 0.005, 0.01, 0.02 and 0.1 at 37 oC for 2 h. The

257

residual activity was measured by the method described above.22

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Genetic stability of the recombinant P. pastoris

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To investigate the stability of inheritance and expression level of the

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recombinant phytase genes in P. pastoris, the mutant colonies were selected on YPDS

261

(YPD with sorbitol) agar plate containing 1,500 µg/mL Zeocin. They were transferred

262

into 10 mL YPD broth and incubated with shaking (250 rpm) at 30 oC.

263

incubation, 1% of culture was inoculated into 250 mL YPD broth with/without 100

264

µg/mL Zeocin.

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expression levels were determined after 10 generations according to Zaghloul et al.26

After 24 h

This procedure was repeated 10 times. Phytase activities and gene

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and Xiong et al. 27. [Plasmid stability (%) = (CFU of YPD broth with Zeocin/CFU of

267

YPD broth without Zeocin) × 100%]

268

Statistical analysis

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One-way analysis of variance (ANOVA) was used to determine the significance of

270

differences within treatments using the Statistical Analysis System ([SAS/STAT],

271

Release 8.0; Carry, NC, USA). For each treatment, three replicates were measured

272

and the mean values were calculated. Values were considered to be significantly

273

different when P 8.0, Figure 6). Therefore, control of pH at

Overexpression of Escherichia coli Phytase in Pichia pastoris and Its

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