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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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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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,

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

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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

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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+,

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Ba2+,

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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

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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

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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.

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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

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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.

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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

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vector (Novagen).

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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

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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

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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

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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

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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

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

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

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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

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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

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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

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according to Zou et al.22

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Determination of protein concentration

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Protein concentrations were determined by dye binding method

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serum albumin (BSA) as standard.

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De-glycosylation

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using bovine

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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

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

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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

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

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

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

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

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was analyzed by SDS–PAGE.

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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

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

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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

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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

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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%.

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

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temperature.

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pH and thermal stability of the purified phytase

About 0.225 nmol of the

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

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

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

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Zou et al. 22

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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

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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+,

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

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

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et al.22

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

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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

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

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measured according to Zou et al.22

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Substrate specificity and kinetic analysis

glycol

tetraacetic

acid

(EGTA),

N-ethylmaleimide

(NEM),

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

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(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

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(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

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

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

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

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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

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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

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

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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

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

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

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

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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

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YPD broth without Zeocin) × 100%]

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Statistical analysis

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

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

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

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

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