РАЗРАБОТКА ХРОМАТОГРАФИЧЕСКОЙ СИСТЕМЫ ДЛЯ ВЫДЕЛЕНИЯ И ОЧИСТКИ L-ФЕНИЛАЛАНИН- АММОНИЙ-ЛИАЗЫ, ПОЛУЧЕННОЙ МЕТОДОМ ГЕННОЙ ИНЖЕНЕРИИ

Бабич О.О.¹, Дышлюк Л.С.², Просеков А.Ю.³

¹Доцент, кандидат технических наук; ²Кандидат биологических наук;

³Профессор, доктор технических наук, Кемеровский технологический институт пищевой промышленности

РАЗРАБОТКА ХРОМАТОГРАФИЧЕСКОЙ СИСТЕМЫ ДЛЯ ВЫДЕЛЕНИЯ И ОЧИСТКИ L-ФЕНИЛАЛАНИН- АММОНИЙ-ЛИАЗЫ, ПОЛУЧЕННОЙ МЕТОДОМ ГЕННОЙ ИНЖЕНЕРИИ

Аннотация Изучены различные методы очистки фермента L-фенилаланин-аммоний-лиазы, полученной методом генной инженерии: осаждение сульфатом аммония, очистка на Ni⁺²–NTA агарозе, гидрофобная хроматография. Установлена зависимость активности очищенного препарата от концентраций солей (ионной силы). Предложена технология очистки фермента методом гидрофобной хроматографии для получения высокой степени очистки белка.

Ключевые слова: L-фенилаланин-аммоний-лиаза, генная инженерия, хроматография, активность, белок.

Babich O.O.¹, Dyshljuk L.S.², Prosekov A.Y.³

¹ Associate professor, Candidate of Technical Sciences; Candidate of Biological Sciences; ³ Professor, Doctor of Technical Sciences, Kemerovo Technological Institute of Food Industry

The article was prepared with support of federal targeted program «Scientific and educational human resources of innovative Russia» for 2009-2013; grant agreement from 14.09.2012 №14.В37.21.1232.

DEVELOPMENT OF CHROMATOGRAPHIC SYSTEM FOR ALLOCATION AND CLEANING OF L-PHENYLALANINE AMMONIA-LYASE RECEIVED BY GENETIC ENGINEERING

Abstract Various methods of purification of enzyme L-phenylalanine ammonia-lyase received by method genetic engineering are studied: sedimentation by ammonium sulfate, cleaning on Ni⁺²–NTA agarose, hydrophobic chromatography. Dependence of activity of the cleared preparation on concentration of salts (ionic force) is established. The technology of purification of enzyme by a method of hydrophobic chromatography for obtaining high extent of purification of protein is offered.

Keywords: L-phenylalanine ammonia-lyase, genetic engineering, chromatography, activity, protein.

Introduction. The importance of enzyme L-phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) in the course of phenylalanine biotransformation results to development of simple and effective methods of its cleaning on various carriers with the maximum preservation of activity in a firm phase [1]. Cleaning, as a rule, leads to decrease in activity of enzyme at the expense of the diffusive resistance, shielding of the active center, conformational modification of protein. On the other hand, activity of the cleared enzymes can remain or increase completely. At the expense of increase of stability of enzyme during cleaning, the quantity of the turned substratum significantly increases [1, 2].

This work is directed on development of method of chromatographic cleaning recombinant L-phenylalanine ammonia-lyase.

Methods. Cultivation of strain producer cells and purification of obtained enzyme by methods of an ion-exchange chromatography and electrodialysis carried out by a technique described in work [3].

In this work studied influence of high concentration of salts (ionic force) on activity of cleared preparation PAL (10,1 mg/ml). For this purpose defined activity of PAL in a standard reactionary mix (1 ml) with various concentration of NaCl, KCl and (NH₄)₂SO₄ (100 mM, 250 mM, 500 mM). After preincubation began reaction by cleared preparation divorced by 50 times of 0,1 M Tris-HCl with the buffer рН 8,5. As control used the activity of enzyme measured in standard conditions.

Further investigated influence of various chemical agents on activity of PAL for the purpose of their application as stabilizers, preservatives and cryoprotectants of this protein. PAL activity measured in a standard reactionary mix (1 ml) with reactants: dithiothreitol, β-mercaptoethanol, D-sorbitol, D-trehalose, polyvinylpyrrolidone, polyethylenimine and sodium dodecylsulfate. After predynkubation began reaction the cleared preparation divorced by 50 times with the buffer of 0,1 M Tris-HCl, рН 8,5. As control used the activity of enzyme measured in standard conditions without additives.

The second cleaning of PAL carried out for the purpose of optimization of the scheme of cleaning and increase of exit of enzyme. For this purpose a biomass resuspended in 150 mM phosphatic buffer (рН 8,0) with 10 mM imidazole. Added inhibitors of proteases in suspension. Cells destroyed by ultrasound at the average power and amplitude equal 4, using an average rod. Sounded suspension (20 ml) on ice of 8 times on 20 sec. with minute intervals for cooling. Homogenate centrifuged at 10000 rpm within 30 min.

The following stage of cleaning - fractionation by the ammonium sulfate, depending on the following factors: ionic force, temperature, рН and initial concentration of protein. In a supernatant brought dry ammonium sulfate to 25% from saturation. After salt dissolution extract was maintained by 15 min on cold. Deposit separated by centrifugation.

In a supernatant concentration of salt led up, adding dry sulfate of ammonium, to 50% from saturation. After 15-minute incubation on cold repeatedly centrifuged, and the received deposit resuspended in the 55% ammonium sulfate to cleaning from EDTA containing in inhibitors, also centrifuged. Then a deposit dissolved in 150 mM phosphatic buffer (рН 8,0) with 10 mM imidazole.

As obtaining protein contains additional hexagistidine sequence in C-end area of a polypeptide chain, the further stage of cleaning is connected with drawing a preparation protein on a column with Ni^{2 +}-NTA agarose. Preparation centrifuged and put on a column with Ni^{2 +}- NTA agarose (7 ml), counterbalanced by the same buffer. Work carried out at +15°C. Column washed out from an untied material the initial buffer (4 volumes of a column), then - 150 mM phosphatic buffer (рН 8,0) with 20 mM imidazole (4 volumes of a column). PAL eluated by the same buffer with the concentration of imidazole increased to 250 mM (3 volumes). For concoction and buffer replacement in a preparation brought 4 M solution (NH₄)₂SO₄ to 50% of saturation and left for the night at +6°C. Next day centrifuged (15000 rpm × 15 min) and a deposit dissolved in 50 mM phosphatic buffer (рН 8,2). Then added saturated solution (NH₄)₂SO₄ to 0,8 M.

Further cleaning carried out by the second sedimentation by ammonium sulfate and method of hydrophobic chromatography. Used the FPLC chromatograph with a length of wave of 280 nanometers (Pharmacia, Sweden), using a column 7mm x 10mm Protein PAK Glass HIC about phenyl-TSK-5PW a sorbent (Nihon Waters Ltd, Japan). After centrifugation (15000 rpm × 15 min) a sample brought in a column counterbalanced of 50 mM phosphatic buffer and 0,8 M (NH₄)₂SO₄ at рН 8,2. Speed of drawing made 0,5 ml/min, washing and elution speed – 1 ml/min. After drawing a sample a column washed within 10 min. PAL eluated by decreasing linear gradient (NH₄)₂SO₄ from 0,8 M to 0 M in 50 mM phosphatic buffer at рН 8,2. Gradient volume – 35 ml. Fractions at the exit from a column united.

Results and discussion. Results of research of influence of high concentration of salts (ionic force) on activity of cleared preparation PAL, presented in tab. 1, testify that activity of enzyme is considerably inhibited at the studied concentration of NaCl (for 26-48%) and KCl (for 26-44%). Inhibition degree is almost identical to both salts. Influence of ammonium sulfate on PAL activity at the same concentration in reactionary mix is insignificant (inhibition for 8-10%) with higher ionic force. This fact obviously is connected with that the inhibition of enzyme is caused more by presence chloride ion in a reactionary mix, than high ionic force. Thus, at measurements of activity of PAL it is desirable to avoid presence chloride ions in a reactionary mix.

Table 1. Influence of NaCl, KCl and (NH₄)₂SO₄ on PAL activity

Reagent	Concentration in a ditch, mM	Activity, Е/mg of protein	Activity, %
Control	0	0,743	100
NaCl	100	0,515	69
	250	0,548	74
	500	0,388	52
KCl	100	0,552	74
	250	0,528	71
	500	0,413	56
(NH4)2SO4	100	0,668	90
	250	0,748	101
	500	0,686	92

It is established that such reagents as dithiothreitol, β-mercaptoethanol, D-sorbitol, D-trehalose, polyvinylpyrrolidone, polyethylenimine and sodium dodecylsulfate inhibit activity of PAL.

Thus, authors decided to refuse application of reducers in cleaning, and as an antioxidant to use EDTA. EDTA at 5 mm of concentration in incubatory mix reduced reaction speed insignificantly (6,0%).

Results of the offered cleaning by method of a hydrophobic chromatography are presented in tab. 2.  

Table 2. Results of cleaning of recombinant PAL on the optimized scheme

Cleaning stage	Specific activity, Е/mg	Protein, mg/ml	Yield, %
Extract	0,254	12,3	100
I fractional sedimentation by ammonium sulfate	0,763	20,2	99
Ni+2–NTA agarose	2,245	5,96	88
II sedimentation by ammonium sulfate	1,930	4,5	63
Hydrophobic chromatography	2,824	2,8	63

Conclusions. The researches directed on development of technology of allocation and cleaning of PAL are conducted, optimum parameters of cleaning are chosen and degree of stability of a preparation is studied. The method of an ion-exchange chromatography is studied. It is established that high concentration of salts (ionic force) influence activity of the cleared preparation. The method of fractionation of enzyme by ammonium sulfate is studied. It is proved that the fullest cleaning of L-phenylalanine ammonia-lyase is reached by fractional sedimentation and a method of a hydrophobic chromatography.