Isolation of the alcohol oxidase from the methylotrophic yeast cells genera Pichia, Hansenula

Zaytcev M.

Tula State University

For trace aliphatic alcohols determination are mainly used gas and HPLC methods, spectrophotometry and refractometry methods. However despite the low limits of detection of alcohols, general shortcomings of these methods are the complexity of the equipment, in some cases - complexity sample preparation and the duration analysis. At the same time enzymatic methods are successfully used for alcohol determine with combine high sensitivity and selectivity with the simplicity of the equipment and processing methods, rapidity and efficiency. Enzyme sensors for ethanol determination may be developed which immobilized enzyme alcohol oxidase (AO). The yeast Pichia pastories, Pichia angusta, Hansenula polymorpha are source of the AO. Development of a technique for isolation and purification of the alcohol oxidase from methylotrophic yeasts with modular system for the separation and purification of proteins which can be used for large-scale production of biological preparations based modern bioanalytical and economical automated equipment is the actual problem.

The methylotrophic yeast P. angusta VKM Y-2518, P. angusta VKM Y-1397, P. angusta VKM Y-2559, H. polymorpha NCYC 495 ln are used as a source of alcohol oxidase. The method of enzyme isolation included the following steps: yeast cells breaking with ultrasonic disrupter, a stepwise proteins precipitation with ammonium sulphate, ion-exchange chromatography on DEAE sepharose with semi-preparative proteins separation system Biologic LP (BIO-RAD), alcohol oxidase concentration. Enzyme purification was monitored at all stages with polyacrylamide gel electrophoresis.

The cells were disrupted with an ultrasonic disintegrator; insoluble cell debris was separated by centrifugation (8000g). The protein fraction was stepwise fractionated ammonium sulfate in a concentration range of ammonium sulfate saturation of 20-100%. Specific activity of enzyme preparations and protein content were determined in the supernatant and the precipitate at all stages of salting. Figure 1 shows the total amount of protein and specific activity of the enzyme preparation at each stage of salting out.

Figure 1: Total protein content and specific activity at different stages AO salting. AO (H. polymorpha NCYC 495ln).

Highest specific activity of AO corresponds to the precipitated proteins fraction at a concentration of 50% ammonium sulfate saturation and 70% ammonium sulfate saturation. A similar distribution was observed to AO for all methylotrophic yeast strains. The fractions with the highest specific activity of AO were combined and used in the final purification step.

Further proteins purification from outside AO performed using ion exchange chromatography. Alcohol oxidase desorption was performed phosphate buffer solution containing potassium chloride (1M), with linearly increasing percentage of KCl in the final solution (0-100%). As the alcohol oxidase molecular weight is a 600kDa collected enzyme can be further concentrated using with concentrating filter (100KDa) on the centrifuge (8000g). The concentrated enzyme is a light - yellow color substance. The specific activity and the protein content of the enzyme alcohol oxidase preparations isolated from various yeast strains are shown in Table 1

Table 1. The specific activity and the protein content for the enzyme alcohol oxidase preparations derived from various methylotrophic yeast strains

№	alcohol oxidase producer strain	total protein content, mg	Specific enzyme activity U/mg of protein
1	P. angusta ВКМ Y-2518	-	-
2	P. angusta ВКМ Y-2559	10	9
3	P. angusta ВКМ Y-1397	9	16
4	H. polymorpha NCYC 495 ln	11	19

Thus the alcohol oxidase specific activity isolated from Hansenula polymorpha NCYC 495 ln higher then specific activity of AO isolated from other studied strains. The high protein content and high activity of the isolated enzyme showed the promising use of this strain for the production of the alcohol oxidase.

The work was supported by the Federal Goal-oriented Program “Scientific and Scientific-Pedagogical Cadres of Innovative Russia” for 2009–2013, agreement No 14.B37.21.0561 and State contract No 16.740.11.0766