Hi Allan,

In addition to former answers, all of them helpful, you have a lot of literature describing classical PPO purification of a number of fruits. See for instance:

Isolation, purification and physicochemical characterization of polyphenoloxidases (PPO) from a dwarf variety of banana (Musa cavendishii, L)MAM GALEAZZI, VC SGARBIERI… - Journal of Food …, 1981 - Wiley Online Library

Purification of polyphenoloxidase from coffee fruits P de Fátima Pereira Goulart, J Donizeti Alves… - Food chemistry, 2003 - Elsevier

Polyphenoloxidase from apple, partial purification and some properties

A Janovitz-Klapp, F Richard, J Nicolas - Phytochemistry, 1989 - Elsevier

Strawberry polyphenoloxidase: purification and characterization

P WESCHE‐EBELING… - Journal of Food …, 1990 - Wiley Online Library

And many others. A diferent point is the appropriate substrate to follow the activity. Sometimes, the affinity is not the more determinant parameter. A high Vm and a simple colorimetric methods coulb be great although the Km is not very low.

If you need more details. just say. Cheers.

Try Ni-NTA (nickel agarose) if you have the enzyme in a C-terminal His tagged plasmid vector. You can then use zymography (Native SDS-PAGE with a substrate in the gel which your enzyme can break down; plus a stain of some sort that binds to the substrate but not the degraded product, e.g. iodine and starch). You could try this after your ammonium sulphate fractionation to get a more pure product maybe.

Allan, You may not like this answer, but basically you won't know until you try. Every protein, even ones that are very similar, behaves differently to some degree. I'm not familiar with the literature on your class of enzyme, but that is the best place to start. Protein purification is part art, part science. If you are purifying from a crude extract affinity tags won't be much use. If you can clone the gene that would be ideal. Zymography is nice for identifying which band in a dirty prep is yours if you have an amenable substrate which given the fruit browning story may exist for you. This can be done under non-native conditions if you wash the sds out of the gel prior to testing for activity. .

Hi Allan,

In addition to former answers, all of them helpful, you have a lot of literature describing classical PPO purification of a number of fruits. See for instance:

Isolation, purification and physicochemical characterization of polyphenoloxidases (PPO) from a dwarf variety of banana (Musa cavendishii, L)MAM GALEAZZI, VC SGARBIERI… - Journal of Food …, 1981 - Wiley Online Library

Purification of polyphenoloxidase from coffee fruits P de Fátima Pereira Goulart, J Donizeti Alves… - Food chemistry, 2003 - Elsevier

Polyphenoloxidase from apple, partial purification and some properties

A Janovitz-Klapp, F Richard, J Nicolas - Phytochemistry, 1989 - Elsevier

Strawberry polyphenoloxidase: purification and characterization

P WESCHE‐EBELING… - Journal of Food …, 1990 - Wiley Online Library

And many others. A diferent point is the appropriate substrate to follow the activity. Sometimes, the affinity is not the more determinant parameter. A high Vm and a simple colorimetric methods coulb be great although the Km is not very low.

If you need more details. just say. Cheers.

Dear all, thanks for your valuable suggestions. I know using a metal chelate chromatography is not suitable for my case as I'm going to extract the enzyme from natural resources (a fruit peel). From what I learnt, I know Ni-NTA is more preferable to purify recombinant proteins.

For your information, this class of enzymes from this fruit peel is not fully studied yet so I actually have no idea on how to start the purification procedures in a proper way. Anyways thank you all again for your suggestions.

I had 2 typos in my line about zymography, one of which could have been very misleading. Many proteins can be analyzed via zymography on SDS PAGE (non native) gels, you just need to refold in the gel which is not hard at all if your protein is amenable to it. Not sure if it will be a helpful technique for you, but I didn't want to leave an error out there.

In my opinion too, Ni-NTA (A kind of affinity chromatography) should be preferred for enriched prcent recovery of enzyme as well as fold purification as far as this specific case is concerned.

To Patrick

About zymography, if eventually used, probably there is no trouble about SDS. Most PPOs are SDS-resistant, and some of them are even activated by the detergent..

Dear Francisco,

Can other detergents such as Triton X-100 of Tween 20 activate PPOs as well? Or it must be specifically SDS? Do you have any idea on this?

My answer is YES and you are the best person to verify it. But you must be systemic, methodical and meticulous in data interpretation. One golden rule, there is no any absolutely set protocol in entomology, you must design your own. Others will only give you information about their usefulness and hurdles overcome.

Dear Ranadeep, thanks for your support. I will try my best as a Bachelors degree student. I know I'm a pioneer and I believe that I'm the pioneer in using this fruit peel because no one used this sample to extract PPOs before. I've verified that. Thanks again.

You have achieved or rather traveled more than half of your goal or journey, and that too the more difficult part. The remaining portion will be definitely more enjoyable. All the best and let me know the end scenario.

During polyphenoloxidase extraction from peel usually the phenolics come out from the disrupted tissues and cause denaturation of protein. Therefore, it is suggestive of using polyvinyl pyrolidone in the extraction buffer. This can help you recover high percentage of active enzymes. You can see the reference: M. R. Karim and F. Hashinaga (2002). Isolation and characterization of limonoid glucosyltransferase from pummelo albedo tissue. Food Chemistry, 76 (4): 431-436.

Dear Allan,

It depends on the source of the PPO. I am afraid you have to try, but as far as I know (I had worked a lot with tyrosinase and some bacterial PPO, and some friends of mine have work in plant PPOs) SDS has a greater effect. With non-ionic detergents, I have seen different patterns. Of course, most PPO are resistant to those detergents, and people use if PPO is bound to tylakoids or cell membranes. Good luck.

Dear all,

Thank you very much for your valuable answers and suggestions. Yes, I will try to run a mixture of protocols based on the suggestions that you all gave me. Thanks for your blessings as well!

It is all right Allan. If you think that I can help you, do not hesitate contact me. PPO are very tricky enzymes.

Your protocol for purification is very good and according to your equipments you can make different steps of purification after salting out using ammonium sulfate such as ion exchange, gel filtration and affinity chromatography. In all cases, you must determine the properties of your enzyme as optimum temperature, pH, metal co-factors and its stabilities. Also, I advise you to read the book of Methods in Enzymology. Good luck

I would extend the excellent suggestion that you use polyvinyl pyrrolidone to counteract the phenolics, and suggest that you could try the insoluble variant, which is poly(vinyl polypyrrolidone). You will have to soak this in your buffer overnight before use, but it means that following cell disruption you can spin it down with other particles during the initial extraction, and your extract will not get so viscous.

did u try ammonium sulphate precipitation of ur enzyme and then used different column chromatography technique, use DEAE-Sepharose

Dear Rohit,

I haven't try anything yet, I just want to gather some information regarding the purification protocol for PPOs. I might try DEAE-Sepharose once I understand the pI value of the extracted enzyme. Thanks for your valuable suggestion.

do one thing go for IEF gel of your crude enzyme extract so that u can get the exact pI of ur enzyme extract then its easy to do purification with known pH of buffer

Your experimental protocol is likely to yield partially purified enzyme, but unless your enzyme is highly unusual in mass, your prep is probably going to have a number of other proteins as well. This probably won't affect your ability to assay activity, but for example may make running an IEF gel problematic. I would look closely at the published protocols that Francisco Solano provided you for possible other approaches.