Removal of feather lipids to improve nutritional value and processing of feathers for animal feed

Removal of feather lipids to improve nutritional value and processing of feathers for animal feed

Summary

The focus of this study is to determine if enzymatic removal of chicken feather lipids can enhance feather meal processing. Keratinolytic enzymes are commonly used to improve feather meal digestibility, so if the lipids are no longer present on the feather, keratinases should have greater access to their substrate. To date on this project, lipids from chicken feathers have been isolated using a Soxhlet apparatus and lipid analytical techniques are being optimized for characterization of the lipid profile. Also, we have been able to clone multiple lipase genes from a feather degrading strain of Streptomyces into Pichia pastoris for heterologous protein expression. Only one lipase has consistently shown activity, but there have been a couple other lipases to show moderate activity at times. Early feather degrading experiments with lipase and keratinase have been inconsistent and will need to be further verified. The project has already participated in two outreach events: one with a visiting high school group and the other being a presentation in the department. 

Objectives/Performance Targets

In the original plan for the research project, there were seven main objectives:
     1. Determine the chemical composition of feather lipids.
     2. Identify potential lipases to hydrolyze triacylglycerides from the feathers.
     3. Isolate, clone and express a wax esterase from feather degrading bacteria.
     4. Determine if keratinase assisted by lipase enhances feather meal preparation.
     5. Determine if keratinase assisted by wax esterase enhances feather meal preparation.
     6. Analyze wax esterase for a synergistic effect when accompanied by lipase on the keratinase-                   mediated feather degradation.
     7. Contribute to the knowledgebase on sustainable feather meal production and share that                        knowledge with fellow researchers and farmers. 

To date, I have been working towards completing the first four objectives. This has mainly encompassed characterizing the lipids on feathers, cloning various genes from Streptomyces and expressing them in yeast, and conducting several feather hydrolysis assays. 

Accomplishments/Milestones

At the start of the project, I first made a trip to a local farm and picked up chicken feathers. After rinsing them in water, I extracted the lipids using a Soxhlet apparatus. The next step is to create a lipid profile, of which I can use either thin-layer chromatography or gas chromatography. Retrieving all the necessary equipment for each technique has proven challenging. The lab has access to a gas chromatographer but a column capable of the high temperatures necessary for waxy lipids took a while to find. I was able to finally locate one, but will still need to go through the rigors of setting up and optimizing the machine to produce consistent results. The lab has a few thin-layer chromatography plates so I am currently in the process of troubleshooting a few protocols using olive oil. Upon developing a reliable technique, I should then be ready to run the feather lipids for analysis.  

Simultaneously, I also began working on cloning different lipase genes into a yeast expression system. The genes were from a strain of Streptomyces capable of hydrolyzing chicken feather. This same strain has been in the lab for a few years and has previously had its draft genome sequenced and annotated. There were four putative lipase/esterase genes with their full sequence determined with one putative lipase containing half of its sequence. It was attempted to recover the remaining half of the partially sequenced gene, but with no success. The four complete genes were successfully cloned and expressed. When screening for activity however, only one construct consistently demonstrated lipase activity. To compensate for the limited number of active lipases, two other strains of Streptomyces that we have in the lab were also used. These strains have been studied more and their sequences are already in the public domain. We used this information to clone additional lipases. Of those lipases that have successfully been cloned, one has demonstrated activity at times.

Having a couple of lipases expressed and demonstrating activity, I tried some preliminary feather hydrolysis experiments. Proteinase K was supplemented with and without lipase and incubated with a chicken feather for a couple of days. Another strategy for this experiment was taking feathers that had their lipids removed and compared their hydrolysis from proteinase K on feathers only rinsed in water. The results in both cases were widely variable and not reproducible. I have begun searching for a feather hydrolysis assay that can be consistent and not rely on too many feather preparation steps that would alter the impact of feather lipids.

A lot of work still remains left to do for the project. Mainly, the lipid profile still needs to be determined. From there, testing these lipids against the cloned lipases for hydrolysis would be the next step. These lipids would be sure to include the many wax esters on the feathers. A consistent keratinase assay will need to be developed, and it will need to consider preparation of the feather, taking account the lipids (unable to rinse in ethanol or autoclave). Effects of the lipase and wax esterase enzymes on feather hydrolysis will then need to be quantified. Finally, the results will need to be summed up and outreach information prepared to share what we have learned.

Overall, the project has been progressing. Ideally, a decent profile of the lipids would have been made by now and more lipases would have been cloned. I anticipate having to try multiple keratinase assays in the coming months to have the consistency and reproducibility required to test the effects of the enzymes. It would be ideal to have more active lipases at this point, but there are other strategies to try to get them active (codon optimization, different expression host, or just new genes).

This project has taught me a lot of things so far. I have a better appreciation for time frames and estimating when things can get done. With that said, learning new techniques is not always so simple. It is great when there are people in the lab and the department who can teach you new things or have equipment they are willing to share. 

Impacts and Contributions/Outcomes

The potential impact the results from this project are large. Chicken feathers are huge by-product from the poultry industry and processing of these feathers has been difficult. If lipases/wax esterases that remove lipids on chicken feathers is capable of enhancing feather hydrolysis, and reduce processing inputs, the benefit can be great. Contrarily, if there is not much or no impact, then future efforts can be focused towards finding the most efficient keratinase and not wasted on creating enzyme cocktails.

So far the project has already participated a couple of outreach activities, one in the community and one in the department. A couple months after writing the project proposal, I volunteered to show a visiting high school group around the lab. This provided me an opportunity to discuss with them my proposed project and inspire the students to explore a future in science. In the beginning of September I gave a presentation to the department describing the project and talking about early experiments being done. This also seemed to go well and provided me an opportunity to share what I have and will be working on for the next year.

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