Bromelain is a plant protease which has a variety of properties, including anti-cancer activity, anti-inflammatory effect, antimicrobial effect, antibiotic potentiation, skin protection, postsurgery recovery and so on. It has a wide range of applications in the medical and food fields. Even though bromelain has great application potential, the traditional production process has issues of inefficiency, unstable source and low enzyme activity. Bplant replaced the traditional technique by synthetic biology and improved both enzymatic activity and stability by directed evolution.
Bromelain is a group of thiol hydrolytic proteases extracted from the tropical plant pineapple. Bromelain mainly exists in the fruit, bud, leaf and stem of pineapple, with a molecular weight of 33000. It belongs to the papain family of cysteine proteases, the proteolytic activity of bromelain is approximately ten times higher than that of papain. Its enzymatic activity is dependent on the thiol group of a cysteine residue within its active site. Currently available bromelain extracts are composed of 80% stem bromelain, 10% fruit bromelain, and 5% other components. Bromelain has a variety of properties, including anti-cancer activity, anti-inflammatory effect, antimicrobial effect, antibiotic potentiation, skin protection, postsurgery recovery and so on[1]. Therefore, it has a wide range of applications in the medical and food fields.
However, the traditional production technology of bromelain has the problems of high cost, low enzyme activity and low yield because it takes pineapple as raw material. At the same time, the processing process of this traditional technology will bring some environmental resources problems. For example, the yield of bromelain is highly dependent on the yield of pineapple, and its source is not stable; Using agricultural products to produce such high value-added products is a kind of occupation and waste of land resources; Moreover, the production process may pollute the environment, which is not in line with sustainable development.[2]
We hope to use engineered bacteria to heterogeneously express bromelain, so as to ameliorate the traditional production methods. The purpose for our study is to improve both enzymatic activity and stability by directed evolution. Here is a brief overview of our approach, you can see further details on the Design page.
Our directed evolution mainly focused on dry experiments. We carried out initial knowledge accumulation, target fragment determination, molecular dynamics thermodynamics and other related knowledge acquisition, and related software learning. Then protein homology modeling and substrate selection were carried out, followed by molecular docking, surface energy calculation and development tree construction. In terms of the model, we conducted protein homology modeling, and selected the double mutation models of p.(S16G,W67L).
Bromelain has been recombinant expressed in Escherichia coli in the current literature[3,4], so we chose E. coli to express it. Sequence corresponding to the bromelain expression is synthesized by the methods mentioned above and cloned as BamHI- XhoI inserts in the pET-32a expression vector. pET-32a is a commonly used T7 promoter based vector which can propagate in Escherichia coli and express the recombinant protein after appropriate .The desired polypeptide can be expressed as a fusion protein with 6xHis tag at the C-terminus for simplified purification. To enhance the expression of the heterologous protein, the strain used in this experiment was the rne131 mutant E. coli BL21 Star (DE3), from Tsingke Biotechnology Co.
We selected His-tag tag to screen bromelain, and then used solid metal affinity chromatography for protein purification. Ni-NTA agarose gel was selected as the filler for affinity chromatography. After a short time of low temperature storage, we eluted and exchanged the stock solution of protein product with protein buffer by gel chromatography technology.
Bromelain can be used in many scenarios, like medical, food fields and fodder processing. Due to the Local problem of shortage of protein feed resources and low efficiency of protein utilization of livestock and poultry in China[5], we choose fodder processing as the main application scenario of our study.
feed like soybeans and corns largely depend on imports.
Bromelain is a plant protease, which can convert protein in feed into peptides and small peptides easily absorbed by animals, improve the conversion rate of feed, so as to reduce the pollution of breeding industry to the environment. Bromelain also has a certain therapeutic effect on diarrhea caused by pathogenic bacteria, avoiding the negative effects of antibiotics, improving the growth performance of animals, and resisting the damage of parasites to animals. And the production of bromelain resources are rich, has the potential to vigorously develop and use, is an ideal antibiotic substitute feed additives.[6,7]
We choose Bacillus subtilis as chasis in this particular field for the reason that it is recognized as GRAS(Generally recognized as safe) by FDA(US Food and Drug Administration) and is particularly attractive as bioplant for heterologous protein production in fermentation industry[8,9]. Because at least eight extracellular proteases produced by Bacillus subtilis can inhibit the expression of source proteins, strain WB800 was selected[10]. We sorted out the commonly used parts of Bacillus subtilis through searching a large number of literature review, and identified the promoter and RBS that need to be improved. Then we selected the shuttle-type plasmid and Tat pathway signal peptide for secretion.
We’ve used Molecular Dynamics Simulations to verify the stability of bromelain reaction with BAEE. The enzyme activity and stability of recombinant bromelain were determined. We also characterized our enzyme products by treating roughage. You can see further details on the Proof of Concept page.
[1] Hikisz P, Bernasinska-Slomczewska J. Beneficial Properties of Bromelain. Nutrients. 2021 Nov 29;13(12):4313. doi: 10.3390/nu13124313. PMID: 34959865; PMCID: PMC8709142.
[2] Fouz N, Amid A, Hashim YZ. Cytokinetic study of MCF-7 cells treated with commercial and recombinant bromelain. Asian Pac J Cancer Prev. 2014 Jan;14(11):6709-14. doi: 10.7314/apjcp.2013.14.11.6709. PMID: 24377593.
[3] Azura Amid, Nurul Azira Ismail, Faridah Yusof, Hamzah Mohd Salleh. Expression, purification, and characterization of a recombinant stem bromelain from Ananas comosus.Process Biochemistry, Volume 46, Issue 12, 2011, Pages 2232-2239, ISSN 1359-5113. doi:10.1016.
[4] George S, Bhasker S, Madhav H, Nair A, Chinnamma M. Functional characterization of recombinant bromelain of Ananas comosus expressed in a prokaryotic system. Mol Biotechnol. 2014 Feb;56(2):166-74. doi: 10.1007/s12033-013-9692-2. PMID: 23921698.
[5] 陶莎,张峭,张晶.2021年饲料市场形势、展望和对策建议[J].中国畜牧杂志,2022,58(05):269-272.DOI:10.19556/j.0258-7033.20220221-03.
[6] 王修启,梁少杰,周加义,高春起,严会超.菠萝蛋白酶在饲料上的研究进展及应用前景[J].饲料工业,2016,37(04):1-4.DOI:10.13302/j.cnki.fi.2016.04.001.
[7] Dutta S, Bhattacharyya D. Enzymatic, antimicrobial and toxicity studies of the aqueous extract of Ananas comosus (pineapple) crown leaf. J Ethnopharmacol. 2013 Nov 25;150(2):451-7. doi: 10.1016/j.jep.2013.08.024. Epub 2013 Sep 26. PMID: 24076462.
[8] 沈卫锋,牛宝龙,翁宏飚,何丽华,孟智启.枯草芽孢杆菌作为外源基因表达系统的研究进展[J].浙江农业学报,2005(04):234-238.
[9] Kang Q, Xiang MJ, Zhang DW. Research progress and industrial application of Bacillus subtilis in systematic and synthetic biotechnology. Chinese Journal of Biotechnology, 2021, 37(3): 923-938.
[10] Zhao L, Ye B, Zhang Q, Cheng D, Zhou C, Cheng S, Yan X. Construction of second generation protease-deficient hosts of Bacillus subtilis for secretion of foreign proteins. Biotechnol Bioeng. 2019 Aug;116(8):2052-2060. doi: 10.1002/bit.26992. Epub 2019 Apr 24. PMID: 30989640.