Part:BBa_K2817004

Myrosinase (horseradish)

Myrosinase is present in cruciferous plants such as broccoli, Brussels sprouts and Celery cabbage. It can convert the precursor glucosinolates in cruciferous plants into sulforaphane, a well-known anticancer substance. Sulforaphane is an activator of Nrf2, which induces Nrf2 activation and translocation into the nucleus, binding to the antioxidant response element (ARE) to promote transcriptional activation of the phase II metabolic enzyme gene. As a result, sulforaphane, on the one hand, can eliminate cancer cells by inducing phase II reaction; on the other hand, it can alleviate inflammation by inducing antioxidant enzymes (such as HO-1 and SOD) to resist oxidative stress. The horseradish enzyme derived from horseradish has good catalytic activity at body temperature and in vivo pH.

Usage and Biology

We inserted this part into plasmid pet-28a. Next, we transformed the plasmid of myrosinase-His into BL21, and cultured at 37 ℃ overnight and diluted to OD = 0.2. After growth for 2 h at 37 ℃, different concentrations of IPTG were added to induce at 16 ℃ for 16 h. Subsequently, bacterial cell lysate was obtained and the expression of myrosinase was detected by SDS-PAGE. (Figure 1). The sequence of the myrosinase enzyme was optimized based on the K12 strain and the position of the strip is correct, so myrosinase was successfully expressed. The myrosinase can convert cruciferous vegetables contained glucosinolates into sulforaphane which has well-known anti-cancer activity. A previous work demonstrated the activity of myrosinase expressed by E. coli Nissle 1917 in vivo and in vitro, the expressed myrosinase in our constructed plasmid should with broad applications in vivo.

Figure 1. SDS-PAGE analyses on bacterial lysate to detect myrosinase. Lane 1: Negative control (cell lysate without IPTG induction); Lane 2: 0.25mM IPTG; Lane3: 0.5mM IPTG, Lane4: 0.75mM IPTG induction for 16h at 16℃.

[1] Ho CL, Tian HQ.et al. 2018 Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention. Nature Biomedical Engineering. 2, pages27–37

[2] Tortorella, S. M., Royce, S. G., Licciardi, P. V. & Karagiannis, T. C. Dietary sulforaphane in cancer chemoprevention: the role of epigenetic regulation and HDAC inhibition. Antioxid. Redox Signal. 22, 1382–1424 (2015).

[3] Wakabayashi N, Dinkova-Kostova AT, Holtzclaw WD, Kang MI, Kobayashi A, Yamamoto M, Kensler TW; Talalay P. Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keapl sensor modified by inducers. Proc Natl Acad Sci U S A. 2004;101(7):2040一2045.

THINKER-CHINA 2023

Description

The number of people suffering from rectal cancer is now increasing worldwide, accounting for 10.7% of the world's total population【1】，we decided to design myrosinase to convert thioglucoside into sulforaphane, and then use sulforaphane to inhibit the cancer cells. Sulforaphane inhibits cancer cells through cell cycle arrest (G2/M phase), upregulation of pro-apoptotic factors (such as caspase 8, p21, p53, and Bax), and downregulation of anti-apoptotic factors (such as Bcl-2 and Hsp90).

Usage and Biology

We used the promoter T7 and terminator pET28a to express myrosinase. Among them,the T7 promoter, which is a strong promoter from T7 phage that can respond specifically to T7 RNA polymerization, is a sequence that initiates transcription of the T7 phage gene. The most commonly used plasmids in the pET series of commercial vectors use the T7 promoter to control gene expression.when it is imported in, it can keep on transcribing, then translating, and eventually produce myrosinase. Besides, the B0015 terminator plays the role of terminating transcription.

Figure1 The design of the myrosinase.

Characterization

We determined the enzyme activity of myrosinase and found that the Km value of myrosinase was 84.15 μM and the Vmax was 15.51 μM/μM protein at 37°C and pH 7.4 (Figure.2). Also, we found that the temperature of 37 degrees Celsius and pH 7.4 were the best reaction conditions for myrosinase (Figure.3).

Figure2 Gel image of myrosinase.

Figure3 The result of expression of myrosinase. (a,Graph of myrosinase Michaelis-Menten Equation. b and c, Optimal Reaction Conditions Testing for myrosinase.)

To characterize the enzyme activity, we used GraphPad Prism software to fit the Michaelis-Menten equation (Figure.1) to determine the maximum rate (Vmax) and Km value of myrosinase reaction. The results showed that the Km value of myrosinase was 84.15 μM and Vmax was 15.51 μM/μM protein at 37°C and pH 7.4. What's more, we prepared reaction buffers (with a fixed substrate concentration of 100 μM) by preparing the reaction buffers (with a fixed substrate concentration of 100 μM) at different pH values (6.3, 7.4, and 8.2) and at different reaction temperatures (25 °C, 37 °C, and 42 °C) to test the best reaction conditions of the enzyme (Figure.3), and the results showed that the temperature of 37 degrees Celsius and pH 7.4 were the best reaction conditions for myrosinase.

In vitro anti-cancer activity and time dependency of myrosinase CT26 colon cancer cell line in mice was seeded at a density of 5000 cells per well in a 24-well plate. The complete culture medium consisted of DMEM basal medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were cultured at 37°C in a 5% CO2 environment until they reached 60-70% confluency. Myrosinase produced by the engineered bacterial strain was incubated with 100 μM sinigrin (black mustard glycoside). Samples were taken at 3, 24, and 72 hours after treatment, and cell viability was assessed using the CCK8 assay. An aliquot of 100 µL of CCK8 solution was added to each well and incubated at 37°C for 3 hours. The absorbance at 450 nm was measured using an Microplate reader. The average absorbance and standard deviation of each group were calculated, and data analysis and plotting were performed using GraphPad Prism. One-way ANOVA was used to analyze the statistical differences in the data, followed by Tukey's post hoc test. A P-value less than 0.05 was considered statistically significant. The results, as shown in Figure 4AB, indicated a significant decrease in CT26 cell viability after co-expression of myrosinase and sinigrin, demonstrating significant in vitro anti-cancer activity of myrosinase that persisted within 72 hours.

Figure4 The results about in vitro anti-cancer activity of myrosinase

Potential application directions

This experiment has demonstrated that has the fastest reaction rate at a temperature of 37 degrees Celsius and pH value of 7.4, which are considered optimal reaction conditions for myrosinase. In the future, this can be applied by combining myrosinase with broccoli juice and subjecting them to a constant temperature machine at around 37 degrees Celsius and pH value of 7.4. This will convert glucosinolates in broccoli into sulforaphane, which can be used to inhibit cancer cells and treat colorectal cancer. This approach addresses the issue of mammals not expressing myrosinase, thus preventing colorectal cancer patients from converting glucosinolates into sulforaphane. It has promising prospects for development.

References

【1】 Worldwide cancer data | World Cancer Research Fund International (wcrf.org)

2019 NEU_CHINA

Characterization

Expressing vector has been constructed using pcold-1 backbone. We demonstrated SDS page and coomassie brilliant blue last year, but the result was not very convincing. So this time, western blotting is used to show the expression of myrosinase. Expressing vector harboring myrosinase gene is transformed into BL21 strain using chemical method. After incubating at 37℃ for 12h, CFU is inoculated to LB medium followed by 12h incubation. The culture is then diluted to OD600=0.2 and let grow to around 0.5. 1 mM IPTG is added to the culture to induce protein expression followed by 12h incubation. Cells are washed and collected, ready for western blotting. The molecular weight of protein myrosinase is 57.46 kDa.

Figure 1. Diagram for myrosinase expressing plasmid in pcold-1. Promoter cspA, a super strong promoter when incubating at low temperature. RBS, ribosome binding site, downstream is gene myrosinase.

Figure 2. Protein expression of myrosinase gene which transformed in BL21 strain. After induction of IPTG, the culture is incubated at 37℃ overnight. The concentration of protein loaded on this two lanes are different.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 139
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 368
  Illegal BsaI site found at 1397
  

MIT_MAHE 2020

Summary

Myrosinase (horseradish) is a present in cruciferous plants such as broccoli, Brussels sprouts and Celery cabbage. It can convert the precursor glucosinolates in cruciferous plants into sulforaphane, a well-known anticancer substance. Results have shown that it reduced nitric oxide, tumor necrosis factor-α and interleukin-6 release and nitric oxide synthase and cyclooxygenase-2 expression in macrophages, acting on nuclear transcription factor NF-κB p65 activation. Moreover, it has been shown to reduce reactive oxygen species release and increased heme-oxygenase-1 expression, thus contributing to the cytoprotective cellular effect during inflammation.

References

1. S. Marzocco , L. Calabrone , S. Adesso , M. Larocca , S. Franceschelli , G. Autore , G. Martelli and R. Rossano , Anti-inflammatory activity of horseradish (Armoracia rusticana) root extracts in LPS-stimulated macrophages, Food Funct., 2015, 6 , 3778 —3788