aLP Lysobacter Enzymogenes Prep

(Modified version from Jonathan Davis’s Thesis, Chapter 2, 1996)

Bachovchin’s Media

For 1L:

              Casamino acids                                        2 g

                 MSG                     (187.14g/mol)            12 g  **(or glutamic acid + NaOH to pH 7.0)

                 K₂HPO₄·H₂O        (174.18g/mol)            1.2 g

                 NaCl                      (58.44 g/mol)            1.2 g

                 MgSO₄·7H₂O       (246.48 g/mol)          0.6 g

                 ZnSO₄·7H₂O        (287.54 g/mol)          0.004 g

                 MnSO₄·H₂O         (169.01g/mol)            0.003 g

                 Fe₂(SO₄)₃               (399.9 g/mol)            0.015 g  (add to each flask individually)

            Adjust to pH 7.0 (with HCl).

            Autoclave.

            Add:   Sucrose (sterile filtered)       60 g  **  

                        MEM vitamins (100x)   1:100 (increases yield by 10%)

Can make stocks of metals, sterilize, and add to 1x media. 

Fe₂SO₄ remains solid even in 1x media, but rest should dissolve.

Prep Protocol:

1.              Grow up L-plates from freezer stock (see box marked lysobacter) à 2 nights at 28C

2.             Pick 1 colony in 100 mL Bachovchin’s media (in 500 mL flask) à grow over 1-2 nights at 74 rpm (28C)  (I had good success at 100 rpm, June2001)

3.             Inoculate 300 mL Bachovchin’s media (in 2800 mL triple-dimpled Fernbach) from the overnight, 1:50 dilution (I inoculated with 10mL)à grow at 28C @ 72-74 rpm (again, I used 100rpm; but watch to prevent frothing!!)

4.             Maintain pH < 9.0

5.             Assay activity until it plateaus (410 nm, Tris-Cl (0.1 M, pH 8.0) + substrate (suc-APA-p-Na, 1.0 mM).  mg/L enzyme = (DOD/min) / (0.42 · mL enzyme used) ; De = 8860.

6.             Growth probably for 2-3 days.  (yield of 100-400mg (or more?) aLP)

                       

Maximum yield after 48-72 hours after inoculation.

Watch pH—tends to increase over time in Bachovchin’s Media.

 

**Sucrose appears to be very important!  10g/L gives yield of only about 30g/L.  80g/L gives about 200 mg/L a-lytic protease; 60g/L gave most aLP (about 400mg/L).  (June 2001)