Use of Nonspecific, Glutamic Acid-Free, Media and High Glycerol or High Amylase as Inducing Parameters for Screening Bacillus Isolates Having High Yield of Polyglutamic Acid

Table 2

Extracellular amylase: protease activity and PGA production by Bacillus strains.

Parameter/property	Bacillus  licheniformis T.		Bacillus  licheniformis F.		Bacillus  subtilis
	24 h	48 h	24 h	48 h	24 h	48 h
Amylase^a units/mL broth	5000	6744	4200	5995	3900	5578
Protease^b units/mL broth	42	50	42	56	42	72
Amylase: protease: 24 h, 48 h	120	134	100	107	93	76
PGA (g/L)^c in broth media with sodium citrate + glycerol (10, 40 g/L)		11 (±3.4)		22 (±1.2)		6 (±1.3)
PGA^c on solid media containing glucose (10–20 g/L)	+	+	(+)	(+)	−	−
PGA production^c on media containing starch (5–20 g/L) and amylase activity^d detected	−, A	−, A	−, A	−, A	+, A	+, A
PGA production^c on synthetic media containing starch (10 g/L), glucose (2.5 g/L); Amylase^d detected	+, A	+, A	−, A	−, A	+, A	+, A
PGA production^c on synthetic media containing starch (10 g/L), glucose (2.5 g/L), NaCl (10–100 g/L); Amylase^d	+, A	+, A	−, A	−, A	+, A	+, A
PGA production^c on synthetic media containing skimmed milk 20% v/v, pH 4–8; Protease detected^e	+, P	+, P	+, P	+, P	+, P	+, P

Amylase/ ^bProtease assay:  Bacillus  subtilis (nonmucoid strain) and Bacillus T. and F. were inoculated in 1% starch broth or 1% skimmed milk broth and incubated at 37°C and amylase/protease activity was assayed.
^cProduction medium (pH = 7): all substrates were added to Bushnell and Hass synthetic medium base (composition g/L: MgSO₄ 0.2, CaCl₂ 0.02, FeCl₃ 0.05, K₂HPO₄ 1, KH₂PO₄ 1, NH₄NO₃ 1, and agar 25.
+/(+) = PGA present/variable. Values in parentheses are the standard deviation. − = PGA absent.
^dA = amylase activity using starch agar (white/brown zone of hydrolysed starch visible after addition of Lugol’s iodine solution).
^eP = protease activity (clear zone against turbid background of skimmed milk agar).