Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 9/30/2004
Publication Date: 7/1/2005
Citation: Smith, M.R., Zahnley, J.C. 2005. Characteristics of the amylase of arthrobacter psychrolactophilus. Journal of Industrial Microbiology and Biotechnology, 32:439-448. Interpretive Summary: Amylases which are efficient at low to moderate temperature are needed to reduce heating costs. We described an amylase produced by a cold-tolerant bacterium that can grow at 0 deg. C. The amylase hydrolyzed soluble starch or uncooked starch granules best at 50 deg. C on a synthetic substrate. The temperature was somewhat higher than we had found previously for soluble starch. Despite the high temperature for optimum activity,the enzyme was as sensitive to thermal inactivation as some psychrophilic enzymes. Substrates protected the amylase from thermal denaturation somewhat. Calcium was required for activity and half-maximal rates of hydrolysis occurred at 0.12 mM synthetic substrate at 22 deg. C. The amylase worked best at pH 5-9. Interfering protease contaminated crude amylase preparations, but they were eliminated by purification.
Technical Abstract: Properties of the extracellular amylase produced by the psychrotrophic bacterium, Arthrobacter psychrolactophilus, were determined for crude preparations and purified enzyme. The hydrolysis of soluble starch by concentrated crude preparations was found to be a non-linear function of time at 30 and 40 degree C. Concentrates of supernatant fractions incubated without substrate exhibited poor stability at 30, 40 or 50 C, with 87% inactivation after 21 hrs at 30 C, 45% inactivation after 40 min at 40 C and 90% inactivation after 10 min at 50 C. Proteases known to be present in crude preparations had a temperature optimum of 50 C, but accounted for a small fraction of the thermal instability. Inactivation at 30, 40 or 50 C was not slowed by adding 20 mg/ml bovine serum albumin or protease inhibitor cocktail to the preparations or the assays to protect against proteases. Purified amylase preparations were almost as thermally sensitive in the absence of substrate as crude preparations. The temperature optimum of the amylase in short incubations with Sigma Infinity Amylase Reagent was about 50 C, and the amylase required calcium ion for activity. The optimal pH for activity was 5.0-9.0 on soluble starch (30 C), and the amylase exhibited a Km with 4-nitrophenyl-a-D-maltoheptaoside-4,6-O-ethylidene of 120 mM at 22 C. The amylase in crude concentrates initially hydrolyzed raw starch at 30 degree C at about the same rate as an equal number of units of barley alpha-amylase, but lost most of its activity after only a few hours.