Purification and biochemical characterization of novel α-amylase and cellulase from Bacillus sp. PM06

Bacillus sp. PM06, previously isolated from sugarcane waste pressmud, could produce dual enzymes alpha-amylase and cellulase. The isolate's crude enzymes were purified homogeneously using ammonium sulfate precipitation followed by High Quaternary amine anion exchange chromatography. Purified enzymes revealed the molecular weights of alpha-amylase and cellulase as 55 and 52 kDa, with a purification fold of 15.4 and 11.5, respectively. The specific activity of purified alpha-amylase and cellulase were 740.7 and 555.6 U/mg, respectively. It demonstrated a wide range of activity from pH 5.0 to 8.5, with an optimum pH of 5.5 and 6.4 for alpha-amylase and cellulase. The optimum temperature was 50 degrees C for alpha-amylase and 60 degrees C for cellulase. The kinetic parameters of purified alpha-amylase were 741.5 +/- 3.75 mu mol/min/mg(,) 1.154 +/- 0.1 mM, and 589 +/- 3.5/(s( )mM), using starch as a substrate. Whereas cellulase showed 556.3 +/- 1.3 mu mol/min/mg, 1.78 +/- 0.1 mM, and 270.9 +/- 3.8/(s( )mM) of Vmax,Km, K-cat/K-m,K- respectively, using carboxymethyl cellulose (CMC) as substrate. Among the various substrates tested, alpha-amylase had a higher specificity for amylose and CMC for cellulase. Different inhibitors and activators were also examined. Ca2+ Mg2+, Co2+, and Mn2+ boosted alpha-amylase and cellulase activities. Cu2+ and Ni2+ both inhibited the enzyme activities. Enzymatic saccharification of wheat bran yielded 253.61 +/- 1.7 and 147.5 +/- 1.0 mg/g of reducing sugar within 12 and 24 h of incubation when treated with purified alpha-amylase and cellulase. A more significant amount of 397.7 +/- 1.9 mg/g reducing sugars was released from wheat bran due to the synergetic effect of two enzymes. According to scanning electron micrograph analysis, wheat bran was effectively broken down by both enzymes.