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 α-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 α-amylase and cellulase as 55 and 52 kDa, with a purification fold of 15.4 and 11.5, respectively. The specific activity of purified α-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 α-amylase and cellulase. The optimum temperature was 50 °C for α-amylase and 60 °C for cellulase. The kinetic parameters of purified α-amylase were 741.5 ± 3.75 µmol/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 µmol/min/mg, 1.78 ± 0.1 mM, and 270.9 ± 3.8/(s mM) of Vmax, Km, Kcat/Km, respectively, using carboxymethyl cellulose (CMC) as substrate. Among the various substrates tested, α-amylase had a higher specificity for amylose and CMC for cellulase. Different inhibitors and activators were also examined. Ca2+ Mg2+, Co2+, and Mn2+ boosted α-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 α-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.

α-Amylase and cellulase production by novel halotolerant Bacillus sp.PM06 isolated from sugarcane pressmud.

A novel Bacillus sp.PM06 isolated from sugarcane waste pressmud was tested for extracellular α-amylase and cellulase enzyme production. The effect of different substrates, nitrogen sources, pH, and temperature on growth and extracellular enzyme production was examined. Bacillus sp.PM06 was able to grow with starch and carboxymethyl cellulose (CMC) as a sole source of carbon and ammonium chloride was found to be the best nitrogen source. Maximum enzyme production was obtained at 48 H for both α-amylase and cellulase. The optimal condition for measuring enzyme activity was found to be pH 5.5 at 50 °C for α-amylase and pH 6.4 at 60 °C for cellulase respectively. It was found that Bacillus sp.PM06 exhibited halotolerance up to 2 M Sodium chloride (NaCl) and Potassium chloride (KCl). The isolate could produce α-amylase in the presence of 2 M NaCl and 1 M KCl. However, the strain produced cellulase even in the presence of 2 M NaCl and KCl. Concomitant production of both enzymes was observed when the medium was supplemented with starch and CMC. A maximum of 31 ± 1.15 U/mL of amylase and 15 ± 1.5 U/mL of cellulase was produced in 48 H. The enzyme was partially purified by Ammonium sulphate (NH4 )2 SO4 precipitation with 2.2 and 2.3-fold purification.