Structural and biochemical properties of a novel pullulanase of Paenibacillus lautus DSM 3035

Abstract

The pullulanase gene (pul PL ), encoding a novel type I pullulanase (Pul PL ), was obtained from a Paenibacillus lautus DSM3035 isolate. The gene has an open reading frame of 2355 bp, After optimizing induction conditions in Escherichia coli, we overexpressed recombinant Pul PL , purified this enzyme, and assayed its function . The level of functional Pul PL -like protein reached its maximum (about 0.28 mg/mL, 15% of total protein) after induction for 16 h at 20°C. Under these optimized harvesting conditions, Pul PL activity was 11.1 U/mL. The purified recombinant enzyme with an apparent molecular mass of about 87.9 kDa was able to specifically attack the a-1,6 linkages in pullulan to generate maltotriose as the major product. The purified Pul PL exhibited optimal activity at pH 7.0 and 40°C. The Pul PL hydrolyzed pullulan, amylopectin, starch, and glycogen, but not amylose. Substrate specificity observations and reaction products identifications indicate that the purified pullulanase from P. lautus DSM3035 is a type I pullulanase. The present report, to our knowledge, the first to identify type I pullulanase in P.lautus, and detail the enzymatic properties of this enzyme after heterologous expression.

Properties of a neutral, thermally stable and surfactant-tolerant pullulanase from worker termite gut-dwelling Bacillus safensis as potential for industrial applications

“…Using molecular technique, subunit molecular weight of the purified pullulanase (Pull) was estimated to be 42.34 kDa. The molecular mass obtained in this study is found to be lower than that of Paenibacillus lautus , 87.9 kDa ( Chen et al., 2016 ). Much higher molecular weight was observed for pullulanase of Klebsiella pneumonia (94 kDa) ( Abdul-Hadi and Al-Bayyar, 2019 ), Bacillus acidopullulyticus (102 kDa) ( Lappalainen et al., 1991 ), Pyrobaculum calidonfontis (111 kDa) ( Rehman et al., 2018 ), white edible mushroom (112 kDa) ( Shehata et al., 2016 ), Bacillus sp (136 kDa) ( Orhan et al., 2014 ).…”

“…The optimum pH 7.0 exhibited by B. safensis pullulanase isolated from the gut of termite is consistent with Paenibacillus lautus pullulanase ( Chen et al., 2016 ), and B. subtilis and Geobacillus thermoleovorans NP 33 pullulanase ( Nisha and Satyanarayana, 2018 ). Significantly, the enzyme exhibited high relative activity of 91 and 94, respectively at acidic pH 5.0 and 6.0 respectively.…”

“…C. thermohydrosulfuricum ( Saha et al., 1988 ) and K. pneumoniae ( Abdul-Hadi and Al-Bayyar (2019) ) pullulanase exhibited maximum stability at pH 3.0–5.0 and 6–7 respectively. Meanwhile, Chen et al. (2016) reported Paenibacillus lautus pullulanase to attain its pH stability at pH 6.5–9.0 with 80% residual activity.…”

“…S-1 ( Kim et al., 1993 ) and K. pnuemoniae ( Abdul-Hadi and Al-Bayyar (2019) ) pullulanase. However, Chen et al. (2016) and Shehata et al.…”

Biochemical Characterization of a Novel Thermostable Type I Pullulanase Produced Recombinantly in Bacillus subtilis

“…The previous articles described the methods for heterologous protein expression and Ni‐NTA purification procedures in our group . Pullulanase activity of Pul GK was measured in triplicate spectrophotometrically at 65 °C using the 3,5‐dinitrosalicylic acid (DNS) method .…”