A mannanase, ManA, of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has carbohydrate binding and docking modules

Abstract

The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or beta-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 degrees C, but was rapidly inactivated at 60 degrees C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.

Fig. 1.

Illustration of module organizations of some mannanases from various microbial sources. O-ManA, Orpinomyces mannanase A; OS-ManA, Orpinomyces mannanase A expressed in S. cerevisiae with NCDM truncated; M-CBM, Orpinomyces mannanase A CBM expressed in E. coli; TrMan1, T. reesei mannanase 1 (Stålbrand et al. 1995); AaMan1, Aspergillus aculeatus mannanase 1 (Christgau et al. 1994); AbCEL4, Agaricus bisporus CEL4 (Tang et al. 2001; Yagüe et al. 1997); P-Mans, mannanases of the anaerobic fungus Piromyces equi (Fannuti et al. 1995; Millward-Sadler et al. 1996).

Fig. 2.

Amino acid sequence alignment of the catalytic domains of fungal mannanases belonging to family 5 glycoside hydrolases of Orpinomyces PC-2 (O-ManA), T. reesei (TrMan1; Stålbrand et al. 1995), Aspergillus aculeatus (AaMan1; Christgau et al. 1994), and Agaricus bisporus (AbCEL4; Yagüe et al. 1997).

Fig. 3.

Alignment of the Orpinomyces mannanase CBM (O-ManA) with those from T. reesei mannanase (T-Man1; Stålbrand et al. 1995), cellobiohydrolase I (T-CbhI; Shoemaker et al. 1983), cellobiohydrolase II (T-CbhII; Teeri et al. 1987), endoglucanase I (T-EngI; Penttilä et al. 1986), endoglucanase II (T-EngII; Saloheimo et al. 1988), and endoglucanase V (T-EngV; Saloheimo et al. 1994). Identical amino acids are in bold.

Fig. 4.

Mannanase production by S. cerevisiae transformed with pManA after galactose induction. An aliquot of an overnight culture grown in DOB medium was used to inoculate raffinose – YPD medium. After growth to an OD₆₀₀ of 1.0, sterile galactose was added. Samples were withdrawn at time points shown in the figure. Levels of OD₆₀₀ (open symbols) and extracellular mannanase activity (filled symbols) are shown for the transformants containing pManA and pYES2.

Fig. 5.

SDS–PAGE analysis of the Orpinomyces ManA produced in S. cerevisiae and ManA CBM produced in E. coli. Gel was stained with Coomassie Brilliant Blue R-250 and destained before photographs were taken. Lane M, low molecular weight standards (Bio-Rad); Lane 1 of Panel A, 40-μg proteins of yeast culture supernatant; Lane 2 of Panel A, 10 μg ManA purified from yeast culture supernatant; Lane 1 of Panel B; 15 μg ManA CBM purified from E. coli cell lysate.

Fig. 6.

Effect of pH on the activity of ManA assayed at 50 °C. For details see “Materials and methods” section.