Enzymes for the cellulose liquefaction: Thermophilic enzymes

The thermophilic microrganisms can be grouped in thermophiles (growth up to 60 °C), extreme thermophiles (65-80 °C) and hyperthermophiles (85-110 °C). The unique stability of the enzymes produced by these microrganisms at elevated temperatures, extreme pH and high pressure (up to 1000 bar) makes them a valuable resource for the industrial

Commercial

mixture

FPU

(U/ml)a

Cellobiase

(U/ml)b

Proteins

(U/ml)c

Source

Supplier

Bio-feed beta L

<5

12

8

T. longibrachiatum T. reesei

Novozymes

(Bagsvaerd,

Denmark)

Cellubrix

(Celluclast)

56

136

43

T. longibrachiatum A. niger

Novozymes

Cellulase 2000L

10

nd

7

T. longibrachiatum T. reesei

Rodhia — Danisco (Vinay, France)

Cellulyve 50L

24

nd

34

T. longibrachiatum T. reesei

Lyven (Colombelles France)

Energex L

<5

19

28

T. longibrachiatum T. reesei

Novozymes

GC220

116

215

64

T. longibrachiatum T. reesei

Genencor-Danisco (Rochester, USA)

GC440

<5

70

29

T. longibrachiatum T. reesei

Genencor

GC880

<5

86

43

T. longibrachiatum T. reesei

Genencor

Novozymes 188

<5

1,116

57

A. niger

Novozymes

Rohament CL

51

28

44

T. longibrachiatum T. reesei

Rhom-AB Enzymes (Rajamaki, Finland)

Spezyme CP

49

nd

41

T. longibrachiatum T. reesei

Genencor

Ultraflo L

<5

20

18

T. longibrachiatum T. reesei

Novozymes

Viscozyme L

<5

23

27

T. longibrachiatum T. reesei

Novozymes

Viscostar 150L

33

111

40

T. longibrachiatum T. reesei

Dyadic (Jupiter, Usa)

A) One FPU (filter paper unit) is the amount of enzyme that forms 1 pmol of reducing sugars/min during the hydrolysis reaction of filter paper Whatman No.1

B) One CBU (cellobiase unit) corresponds to the amount of enzyme which forms 2 pmol of glucose/min from cellobiose

Table 4. Commercial cellulases

Commercial mixture

B-glucosidase

activity(U/ml)a

pH

Temperature (°C)

Source

Supplier

Biocellulase A

32

5

55

A. niger

Quest Intl. (Sarasota, Fl)

Cellulase AP 30 K

60

4.5

60

A. niger

Amano Enzyme Inc.

Table 5. Commercial cellulases able to work at temperature ranging from 50 to 60°C.

bioprocesses that run at harsh conditions (Demain et al., 2005). Of special interest is the thermoactivity and thermostability of these enzymes in the presence of high concentrations of organic solvents, detergents and alcohols. On the whole, thermophilic enzymes have an increased resistance to many denaturing conditions such as the use of detergents which can be often the unique efficient mean to obviate the irreversible adsorption of cellulases on the substrates. Furthermore, the utilization of high operation temperatures, which cause a decrease in viscosity and an increase in the diffusion coefficients of substrates, have a significant influence on the cellulose solubilization. It is worth noting that, differently from the mesophilic enzymes, most thermophilic cellulases did not show inhibition at high level of reaction products (e. g. cellobiose and glucose). As consequence, higher reaction rates and higher process yields are expected (Bergquist et al., 2004). The high process temperature also reduces any contamination of the fermentation medium.

Several cellulose degrading enzymes from various thermophilic organisms have been investigated. These include cellulases mainly isolated from anaerobic bacteria such as Anaerocellum thermophilum (Zverlov et al., 1998), Clostridium thermocellum (Romaniec et al., 1992), Clostridium stercorarium (Bronnenmeier et al., 1991; Bronnenmeier & Staudenbauer, 1990) and Caldocellum saccharolyticum (Te’o V et l., 1995), Pyrococcus furiosus (Ma & Adams,

1994) , Pyrococcus horikoshi (Rahman et al.,1998), Rhodothermus strains (Hreggvidsson et al., 1996), Thermotoga sp., (Ruttersmith et al., 1991), Thermotoga marittima (Bronnenmeier et al.,

1995) , Thermotoga neapolitana (Bok et al., 1998).

Xylanase have been detected in Acidothermus cellulolyticus in different Thermus, Bacillus, Geobacillus, Alicyclobacillus and Sulfolobales species (Sakon et al., 1996).

Although many cellulolytic anaerobic bacteria such as Clostridium thermocellum produce cellulases with high specific activity, they do not produce high enzymes quantities. Since the anaerobes show limited growth, most researches on thermostable cellulases production have been addressed to aerobic species. Several mesophilic or moderately thermophilic fungal strains are also known to produce enzymes stable and active at high temperatures. These enzymes are produced from species such as Chaetomium thermophila (Venturi et al., 2002), Talaromyces emersonii (Grassick et al., 2004), Thermoascus aurantiacus (Parry et al., 2002). They may be stable at temperatures around 70 °C for prolonged periods. Table 6 summarizes some of thermostable enzymes isolated from Archea, Bacteria and Fungi. During the last decade several efforts have been devoted to develop different mixtures of selected thermostable enzymes. In 2007, mixtures of thermostable enzymes, including cellulases from Thermoascus auranticus, Thrichoderma reseei, Acremonium thermophilum and Thermoascus auranticus, have been produced by ROAL, Finland (Viikari et al., 2007). Multienzyme mixtures were also reconstituted using purified Chrysosporium lucknowense enzymes (Gusakov et al., 2005).

Despite the noticeable advantages of thermostable enzymes, cultivation of thermophiles and hyperthermophyles requires special and expensive media, and it is hampered by the low specific growth rates and product inhibition (Krahe et al., 1996; Schiraldi et al., 2002;Turner et al., 2007). Large scale commercial production of thermostable enzymes still remains a challenge also dependent on the optimization of their production from mesophilic microorganisms.