Biofuel Cellulases: Diversity, Distribution and Industrial Outlook

Lavika Jain and Deepti Agrawal

Introduction

Presently, the global economy is driven by fossil fuels, which predominantly include oil, coal and natural gas. However, some of the major drawbacks associated with these fossil fuels include their dynamic prices, monopoly vs dependency, exhaustive nature, negative impact on terrestrial and aquatic ecosystems, growing environmental concerns on greenhouse gas (GHG) emissions, etc. (Owusu and Asumadu-Sarkodie 2016). As a sustainable alternative to these energy sources, thrust has been made to utilize renewable sources of energy. Besides harnessing the solar and wind power, dedicated efforts have also been made towards the development of biomass-to-biofuels conversion technologies that are cost and performance competitive with petroleum-based fuels. Since a variety of processes can be employed for technological exploitation of biomass offering wide product range, this form of feedstock seems more lucrative.

Biomass feedstock includes first-generation feedstock or food feedstock such as vegetable oil, plant starch, edible sugars, grains, vegetable fats, second-generation feedstock or non-food biomass such as dedicated energy crops, agricultural and forest waste and third-generation feedstock representing mostly algal biomass. Second- generation feedstock, or more precisely lignocellulosic feedstock, has especially gained considerable importance due to its abundant and sustainable supply, inexpensive and renewable nature, effective land usage, low GHG emissions and no controversy on food vs fuel (Saini et al. 2015). Typically, one of the dominant structural components of lignocellulosic feedstock is cellulose.

Cellulose represents the most abundant polysaccharide on the earth. The significance of this linear water insoluble homo-polymer increases further as (3-D-glucose forms its backbone. This fermentable sugar serves as the raw material for the production of versatile products such as biofuels and bio-based chemicals/petrochemicals. However, the cumulative effect of hydrogen bonding and Van der Waals interaction which confers crystallinity (Arantes and Saddler 2010), inefficient protonation of ano- meric oxygen and exo-anomeric effect (Loerbroks et al. 2013) contributes resistance to hydrolysis of this robust biopolymer.

Total cleavage of (3-1,4-glycosidic linkage is the prerequisite step for production of fermentable sugars at competitive or affordable cost. Several chemical and biological processes such as acid pretreatment, solid acid catalyst, ionic liquids, enzymatic hydrolysis and their combinations have been evaluated for the past three decades for complete depolymerization of cellulose (Wahlstronm and Suurnakki 2015). Use of a greener approach like enzymatic hydrolysis offers several advantages such as high product yields, milder operating conditions of temperature and pH at ambient pressures, lower energy inputs, better selectivity, etc.

Biofuel Cellulases: A Brief Description

Cellulases are the group of enzymes belong to glycoside hydrolase family, which cleaves the glycosidic bonds between two or more carbohydrate moieties. Particularly, biofuel cellulases comprise three essential enzyme components which act sequentially and synergistically to release glucose as a final product.

Endoglucanases or EG (EC 3.2.1.4)

They primarily attack in the middle of the cellulosic chain, especially flexible amorphous sites that predominantly contain bent and hydrated disordered regions. The final products of their bio-catalytic activity are oligosaccharides with varying lengths. Thus, their primary responsibility is the reduction in the degree of polymerization of lignocellulosic and cellulosic substrates. Structurally, this group of enzymes have open cleft capable of binding anywhere along the length of the cellulose molecule and that runs along the active site, which hydrolyses (3-1,4-glycosidic linkage (Bianchetti etal. 2013).

Cellobiohydrolyases or CBH or Exo-l,4-p-glucanases (EC 3.2.1.91)

They are processive (the ability of an enzyme to continuously perform its catalytic function without the substrate dissociation) enzymes which preferentially attack at the end of crystalline cellulose chain, sequentially cleaving cellobiose moieties. There are two classes of cellobiohydrolyases:

  • 1. Type I or CBHI - This class attacks the reducing ends of cellulose chains
  • 2. Type II or CBHII - This class attacks the non-reducing ends of cellulose chains

Unlike the endoglucanases, the active site of CBH is located in a tunnel (Wilson and Kostylev 2012). During the catalytic activity, the cellulose chain slides into the tunnel progressively, wherein every second glycosidic bond is exposed to the active site of the enzyme, thereby cleaving and liberating cellobiose units at the other end.

Cellobiases or |3-glucosidases / or BG (EC 3.2.1.21)

They hydrolyse cellobiose and short (soluble) cello-oligosaccharides to glucose. These enzymes primarily accelerate the extent of cellulose hydrolysis by relieving the EG and CBH, which suffer from product inhibition (due to cellobiose/cellodextrin accumulation), a characteristic feature of these cellulases.

Besides these three critical components of biofuel cellulases, there are key auxiliary non-hydrolytic enzymes (AE) which critically enhance the cellulose accessibility for cellulases. Some important enzymes which belong to this category include biocatalysts from the copper-dependent AA9 and AA10 families with oxidoreductase activity (Levasseur et al. 2013), swollenin (Gourlay et al. 2013) and cellobiose dehydrogenase or CDH (Kracher and Ludwig 2016).

Diversity in Microbial Cellulases

In terrestrial ecosystems, several eukaryotes and prokaryotes have evolved bio- catalytic mechanisms to harness the energy stored in the lignocellulosic biomass in the form of polysaccharides. Constant efforts have been made by the researchers to explore novel biomass-degrading microbes that can secrete a variety of lignocellu- lolytic enzymes. Evidence has been provided where these microbes could efficiently hydrolyse the cellulosic biomass to produce cheaper fermentable sugars for second- generation biofuels and biochemicals.

Table 18.1 highlights some of the microbial biofuel cellulases from fungal, bacterial and actinomycetes origin which have been isolated globally and tested for their biofuel applications.

Microbial cellulases exhibit two paradigms for depolymerizing cellulose, namely, complexed and non-complexed (Saini et al. 2015). Complexed systems, which are popularly known as cellulosomes, is the characteristic feature of anaerobic bacteria wherein protein scaffolds harbouring multi-enzyme complex proturbences from the cell surface and is stabilized by non-covalent cohesion-dockerin interactions.

TABLE 18.1

Microorganisms Secreting Cellulases for Biofuel Applications

Major group

Microorganism

References

Fungi

Aspergillus niger

Adav et al. (2010)

Chrysoporthe cubensis

Dutra et al. (2017)

Myceliophthora thermophila

Pereira et al. (2015)

Phanerochaete chrysosporium

Adav et al. (2012)

Penicillium echinulatum

Schneider et al. (2016)

Trichoderma reesei

Bischof et al. (2016)

Talaromyces verruculosus

Jain and Agrawal (2018)

Bacteria

Acidothermus cellulolyticus

Barabote et al. (2009)

Bacillus subtilis

Deka et al. (2013)

Geobacillus sp.

Potprommanee et al. (2017)

Caldicellulosiruptor

Zurawski et al. (2015)

Clostridium thermocellum

Nislia et al. (2017)

Brevibacterium JXL

Liang et al. (2009)

Paenibacillus sp. strain B39

Wang et al. (2008)

Actinomycetes

Cellulomonas ftrni

Kane and French (2018)

Streptomyces

Hsu et al. (2011)

Thermobifida fusca

del Pulgar and Saadeddin (2013)

Non-complexed system symbolize extracellular enzymes, which are secreted by microbes as discrete enzymes with independent functioning. They are predominantly found in aerobic bacteria, actinomycetes and fungi (Payne et al. 2015).

Furthermore, with the advent of sophisticated research, analytical tools such as the whole genome, transcriptome and secretome analysis, the vast repertoire of genes encoding for plant cell wall degrading enzymes in these microbes has also been elucidated.

The Molecular Structure of Cellulases and Mechanistic Action

Cellulases are multi-modular biocatalysts composed of structurally and functionally distinct units that are often folded independently. These discrete structures are often referred to as either domains or modules (Maki et al. 2009). Most commonly, cellulases consist of one catalytic domain (CD) and one carbohydrate binding module (CBM), which are bridged by a relatively long (30-44 amino acids) linker peptide which is often glycosylated.

The primary role of the CBM is to accommodate the cellulases and increase the effective concentration of cellulose surface and facilitate the CD to remain in close proximity with the substrate for prolonged periods, thereby enabling efficient hydrolysis of the substrate (Shoseyov et al. 2006). CBMs are distributed across 49 families, ranging from small peptides (30-40 amino acids) to modules (-200 amino acids) as reviewed by Shoseyov et al. (2006). Several pieces of evidence have been provided by the researchers where an additional and unique role of CBMs has been revealed, that is, non-hydrolytic disruption of cellulose (Reyes-Ortiz et al. 2013).

Linker peptide, which connects CD and CBM, is highly divergent and exhibits low sequence conservation or apparent homology. However, irrespective of their source of origin, most of the linker peptides are rich in proline, glycine and hydroxyl amino acids, namely, threonine and serine (Sammond et al. 2012). Their importance in efficient cellulose hydrolysis is paramount as their precise length provides sufficient spatial separation of the two domains (CD and CBM), besides its flexibility.

CDs catalyse the cellulose hydrolysis and have been classified into 12 families based on hydrophobic cluster analysis (HCA) and amino acid homology (Ohmiya et al. 1997). Cleavage of the (3-1,4-glycosidic bond is accomplished by two different mechanisms, namely, inverting and retaining.

Inverting Mechanism

It is a one-step displacement mechanism where the charged environment of the catalytic site activates the water molecule to act as a nucleophile, while an acidic amino acid residue, namely, glutamic or aspartic acid, acts as proton donor. The mechanism derives its name from the fact that during the hydrolysis, there is linkage inversion (p to a configuration) at the anomeric carbon of the sugar.

Retaining Mechanism

It works via double displacement and proceeds in two steps. In the first step, glyco- sylation, a covalently bound intermediate is formed through nucleophilic attack of the acidic amino acid on the glycosidic bond. In the second step, deglycosylation, water molecule frees the hydrolysis product from the enzyme and recharges the proton donor.

Designation and Distribution of Cellulases in Glycoside-Hydrolase (GH) Families

The Carbohydrate-Active Enzymes database (CAZy; www.cazy.org) comprehensively describes the designation and distribution of cellulases, which belong to the GH superfamily. GH primarily attacks the glycosidic linkages between carbohydrates or between carbohydrates and non-carbohydrate moieties. Cellulases, in general, are phylogenetically distributed in 14 different families of GH, which are distinctly different from one another based on their mechanism of action (inverting or retaining), protein structure and specificity to attack the glycosidic linkage. Among the three essential components of the cellulolytic complex, the following is the distribution.

CBHI

These enzymes are phylogenetically distributed in two families, namely, GH7 and GH48 (Tables 18.2 and 18.3). If GH7 is characteristic of aerobic fungi, bacterial CBHI mostly belongs to the GH48 family. CBHI from anaerobic fungi also belong to the GH48 family.

TABLE 18.2

Distribution of Representative Fungal Cellulases Across Different GH Families Based on CAZy Classification

Enzymes

CAZy

Classification

(Family)

Structure/

Mechanism

Fungi

References

CBHI

GH7

p-jelly roll; Retaining

Aspergillus niger fi!TCC 1015 Trichoderma reesei QM9414 Penicillium oxalicum 114-2 Phanerockaete chrysosporium

Gong et al. (2015) Gong et al. (2015) Gong et al. (2015) Adav et al. (2012)

GH48

(ala.) 6; Inverting

Piromyces sp.

Steenbakkers et al. (2002)

EG

GH5

(p/0t)8;

Retaining

Aspergillus niger ATCC 1015 Trichoderma reesei QM9414 Penicillium oxalicum 114-2

Gong et al. (2015) Gong et al. (2015) Gong et al. (2015)

GH6

p/O. Barrel; Inverting

Humicola insolens (Structurally similar to CBH but functions as EG)

Davies et al. (2000)

GH7

p-jelly roll; Retaining

Trichoderma reesei QM9414 Penicillium oxalicum 114-2

Gong et al. (2015) Gong et al. (2015)

GH12

p-jelly roll; Retaining

Aspergillus niger fiCrCC 1015 Trichoderma reesei QM9414 Penicillium oxalicum 114-2 Phanerockaete chrysosporium

Gong et al. (2015) Gong et al. (2015) Gong et al. (2015) Adav et al. (2012)

GH45

p6-Barrel;

Inverting

Trichoderma reesei QM9414 Penicillium oxalicum 114-2 Myceliophthom thermophila

Gong et al. (2015) Gong et al. (2015) Kolbusz et al. (2014)

GH61a

-

Phanerockaete chrysosporium

Adav et al. (2012)

BG

GH1

(p/a)8;

Retaining

Aspergillus niger ATCC 1015 Trichoderma reesei QM9414 Penicillium oxalicum 114-2 Phanerockaete chrysosporium

Gong et al. (2015) Gong et al. (2015) Gong et al. (2015) Adav et al. (2012)

GH3

(p/a)8;

Retaining

Aspergillus niger fiCTCC 1015 Trichoderma reesei QM9414 Penicillium oxalicum 114-2 Phanerockaete chrysosporium Rasamsonia emersonii

Gong et al. (2015) Gong et al. (2015) Gong et al. (2015) Adav et al. (2012) Gudmundsson et al. (2016)

Note: GH61a has been reclassified as the AA9 family.

CBHII

These enzymes are phylogenetically distributed in two families, namely GH6 and GH9 (Tables 18.2 and 18.3). However, the GH9 family is predominantly found in bacteria rather than fungi.

EG

Endoglucanases belong to 17 different GH families; however, GH8, GH44 and GH51 are the characteristic features of bacterial EGs whereas the GH9 type is found mostly in plants and bacteria.

TABLE 18.3

Distribution of Representative Bacterial and Actinomycetes Cellulases Across Different GH Families Based on CAZy Classification

Enzymes

CAZy

Classification

(Family)

Structure/

Mechanism

Bacteria and Actinomycetes

References

CBHI

GH48

(a/a)6; Inverting

Thermobifida fusca

Wilson etal. (2012)

CBHII

GH6

p/a Barrel; Inverting

Thermobifida fusca

Wilson et al. (2012)

EG

GH5

(p/a)8; Retaining

Fibrobacter succinogenes S85

Iyo et al. (1996)

GH6

p/а Barrel:;lnverting

Acidothermus

cellulolyticus

Barabote (2009)

GH8

(a/a)6; Inverting

Bacillus circulans

Hakamada et al. (2002)

GH9

(a/a)6; Inverting

Caldicellulosiruptor

Brunecky(2017)

GH44

(p/a)8; Retaining

Ruminococcus

flavefaciens

Rincon et al. (2001)

GH45

рб-Barrel; Inverting

Cellvibrio japonicus

Brumm (2013)

GH48

(a/a)6; Inverting

Caldicellulosiruptor

Brunecky(2017)

GH51

(p/a)8; Retaining

Fibrobacter succinogenes S85

Malburg et al. (1997)

GH74

7-fold-p- propeller; Inverting

Acidothermus

cellulolyticus

Barabote (2009)

BG

GH1

(p/a)8; Retaining

Mucilaginibacter L294 Pedobacter 048

Lopez-Mondejar et al. (2016)

GH3

(p/a)8; Retaining

Mucilaginibacter L294 Pedobacter 048

Lopez-Mondejar et al. (2016)

GH5

(p/a)8; Retaining

Acidothermus

cellulolyticus

Barabote (2009)

GH9

(a/a)6; Inverting

Clostridium

thermocellum

Schubot et al. (2004)

GH12

p-jelly roll; Retaining

Streptomyces sp.

Book et al. (2016)

BG

The distribution of (3-glucosidase enzymes could be seen in six different families of GH. However, fungal BGs belong to either the GH1 or GH3 family. But bacterial BGs show a ubiquitous distribution as shown in Table 18.3.

Tables 18.2 and 18.3 show some representative microbes belonging to various GH families, which are principally known to degrade lignocellulosic biomass, as investigated by multiple researchers.

 
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