Thermostable Cellulases / Xylanases From Thermophilic and Hyperthermophilic Microorganisms: Current Perspective

The bioconversion of lignocellulose into monosaccharides is critical for ensuring the continual manufacturing of biofuels and value-added bioproducts. Enzymatic degradation, which has a high yield, low energy consumption, and enhanced selectivity, could be the most efficient and environmentally friendly technique for converting complex lignocellulose polymers to fermentable monosaccharides, and it is expected to make cellulases and xylanases the most demanded industrial enzymes. The widespread nature of thermophilic microorganisms allows them to proliferate on a variety of substrates and release substantial quantities of cellulases and xylanases, which makes them a great source of thermostable enzymes. The most significant breakthrough of lignocellulolytic enzymes lies in lignocellulose-deconstruction by enzymatic depolymerization of holocellulose into simple monosaccharides. However, commercially valuable thermostable cellulases and xylanases are challenging to produce in high enough quantities. Thus, the present review aims at giving an overview of the most recent thermostable cellulases and xylanases isolated from thermophilic and hyperthermophilic microbes. The emphasis is on recent advancements in manufacturing these enzymes in other mesophilic host and enhancement of catalytic activity as well as thermostability of thermophilic cellulases and xylanases, using genetic engineering as a promising and efficient technology for its economic production. Additionally, the biotechnological applications of thermostable cellulases and xylanases of thermophiles were also discussed.

Enzymatic depolymerization of wheat straw polysaccharides

The purpose of this study is to develop a technology for enzymatic processing for depolymerization of polysaccharides in wheat straw to obtain the maximum yield of glucose and sorbitol. Cellulolytic enzymes endo-1,4-β-glucanase (EC 3.2.1.4) and cellobiose (1,3-β-glucosidase) (CF 3.2.1.21) were isolated and studied in local strains Tr. viride 121, which are grown under deep cultivation conditions. A technology has been developed for obtaining a complex preparation “Cellozyme G20x” with a high yield and specific activity of cellulase, xylanase, β-glucanase and pectinase, and a scheme for purification from cellulases by precipitation, ultrafiltration, and freeze drying is not inferior in efficiency to commercial preparations. The physicochemical properties of the preparation “Cellozyme G20x” have been studied, the optimal parameters of the action and stability of the enzyme preparation have been established. The efficiency of Cellozyme G20x for hydrolysis of straw polysaccharides was 35-40% in terms of glucose yield.

New Insights into the Structure of Kappa/Beta-Carrageenan: A Novel Potential Inhibitor of HIV-1

New insights into the structure of the hybrid κ/β-carrageenan (κ/β-CRG) of the red alga Tichocarpus crinitus have been obtained. Carrageenan oligosaccharides were prepared through the chemical and enzymatic depolymerization of κ/β-CRG with κ-carrageenase and its the enzyme-resistant fraction. The composition and distribution of the repetition units of κ/β- CRG were investigated by using the negative ion tandem MALDI-TOFMS and ESIMS method, which made it possible to prove and characterize the hybrid structure of this polysaccharide. An analysis revealed the blockwise distribution of the long β-blocks along the polysaccharide chain, with the inclusion of κ/β, μ/ν-blocks and some ι-blocks. Furthermore, the desulfated κ/β-CRG was shown to contain of –G–D– repeating units up to 3.5 kDa. Previous studies have demonstrated that CRGs suppress the replication of several viruses. Here, we established that κ/β-CRG and its oligosaccharides significantly inhibit the transduction efficiency of replication-defective lentiviral particles pseudotyped with the envelope proteins of three different viruses. We found that the polysaccharide and its oligosaccharides strongly reduced the transduction efficiency of lentiviral particles pseudotyped with GP160—the envelope protein of the human immunodeficiency virus HIV-1—when added to T-lymphocyte Jurkat cells. The CRG oligosaccharides displayed significantly higher antiviral activity.

Enzymatic depolymerization of highly crystalline polyethylene terephthalate enabled in moist-solid reaction mixtures

Less than 9% of the plastic produced is recycled after use, contributing to the global plastic pollution problem. While polyethylene terephthalate (PET) is one of the most common plastics, its thermomechanical recycling generates a material of lesser quality. Enzymes are highly selective, renewable catalysts active at mild temperatures; however, they lack activity toward the more crystalline forms of PET commonly found in consumer plastics, requiring the energy-expensive melt-amorphization step of PET before enzymatic depolymerization. We report here that, when used in moist-solid reaction mixtures instead of the typical dilute aqueous solutions or slurries, the cutinase from Humicola insolens can directly depolymerize amorphous and crystalline regions of PET equally, without any pretreatment, with a 13-fold higher space-time yield and a 15-fold higher enzyme efficiency than reported in prior studies with high-crystallinity material. Further, this process shows a 26-fold selectivity for terephthalic acid over other hydrolysis products.

Depolymerization of hyaluronan using PEGylated human recombinant hyaluronidase promotes nanoparticle tumor penetration

Aim: Delivery of nanoparticles (NPs) to tumors can be impeded by high levels of hyaluronan (HA) in the stroma. Enzymatic depolymerization of HA with PEGylated hyaluronidase (PEGPH20) improves the delivery of antibodies to tumors. However, it is unknown whether NP delivery is enhanced by this strategy. Methods: The impact of PEGPH20 pretreatment on the uptake and tumor penetration of model PEGylated polystyrene NPs was studied in mice with orthotopic breast cancers. Results: Tumor oxygenation and NP penetration, but not overall tumor uptake, of 50 nm NPs, was significantly enhanced by PEGPH20 pre-administration. Conclusion: PEGPH20 has the potential to improve intratumoral penetration of NP-based drug delivery systems and enhance access to cancer cells in poorly vascularized regions of the tumor.

Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum

Fusarium graminearum produces an α-l-fucosidase, FgFCO1, which so far appears to be the only known fungal GH29 α-l-fucosidase that catalyzes the release of fucose from fucosylated xyloglucan. In our quest to synthesize bioactive glycans by enzymatic catalysis, we observed that FgFCO1 is able to catalyze a transglycosylation reaction involving transfer of fucose from citrus peel xyloglucan to lactose to produce 2′-fucosyllactose, an important human milk oligosaccharide. In addition to achieving maximal yields, control of the regioselectivity is an important issue in exploiting such a transglycosylation ability successfully for glycan synthesis. In the present study, we aimed to improve the transglycosylation efficiency of FgFCO1 through protein engineering by transferring successful mutations from other GH29 α-l-fucosidases. We investigated several such mutation transfers by structural alignment, and report that transfer of the mutation F34I from BiAfcB originating from Bifidobacterium longum subsp. infantis to Y32I in FgFCO1 and mutation of D286, near the catalytic acid/base residue in FgFCO1, especially a D286M mutation, have a positive effect on FgFCO1 transfucosylation regioselectivity. We also found that enzymatic depolymerization of the xyloglucan substrate increases substrate accessibility and in turn transglycosylation (i.e., transfucosylation) efficiency. The data include analysis of the active site amino acids and the active site topology of FgFCO1 and show that transfer of point mutations across GH29 subfamilies is a rational strategy for targeted protein engineering of a xyloglucan-active fungal α-l-fucosidase.