Endo-β-1,3-glucanase plays an essential role in the deconstruction of β-1,3-d-glucan polysaccharides through hydrolysis. The gene (1650-bp) encoding a novel, bi-modular glycoside hydrolase family 64 (GH64) endo-β-1,3-glucanase (GluY) with a ricin-type β-trefoil lectin domain (RICIN)-like domain from Cellulosimicrobium funkei HY-13 was identified and biocatalytically characterized. The recombinant enzyme (rGluY: 57.5 kDa) displayed the highest degradation activity for laminarin at pH 4.5 and 40 °C, while the polysaccharide was maximally decomposed by its C-terminal truncated mutant enzyme (rGluYΔRICIN: 42.0 kDa) at pH 5.5 and 45 °C. The specific activity (26.0 U/mg) of rGluY for laminarin was 2.6-fold higher than that (9.8 U/mg) of rGluYΔRICIN for the same polysaccharide. Moreover, deleting the C-terminal RICIN domain in the intact enzyme caused a significant decrease (>60%) of its ability to degrade β-1,3-d-glucans such as pachyman and curdlan. Biocatalytic degradation of β-1,3-d-glucans by inverting rGluY yielded predominantly d-laminaripentaose. rGluY exhibited stronger growth inhibition against Candida albicans in a dose-dependent manner than rGluYΔRICIN. The degree of growth inhibition of C. albicans by rGluY (approximately 1.8 μM) was approximately 80% of the fungal growth. The superior anti-fungal activity of rGluY suggests that it can potentially be exploited as a supplementary agent in the food and pharmaceutical industries.

Endo-β-1,4-xylanase is a key enzyme in the degradation of β-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-β-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-β-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg2+, Ca2+, or Cu2+ but also significantly suppressed by 1 mM Ni2+, Zn2+, or Fe2+. However, its enzyme activity was upregulated (>1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-β-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-β-1,4-xylanase.

Endo-β-1,4-glucanase is a crucial glycoside hydrolase (GH) involved in the decomposition of cellulosic materials. In this study, to discover a novel cold-adapted β-1,4-D-glucan-degrading enzyme, the gene coding for an extracellular endo-β-1,4-glucanase (GluL) from Lichenicola cladoniae PAMC 26568, an Antarctic lichen (Cladonia borealis)-associated bacterium, was identified and recombinantly expressed in Escherichia coli BL21. The GluL gene (1044-bp) encoded a non-modular polypeptide consisting of a single catalytic GH8 domain, which shared the highest sequence identity of 55% with that of an uncharacterized protein from Gluconacetobacter takamatsuzukensis (WP_182950054). The recombinant endo-β-1,4-glucanase (rGluL: 38.0 kDa) most efficiently degraded sodium carboxymethylcellulose (CMC) at pH 4.0 and 45°C, and showed approximately 23% of its maximum degradation activity even at 3°C. The biocatalytic activity of rGluL was noticeably enhanced by >1.3-fold in the presence of 1 mM Mn²⁺ or NaCl at concentrations between 0.1 and 0.5 M, whereas the enzyme was considerably downregulated by 1 mM Hg²⁺ and Fe²⁺ together with 5 mM N-bromosuccinimide and 0.5% sodium dodecyl sulfate. rGluL is a true endo-β-1,4-glucanase, which could preferentially decompose D-cellooligosaccharides consisting of 3 to 6 D-glucose, CMC, and barley β-glucan, without other additional glycoside hydrolase activities. The specific activity (15.1 U mg^–1) and k_cat/K_m value (6.35 mg^–1 s^–1mL) of rGluL toward barley β-glucan were approximately 1.8- and 2.2-fold higher, respectively, compared to its specific activity (8.3 U mg^–1) and k_cat/K_m value (2.83 mg^–1 s^–1mL) toward CMC. The enzymatic hydrolysis of CMC, D-cellotetraose, and D-cellohexaose yielded primarily D-cellobiose, accompanied by D-glucose, D-cellotriose, and D-cellotetraose. However, the cleavage of D-cellopentaose by rGluL resulted in the production of only D-cellobiose and D-cellotriose. The findings of the present study imply that rGluL is a novel, acidic, and cold-adapted GH8 endo-β-1,4-glucanase with high specific activity, which can be exploited as a promising candidate in low-temperature processes including textile and food processes.

Poly(3-hydroxybutyrate) (PHB) is a versatile thermoplastic with superior biodegradability and biocompatibility that is intracellularly accumulated by numerous bacterial and archaeal species. Priestia sp. strain JY310 that was able to efficiently biotransform reducing sugars in d-xylose-rich rice husk hydrolysate (reducing sugar_RHH) to PHB was isolated from the soil of a rice paddy. Reducing sugar_RHH including 12.5% d-glucose, 75.3% d-xylose, and 12.2% d-arabinose was simply prepared using thermochemical hydrolysis of 3% H₂SO₄-treated rice husk for 15 min at 121 °C. When cultured with 20 g/L reducing sugar_RHH under optimized culture conditions in a batch bioreactor, Priestia sp. strain JY310 could produce PHB homopolymer up to 50.4% of cell dry weight (6.2 g/L). The melting temperature, heat of fusion, and thermal decomposition temperature of PHB were determined to be 167.9 °C, 92.1 J/g, and 268.1 °C, respectively. The number average and weight average molecular weights of PHB with a broad polydispersity index value (4.73) were estimated to be approximately 16.2 and 76.8 kg/mol, respectively. The findings of the present study suggest that Priestia sp. strain JY310 can be exploited as a good candidate for the low-cost production of low molecular weight PHB with improved biodegradability and reduced brittleness from inexpensive agricultural waste hydrolysates.

Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5–11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-β-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N′-diacetyl-β-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N′-diacetyl-β-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.

Endo-β-1,3-glucanase is a glycoside hydrolase (GH) that plays an essential role in the mineralization of β-glucan polysaccharides. In this study, the novel gene encoding an extracellular, non-modular GH16 endo-β-1,3-glucanase (GluH) from Hymenobacter siberiensis PAMC 29290 isolated from Arctic marine sediment was discovered through an in silico analysis of its whole genome sequence and subsequently overexpressed in Escherichia coli BL21. The 870-bp GluH gene encoded a protein featuring a single catalytic GH16 domain that shared over 61% sequence identity with uncharacterized endo-β-1,3-glucanases from diverse Hymenobacter species, as recorded in the National Center for Biotechnology Information database. The purified recombinant endo-β-1,3-glucanase (rGluH: 31.0 kDa) demonstrated peak activity on laminarin at pH 5.5 and 40°C, maintaining over 40% of its maximum endo-β-1,3-glucanase activity even at 25°C. rGluH preferentially hydrolyzed D-laminarioligosaccharides and β-1,3-linked polysaccharides, but did not degrade D-laminaribiose or structurally unrelated substrates, confirming its specificity as a true endo-β-1,3-glucanase without ancillary GH activities. The biodegradability of various substrate polymers by the enzyme was evaluated in the following sequence: laminarin > barley β-glucan > carboxymethyl-curdlan > curdlan > pachyman. Notably, the specific activity (253.1 U mg^–1) and catalytic efficiency (k_cat/K_m: 105.72 mg^–1 s^–1 mL) of rGluH for laminarin closely matched its specific activity (250.2 U mg^–1) and k_cat/K_m value (104.88 mg^–1 s^–1 mL) toward barley β-glucan. However, the k_cat/K_m value (9.86 mg^–1 s^–1 mL) of rGluH for insoluble curdlan was only about 9.3% of the value for laminarin, which correlates well with the observation that rGluH displayed weak binding affinity (< 40%) to the insoluble polymer. The biocatalytic hydrolysis of D-laminarioligosaccharides with a degree of polymerization between 3 and 6 and laminarin generally resulted in the formation of D-laminaribiose as the predominant product and D-glucose as the secondary product, with a ratio of approximately 4:1. These findings suggest that highly active rGluH is an acidic, cold-adapted D-laminaribiose- and D-glucose-releasing GH16 endo-β-1,3-glucanase, which can be exploited as a valuable biocatalyst for facilitating low temperature preservation of foods.