Alginate lyase is a biocatalyst that degrades alginate to produce oligosaccharides, which have many bioactive functions and could be used as renewable biofuels. Here we report a simple and sensitive plate assay for screening alginate lyase-excreting microorganisms from brown algae. Brown algae Laminaria japonica, Sargassum horneri and Sargassum siliquatrum were cultured in sterile water. Bacteria growing on the surface of seaweeds were identified and their capacity of excreting alginate lyase was analyzed. A total of 196 strains were recovered from the three different algae samples and 12 different bacterial strains were identified capable of excreting alginate lyases. Sequence analysis of the 16S rRNA gene revealed that these alginate lyase-excreting strains belong to eight genera: Paenibacillus (4/12), Bacillus (2/12), Leclercia (1/12), Isoptericola (1/12), Planomicrobium (1/12), Pseudomonas (1/12), Lysinibacillus (1/12) and Sphingomonas (1/12). Further analysis showed that the LJ-3 strain (Bacillus halosaccharovorans) had the highest enzyme activity. To our best knowledge, this is the first report regarding alginate lyase-excreting strains in Paenibacillus, Planomicrobium and Leclercia. We believe that our method used in this study is relatively easy and reliable for large-scale screening of alginate lyase-excreting microorganisms.

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Screening of alginate lyase-excreting microorganisms from the surface of brown algae.

Cold-adapted enzymes produced by organisms inhabiting permanently low temperature environments are typically characterized by a high activity at low to moderate temperatures and a poor thermal stability. Such characteristics make these enzymes highly attractive for various applications where they can enable more efficient, cost-effective, and environmentally friendlier processes than higher temperature-adapted enzymes. In this chapter, the biotechnological aspects of cold-adapted enzymes and their application in industry are reviewed and discussed with a focus on cleaning/detergents, food and beverages, molecular biology, biomedicine, pharmaceuticals, cosmetics, textiles, biofuels, and materials applications.

Biotechnological Aspects of Cold-Active Enzymes

Enzyme reaction products and by-products from pretreatment steps can inhibit endoglucanases and are major factors limiting the efficiency of enzymatic lignocellulosic biomass hydrolysis. The gene encoding the endoglucanase from Scytalidium thermophilum (egst) was cloned and expressed as a soluble protein in Pichia pastoris GS115. The recombinant enzyme (Egst) was monomeric (66 kDa) and showed an estimated carbohydrate content of 53.3% (w/w). The optimum temperature and pH of catalysis were 60–70 °C and pH of 5.5, respectively. The enzyme was highly stable at pH 3.0–8.0 with a half-life in water of 100 min at 65 °C. The Egst presented good halotolerance, retaining 84.1 and 71.4% of the control activity in the presence of 0.5 and 2.0 mol L−1 NaCl, respectively. Hydrolysis of medium viscosity carboxymethylcellulose (CMC) by Egst was stimulated 1.77-, 1.84-, 1.64-, and 1.8-fold by dithiothreitol, β-mercaptoethanol, cysteine, and manganese at 10, 10, 10, and 5 mmol L−1 concentration, respectively. The enzyme hydrolyzed CMC with maximal velocity and an apparent affinity constant of 432.10 ± 16.76 and 10.5 ± 2.53 mg mL−1, respectively. Furthermore, the Egst was tolerant to reaction products and able to act on pretreated fractions sugarcane bagasse demonstrating excellent properties for application in the hydrolysis of lignocellulosic biomass.

Overexpression of a Cellobiose-Glucose-Halotolerant Endoglucanase from Scytalidium thermophilum

A growing demand in novel food products for well-being and preventative medicine has attracted global attention on nutraceutical prebiotics. Various plant agro-processes produce large amounts of residual biomass considered “wastes”, which can potentially be used to produce nutraceutical prebiotics, such as manno-oligosaccharides (MOS). MOS can be produced from the degradation of mannan. Mannan has a main backbone consisting of β-1,4-linked mannose residues (which may be interspersed by glucose residues) with galactose substituents. Endo-β-1,4-mannanases cleave the mannan backbone at cleavage sites determined by the substitution pattern and thus give rise to different MOS products. These MOS products serve as prebiotics to stimulate various types of intestinal bacteria and cause them to produce fermentation products in different parts of the gastrointestinal tract which benefit the host. This article reviews recent advances in understanding the exploitation of plant residual biomass via the enzymatic production and characterization of MOS, and the influence of MOS on beneficial gut microbiota and their biological effects (i.e., immune modulation and lipidemic effects) as observed on human and animal health.

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Enzymatic Conversion of Mannan-Rich Plant Waste Biomass into Prebiotic Mannooligosaccharides.

Wines in their variety and styles owe a lot to many endogenous (grape fruit-generated) and exogenous (associated microbes-generated) enzymes. The role of enzymes begins with fruit pressing and juice extraction and continues to the time of bottling, thereby bringing about enormous chemical changes in the wine. Extensive understanding and data generated over the years enables winemakers to control various traits of wines through judicious use of commercial preparation of suitable enzymes. This chapter summarizes the unique physicochemical conditions during the various stages of winemaking (red and white), the important enzymes used (pectinases, glucanases, glycosidases, acid urease, glucose oxidase, acid proteases, and laccases) and their desirable features that help facilitate various practices and regulating attributes thereby determining the quality of the end product. In particular, the role of specific enzymes in the extraction, filtration and clarification of juice; and their use in the release of aromas from sugar-bound precursors, control of ethyl carbamate formation, reduction of alcohol levels, removal of haze-forming proteins, and prevention of the oxidative browning of wine have been discussed.

Wine Enzymes: Potential and Practices

Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake, and investigations into its prebiotic activity

Bos frontalis, which consumes 
bamboo and weeds, may have evolved unique gastrointestinal microorganisms that digest cellulase. A Paenibacillus sp. YD236 strain was isolated from B. frontalis feces, from which a GH8 endoglucanase gene, pglue8 (1107 bp, 54.5 % GC content), encoding a 368-residue polypeptide (PgluE8, 40.4 kDa) was cloned. PgluE8 efficiently hydrolyzed barley-β-d-glucan followed by CMC-Na, soluble starch, laminarin, and glucan from black yeast optimally at pH 5.5 and 50 °C, and retained 78.6, 41.6, and 34.5 % maximum activity when assayed at 20, 10, and 0 °C, respectively. Enzyme activity remained above 176.6 % after treatment with 10.0 mM β-mercaptoethanol, and was 83.0, 78, and 56 % after pre-incubation in 30 % (w/v) NaCl, 16.67 mg/mL trypsin, and 160.0 mg/mL protease K, respectively. Cys23 and Cys364 residues were critical for PgluE8 activity. pglue8, identified from B. frontalis feces for the first time in this study, is a potential alternative for applications including food processing, washing, and animal feed preparation.

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NaCl-, protease-tolerant and cold-active endoglucanase from Paenibacillus sp. YD236 isolated from the feces of Bos frontalis.

The invention discloses low temperature xylosidase HJ14GH43 and a salt-tolerant mutant thereof. The amino acid sequence of the xylosidase is as shown in SEQ ID No.1, and the nucleotide sequence of a xylosidase gene hJ14GH43 for coding xylosidase HJ14GH43 is as shown in SEQ ID No.2; the amino acid sequence of the salt-tolerant mutant is as shown in SEQ ID No.3. The optimum pH of xylosidase HJ14GH43 is 7.0, and the optimum temperature is 25 DEG C. According to the low temperature xylosidase HJ14GH43 and the salt-tolerant mutant thereof, site-directed mutation is conducted on the basis of xylosidase HJ14GH43, valine at the 322 locus is mutated to be aspartic acid, the mutant V322D is obtained, and the activity and stability of the mutant V322D in NaCl are both improved. The HJ14GH43 and the salt-tolerant mutant thereof can be applied to food industries and aquatic feed.

Low temperature xylosidase HJ14GH43 and salt-tolerant mutant thereof

The invention relates to the technical field of genetic engineering, in particular to salt-resistant ethanol-resistant trypsin-resistant xylosidase JB13GH39 and a preparation method thereof. The amino acid sequence of the xylosidase JB13GH39 is shown as SEQ No.1. The xylosidase JB13GH39 has the following characteristics that the optimum pH is 4.5; the remaining enzyme activity of the xylosidase JB13GH39 reaches 70% or above after the xylosidase JB13GH39 is processed for 1 h with a buffer solution with the pH of 4.0-9.0; the optimum temperature is 50 DEG C, the enzyme activity is achieved within the temperature range of 0 DEG C to 70 DEG C, and the enzyme activity is 52.8% at 20 DEG C; the xylosidase JB13GH39 is stable at 37 DEG C and 60 DEG C and is quickly deactivated at 70 DEG C; trypsin and most metal ions have no influence or have a slight influence on the activity of the xylosidase JB13GH39; the xylosidase JB13GH39 can hydrolyze xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose, and the hydrolysate mainly is xylose. The xylosidase can be applied to the industries of feed, food, biological energy sources and the like.

Salt-resistant ethanol-resistant trypsin-resistant xylosidase JB13GH39 and preparation method thereof

The invention relates to the technical field of genetic engineering and protein transformation, in particular to a xylanase thermohaline modified mutant and application thereof .The amino acid sequence of the mutant S27B05 is shown as SEQ ID NO.1 .Compared with wide enzymes, activity at 0 DEG C and 80 DEG C, heat stability at 37 DEG C, activity in 10.0 mM beta-Mercaptoethanol, CoCl2, MnSO4, Pb(CH3COOH)2, ZnSO4 and FeSO4, activity in NaCl of 15.0-30.0% (w/v) and stability in NaCl of 30.0% (w/v) of the mutation xylanase S27B05 are improved .The mutation xylanase S27B05 can be applied to technical fields such as food industry, aquatic feed, low-temperature and high-salt environment biont.

Wu Qian

Papers

Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake, and investigations into its prebiotic activity

NaCl-, protease-tolerant and cold-active endoglucanase from Paenibacillus sp. YD236 isolated from the feces of Bos frontalis.

Low temperature xylosidase HJ14GH43 and salt-tolerant mutant thereof

Salt-resistant ethanol-resistant trypsin-resistant xylosidase JB13GH39 and preparation method thereof

Xylanase thermohaline modified mutant and application thereof