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Chitosanases play an important role in chitosan degradation, forming enzymatic degradation products with several biological activities. Although many chitosanases have been discovered and studied, the enzymes with special characteristics are still rather rare. In this study, a new chitosanase, CsnM, with an apparent molecular weight of 28 kDa was purified from the marine bacterium Pseudoalteromonas sp. SY39. CsnM is a cold-adapted enzyme, which shows highest activity at 40 °C and exhibits 30.6% and 49.4% of its maximal activity at 10 and 15 °C, respectively. CsnM is also a thermo-tolerant enzyme that recovers 95.2%, 89.1% and 88.1% of its initial activity after boiling for 5, 10 and 20 min, respectively. Additionally, CsnM is an endo-type chitosanase that yields chitodisaccharide as the main product (69.9% of the total product). It’s cold-adaptation, thermo-tolerance and high chitodisaccharide yield make CsnM a superior candidate for biotechnological application to produce chitooligosaccharides.

The chitosanase production by Paenibacillus ehimensis was studied in submerged cultures and the chitosan hydrolysis was evaluated by using these enzymes without purification. The bacterium produced inducibles enzymes after 12 h of growth in a culture medium containing 0.2% (w/v) of soluble chitosan as carbon source. The enzyme production was strongly repressed by the presence of glucose. The production started as soon as the available sugars finished in the culture medium. The maximum level of chitosanase activity was 500 U.L-1 at 36°C after 36 h incubation. The crude enzyme was optimally active at pH 6.0 and 55°C and in these conditions, the enzyme presented good stability (6 days). The enzyme without purification was used to hydrolyze the chitosan which resulted chitooligosaccharides between 20 and 30 min of reaction.A produção de quitosanases pelo Paenibacillus ehimensis foi estudada em culturas submersas e a hidrólise da quitosana foi realizada utilizando essas enzimas sem purificação. As enzimas foram obtidas após 12 horas de crescimento desta bactéria em meio de cultivo contendo 0,2% (p/v) de quitosana solúvel como fonte de carbono. A produção das enzimas foi fortemente reprimida na presença de glicose, sendo obtida após o consumo total dos açúcares disponibilizados no referido meio de cultivo. A máxima atividade quitosanolítica foi obtida após 36 horas de cultivo a 36ºC, atingindo valores de 500 U.L-1. As enzimas utilizadas no extrato bruto apresentaram melhores atividades quando submetidas a condições de pH e temperatura de 6,0 e 55ºC, respectivamente, e nessas condições permaneceram estáveis durante 6 dias. Essas enzimas sem serem submetidas aos processos de purificação foram utilizadas para hidrolisar a quitosana, obtendo os quito-oligossacarídeos entre 20 e 30 minutos de reação.

Silver nanoparticles (AgNPs) are of great interest due to their antimicrobial, optical and catalytical properties. Green synthesis of AgNPs is fundamental for some applications such as biomedicine and catalysis. Natural polymers, such as chitosan, have been proposed as reducing and stabilizing agents in the green synthesis of AgNPs. Physico-chemical properties of chitosan have a great impact on its technological and biological properties. In this paper, we explore the effect of chitosan molecular weight (Mw) on the thermal AgNPs production using two sample sets of low Mw chitosans (F1 > 30 kDa, F2: 30–10 kDa and F3: 10–5 kDa) produced by enzymatic depolymerization of a parent chitosan with chitosanase and lysozyme. Both polymer sets were able to effectively reduce Ag+ to Ag0 as the presence of the silver surface plasmon resonance (SRP) demonstrated. However, the ability to stabilize the nanoparticles depended not only on the Mw of the polymer but particularly on the polymer pattern which was determined by the enzyme used to depolymerize the parent chitosan. Low Mw chitosan samples produced by lysozyme were more effective than those produced by chitosanase to stabilize the AgNPs and smaller and less polydisperse nanoparticles were visualized by transmission electron microscopy (TEM). With some polymer sets, more than 80% of the AgNPs produced were lower than 10 nm which correspond to quantum dots. The preparation method described in this paper is general and therefore, it may be extended to other noble metals, such as palladium, gold or platinum.

Chitooligosaccharides (CHOS) are homo- or heterooligomers of N-acetylglucosamine and D-glucosamine. CHOS can be produced using chitin or chitosan as a starting material, using enzymatic conversions, chemical methods or combinations thereof. Production of well-defined CHOS-mixtures, or even pure CHOS, is of great interest since these oligosaccharides are thought to have several interesting bioactivities. Understanding the mechanisms underlying these bioactivities is of major importance. However, so far in-depth knowledge on the mode-of-action of CHOS is scarce, one major reason being that most published studies are done with badly characterized heterogeneous mixtures of CHOS. Production of CHOS that are well-defined in terms of length, degree of N-acetylation, and sequence is not straightforward. Here we provide an overview of techniques that may be used to produce and characterize reasonably well-defined CHOS fractions. We also present possible medical applications of CHOS, including tumor growth inhibition and inhibition of TH2-induced inflammation in asthma, as well as use as a bone-strengthener in osteoporosis, a vector for gene delivery, an antibacterial agent, an antifungal agent, an anti-malaria agent, or a hemostatic agent in wound-dressings. By using well-defined CHOS-mixtures it will become possible to obtain a better understanding of the mechanisms underlying these bioactivities.

Chitosanases and proteases have received much attention due to their wide range of applications. Four kinds of chitinous materials, squid pens, shrimp heads, demineralized shrimp shells and demineralized crab shells, were used as the sole carbon and nitrogen (C/N) source to produce chitosanases, proteases and α-glucosidase inhibitors (αGI) by four different strains of Paenibacillus. Chitosanase productivity was highest in the culture supernatants using squid pens as the sole C/N source. The maximum chitosanase activity of fermented squid pens (0.759 U/mL) was compared to that of fermented shrimp heads (0.397 U/mL), demineralized shrimp shells (0.201 U/mL) and demineralized crab shells (0.216 U/mL). A squid pen concentration of 0.5% was suitable for chitosanase, protease and αGI production via Paenibacillus sp. TKU042. Multi-purification, including ethanol precipitation and column chromatography of Macro-Prep High S as well as Macro-Prep DEAE (diethylaminoethyl), led to the isolation of Paenibacillus sp. TKU042 chitosanase and protease with molecular weights of 70 and 35 kDa, respectively. For comparison, 16 chitinolytic bacteria, including strains of Paenibacillus, were investigated for the production of chitinase, exochitinase, chitosanase, protease and αGI using two kinds of chitinous sources.

Penelitian ini merupakan sebagian dari rangkaian penelitian mengenai eksplorasi enzim kitinolitik dari mikroba lingkungan laut, khususnya dari limbah udang. Tujuan penelitian ini adalah untuk mengidentifikasi isolat bakteri KPU 2123 dari limbah udang, mengkarakterisasi dan mengaplikasikan enzim kitosanase yang dihasilkan oleh bakteri tersebut untuk produksi oligomer kitosan dan menguji bioaktivitas oligomer kitosan tersebut sebagai antitumor dan antibakteri. Karakterisasi enzim dilakukan dengan menguji aktivitas enzim pada berbagai suhu dan pH. Selain itu juga ditentukan besarnya aktivitas yang tersisa setelah enzim diinkubasi pada suhu dan lama waktu tertentu. Pengaruh ion logam terhadap aktivitas enzim juga dilihat dengan mereaksikan enzim dengan 1 mM ion logam dalam bentuk larutan khlorida. Hasil penelitian menunjukkan bahwa berdasarkan analisis gen 16S-rRNA, isolat bakteri KPU 2123 memiliki kemiripan 95% dengan Stanotrophomonas maltophilia. Enzim kitosanase dari isolat ini bekerja optimal pada suhu 50 ºC dan pH 6. Enzim ini cukup stabil pada suhu 37 ºC selama 120 menit. Penambahan ion logam berpengaruh terhadap aktivitas enzim. Ion logam Zn²+ (sebagai garam klorida 1 mM) menghambat 100% aktivitas enzim tersebut. Penggunaan enzim kitosanase dalam menghidrolisis substrat kitosan, menghasilkan oligomer kitosan yang mengandung tetramer, pentamer dan heksamer Oligor kitosan tersebut mampu menghambat pertumbuhan bakteri Staphylococcus aureus sebesar 10,06% dan dapat menyebabkan kematian sel HeLa dengan LC50 pada dosis 120 ppm.

Chitosanases hydrolyze chitosan into chitooligosaccharides (COSs) with various biological activities, which are widely employed in many areas including plant disease management. In this study, the novel chitosanase Aq Csn1 belonging to the glycoside hydrolase family 46 (GH46) was cloned from Aquabacterium sp. A7-Y and heterologously expressed in Escherichia coli BL21 (DE3). Aq Csn1 displayed the highest hydrolytic activity towards chitosan with 95% degree of deacetylation at 40 °C and pH 5.0, with a specific activity of 13.18 U/mg. Product analysis showed that Aq Csn1 hydrolyzed chitosan into (GlcN) 2 and (GlcN) 3 as the main products, demonstrating an endo-type cleavage pattern. Evaluation of antagonistic activity showed that the hydrolysis products of Aq Csn1 suppress the mycelial growth of Magnaporthe oryzae and Phytophthora sojae in a concentration-dependent manner, and the inhibition rate of P. sojae reached 39.82% at a concentration of 8 g/L. Our study demonstrates that Aq Csn1 and hydrolysis products with a low degree of polymerization might have potential applications in the biological control of agricultural diseases. • Aq Csn1 was identified as a novel chitosanase belonging to the GH46 family. • Aq Csn1 was able to hydrolyze chitosan into (GlcN) 2 and (GlcN) 3. • Hydrolysis product possessed significant antagonistic effects against P. sojae. [ABSTRACT FROM AUTHOR]

Informasi pasar dunia menunjukkan bahwa 50% keperluan dunia akan produk turunan kitin, digunakan untuk produk suplemen kesehatan. Untuk menunjang proses produksi kitosan yang bersifat ramah lingkungan dan aman untuk konsumsi manusia, maka enzim pendegradasi kitin/ kitosan sangat berperan. Penelitian ini bertujuan untuk mendapatkan bakteri penghasil enzim pendegradasi kitosan dari spons. Spons dipilih karena biota laut tersebut dikenal kaya akan senyawa bioaktif dan sebagian besar masa tubuhnya didominasi oleh bakteri. Dari 24 spons, telah berhasil diisolasi 86 isolat bakteri dan 22 di antaranya menghasilkan enzim pendegradasi kitin, termasuk di antaranya enzim kitosanase. Berdasarkan pada nilai indeks kitinolitik (IK) dan waktu produksi enzim, isolat KBJ 12 SB telah dipilih sebagai isolat penghasil kitosanase. lsolat tersebut menghasilkan enzim kitosanase maksimal pada hari ke‑5, dengan aktivitas enzim sebesar 0,797 U/mg. Enzim kitosanase yang dihasilkan bekerja optimal pada suhu 60 0C dan pH 8. Enzim stabil pada suhu 370C dengan sisa aktivitas > 50% ketika diinkubasi selama 90 menit. Hasil identifikasi menunjukkan bahwa bakteri KBJ 12 SB memiliki sifat mirip dengan jenis Bacillus sp.

The bioactivities of chitooligosaccharides are markedly influenced by the degree of acetylation, degree of polymerization or molecular weight and pattern of acetylation. Thus, it is crucial to identify reproducible processes that will give rise to well-defined chitooligosaccharides and establish methods for their posterior physicochemical characterization in order to advance in the knowledge of their bioactivity. Chitooligosaccharides were prepared by two different processes. The first used chitosanase enzymatic hydrolysis and the second consisted of a two-step procedure based on chemical hydrolysis followed by chitosanase hydrolysis. Chitooligosaccharides produced in the second process were composed of 63 % of fully deacetylated sequences and inhibited the growth of Escherichia coli and Listeria monocytogenes . Better antibacterial activity was found for those obtained in the first process composed of 27 % of fully deacetylated sequences. Therefore, a low percentage of free amino groups and the presence of acetylated sequences are necessary in these molecules to exert good antibacterial capacity. [ABSTRACT FROM AUTHOR]

Chitosanases belong to glycoside hydrolase families 5, 7, 8, 46, 75 and 80 and hydrolyse glucosamine polymers produced by partial or full deacetylation of chitin. Herein, we determined the crystal structure of chitosanase from the β-proteobacterium, Mitsuaria chitosanitabida, (McChoA) at 1.75 Å resolution; the first structure of a family 80 chitosanase. McChoA is a 34 kDa extracellular protein of 301 amino acids that fold into two (upper and lower) globular domains with an active site cleft between them. Key substrate-binding features are conserved with family 24 lysozymes and family 46 chitosanases. The distance between catalytic residues E41 and E61 (10.8 Å) indicates an inverting type mechanism. Uniquely, three disulphide bridges and the C terminus might contribute to enzyme activity. [ABSTRACT FROM AUTHOR]

The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity. [ABSTRACT FROM AUTHOR]

Ｓｃｒｅｅｎｉｎｇ ｅｎｄｏｐｈｙｔｉｃ ｓｔｒａｉｎｓ ｗｉｔｈ ｐｈｏｓｐｈａｔｅ-ｄｉｓｓｏｌｖｉｎｇ ａｎｄ ｅｘｐｌｏｒｉｎｇ ｔｈｅ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｇｒｏｗｔｈ ｐｒｏｍｏ- ｔｉｎｇ ａｎｄ ｒｅｓｉｓｔａｎｃｅ ｗｅｒｅ ｃｏｎｄｕｃｉｖｅ ｔｏ ｂｒｏａｄ ｔｈｅ ｐｈｏｓｐｈａｔｅ ｄｉｓｓｏｌｖｉｎｇ ｍｉｃｒｏｂｉａｌ ｒｅｓｏｕｒｃｅｓ, ｔｏ ｄｅｖｅｌｏｐ ｍｉｃｒｏｂｉａｌ ｆｅｒｔｉｌｉｚ-ｅｒｓ, ｔｏ ｉｍｐｒｏｖｅ ｓｏｉｌ ｐｈｏｓｐｈｏｒｕｓ ｎｕｔｒｉｔｉｏｕｓ ａｎｄ ｔｏ ｉｎｃｒｅａｓｅ ａｇｒｉｃｕｌｔｕｒａｌ ｙｉｅｌｄ. Ｗｅ ｉｓｏｌａｔｅｄ ５０ ｅｎｄｏｐｈｙｔｉｃ ｂａｃｔｅｒｉａ ｆｒｏｍ ｉｎｔｅｒｉｏｒ ｔｉｓｓｕｅｓ ｏｆ Ｃｉｎｎａｍｏｍｕｍ ｌｏｎｇｅｐａｎｉｃｕｌａｔｕｍ, ａｎｄ ２４ ｐｈｏｓｐｈａｔｅ ｄｉｓｓｏｌｖｉｎｇ ｅｎｄｏｐｈｙｔｉｃ ｂａｃｔｅｒｉａ ｓｔｒａｉｎｓ ｗｅｒｅ ｓｃｒｅｅｎｅｄ ｕｓｉｎｇ ｐｈｏｓｐｈａｔｅ ｓｏｌｕｂｉｌｉｚｉｎｇ ｚｏｎｅ (ＰＫＯ) ｉｎｏｒｇａｎｉｃ ｃｕｌｔｕｒｅ ｍｅｄｉｕｍ, ｓｕｂｓｅｑｕｅｎｔｌｙ, ｔｈｅ ｃａｐａｃｉｔｙ ｏｆ ｄｉｓｓｏｌｖｉｎｇ ｐｈｏｓｐｈｏｒｕｓ ｏｆ ２４ ｓｔｒａｉｎｓ ｗｅｒｅ ｄｅｔｅｒｍｉｎｅｄ ｂｙ ｐｈｏｓｐｈｏｒ-ｍｏｌｙｂｄａｔｅ ｂｌｕｅ ｃｏｌｏｒ ｍｅｔｈｏｄｓ, ａｎｄ ｔｈｅ ｃｈａｒａｃｔｅｒｉｓｔｉｃｓ ｂｅｎｅｆｉｔｅｄ ｔｏ ｐｌａｎｔ-ｍｉｃｒｏｂｅ ｉｎｔｅｒａｃｔｉｏｎ-ｉｎｄｏｌｅａｃｅｔｉｃ ａｃｉｄ (ＩＡＡ), ｓｉｄｅｒｏｐｈｏｒｅ, １-ａｍｉｎｏｃｙｃｌｏｐｒｏｐａｎｅ-１-ｃａｒｂｏｘｙｌｉｃ ａｃｉｄ ｄｅａｍｉｎａｓｅ (ＡＣＣ) ａｎｄ ｃｈｉｔｏｓａｎａｓｅ ａｃｔｉｖｉｔｉｅｓ ｗｅｒｅ ａｌｓｏ ｅｖａｌｕａｔｅｄ. Ｔｈｅ ｒｅｓｕｌｔｓ ｓｈｏｗｅｄ ｔｈａｔ ２４ ｓｔｒａｉｎｓ ｃｏｕｌｄ ｒｅｌｅａｓｅ ｐｈｏｓｐｈａｔｅ ｆｒｏｍ ｔｒｉｃａｌｃｉｕｍ ｐｈｏｓｐｈａｔｅ ｗｉｔｈ ｔｈｅ ｒａｎｇｅ ｏｆ ｄｉｓｓｏｌｖｉｎｇ ｐｈｏｓｐｈａｔｅ ｆｒｏｍ ５１.２６ μｇ‧ｍＬ-１ ｔｏ ２３７.０ μｇ‧ｍＬ-１, ａｎｄ ｔｈｅ ｍａｘｉ- ｍｕｍ ｐｈｏｓｐｈａｔｅ ｃｏｎｔｅｎｔ ｉｎ ｔｈｅ ｓｏｌｕｔｉｏｎ ｗａｓ ｏｂｔａｉｎｅｄ ｗｉｔｈ ｓｔｒａｉｎ ＹＧ６０. Ａｍｏｎｇ ２４ ｓｔｒａｉｎｓ, ６ ｓｔｒａｉｎｓ (ＹＧ６０, ＹＧ４３, ＹＧ３６, ＹＧ２５, ＹＧ４９, ＹＧ４４) ｓｈｏｗｅｄ ｔｈｅ ｈｉｇｈｅｒ ｐｈｏｓｐｈａｔｅ ｓｏｌｕｂｉｌｉｚａｔｉｏｎ ｃａｐａｃｉｔｙ (２３７. ０８, ２１１. ５３, １８０. ６８, １６６.８５, １５１.２８, １５０.２０ μｇ‧ｍＬ-１ ｒｅｓｐｅｃｔｉｖｅｌｙ). Ｆｕｒｔｈｅｒｍｏｒｅ, ２４ ｓｔｒａｉｎｓ ｃｏｕｌｄ ｄｅｃｒｅａｓｅ ｔｈｅ ｐＨ ｏｆ ｔｈｅ ｍｅｄｉｕｍ ｗｉｔｈ ｔｈｅ ｐＨ ｖａｌｕｅ ｒａｎｇｅ ｆｒｏｍ ４.５ ｔｏ ５.５, ａｎｄ ｔｈｅ ｒｅｌａｔｉｏｎｓｈｉｐ ｂｅｔｗｅｅｎ ｐＨ ａｎｄ ｐｈｏｓｐｈｏｒｕｓ ｓｏｌｕｂｉｚｉｔｉｏｎ ｗａｓ ａｌｓｏ ｄｉｓｃｕｓｓｅｄ; ｈｏｗｅｖｅｒ, ｔｈｅｒｅ ｗｅｒｅ ｎｏ ｃｌｏｓｅ ｒｅｌａｔｉｏｎｓｈｉｐ ｂｅｔｗｅｅｎ ｔｈｅｍ. Ｔｈｅ ｆｕｒｔｈｅｒ ｅｘｐｅｒｉｍｅｎｔａｌ ｒｅｓｕｌｔｓ ｓｈｏｗｅｄ ｔｈａｔ ｍｏｓｔ ｏｆ ｔｈｅ ６ ｓｔｒａｉｎｓ ｗｉｔｈ ｈｉｇｈｅｒ ｐｈｏｓｐｈａｔｅ ｄｉｓｓｏｌｖｉｎｇ ｃａｐａｃｉｔｙ ｈａｄ ｔｈｅ ａｂｉｌｉｔｙ ｔｏ ｐｒｏｄｕｃｅ ＩＡＡ ａｎｄ ｓｉｄｅｒｏｐｈｏｒｅｓ, ａｎｄ ｈａｄ ＡＣＣ ｄｅａｍ-ｉｎａｓｅ ａｎｄ ｃｈｉｔｏｓａｎａｓｅ ａｃｔｉｖｉｔｙ. Ａｍｏｎｇ ｔｈｅｍ, ＹＧ４３, ＹＧ６０ ａｎｄ ＹＧ２５ ｐｏｓｓｅｓｓｅｄ ｈｉｇｈｅｒ ＩＡＡ ｐｒｏｄｕｃｉｎｇ ｃａｐａｃｉｔｙ (２２.５５, １８.７５ ａｎｄ １６.４１ μｇ‧ｍＬ-１ ｒｅｓｐｅｃｔｉｖｅｌｙ); ＹＧ４３ ａｎｄ ＹＧ６０ ｐｏｓｓｅｓｓｅｄ ｈｉｇｈｅｒ ｓｉｄｅｒｏｐｈｏｒｅｓ ｏｆ Ａｓ/ Ａｒ<０.６ (０.４５９, ０.５７９ ｒｅｓｐｅｃｔｉｖｅｌｙ ); ＹＧ４３, ＹＧ６０ ａｎｄ ＹＧ２５ ｈａｄ ｓｔｒｏｎｇ ＡＣＣ ｄｅａｍｉｎａｓｅ ａｃｔｉｖｉｔｙ (０.１９４, ０.２２４, ０.２０８ Ｕ‧ｍｇ-１),ＹＧ４３ ａｎｄ ＹＧ６０ ｈａｄ ｓｔｒｏｎｇ ｃｈｉｔｏｓａｎａｓｅ ａｃｔｉｖｉｔｙ (２.９６８ Ｕ,２.５０２ Ｕ). Ｉｎ ｔｅｒｍｓ ｏｆ ａｌｌ ｔｈｅ ｐｒｏｐｅｒｔｉｅｓ ｏｆ ｄｉｓ-ｓｏｌｖｉｎｇ ｐｈｏｓｐｈａｔｅ, ｓｅｃｒｅｔｉｎｇ ＩＡＡ, ｓｉｄｅｒｏｐｈｏｒｅｓ, ＡＣＣ ｄｅａｍｉｎａｓｅ ａｎｄ ｃｈｉｔｏｓａｎａｓｅ, ｓｔｒａｉｎｓ ＹＧ４３, ＹＧ６０ ａｎｄ ＹＧ２５ ｉｓｏｌａｔｅｄ ｆｒｏｍ ｉｎｔｅｒｉｏｒ ｔｉｓｓｕｅｓ ｏｆ Ｃ. ｌｏｎｇｅｐａｎｉｃｕｌａｔｕｍ ｈａｖｅ ａｂｕｎｄａｎｔ ｂｉｏｌｏｇｉｃａｌ ｃｈａｒａｃｔｅｒｉｓｔｉｃｓ ｒｅｌａｔｅｄ ｔｏ ｐｌａｎｔ ｇｒｏｗｔｈ ｐｒｏｍｏｔｉｏｎ, ｓｔｒｅｓｓ ｈｏｍｅｏｓｔａｓｉｓ ｒｅｇｕｌａｔｉｏｎ ａｎｄ ｂｉｏｃｏｎｔｒｏｌ ａｃｔｉｖｉｔｙ. Ｔｈｅｙ ａｒｅ ｐｏｓｓｉｂｌｅ ｔｏ ｂｅ ｆｕｒｔｈｅｒ ｄｅｖｅｌｏｐｅｄ ａｓ ｅｘｃｅｌｌｅｎｔ ｓｔｒａｉｎｓ ｆｏｒ ａｐｐｌｉｃａｔｉｏｎ. [ABSTRACT FROM AUTHOR]

A novel high-throughput method was established for rapid screening of a large numbers of Aspergillus fumigatus ( A. fumigatus) mutants with high chitosanase production under acidic culture condition by exploiting the fact that iodine can be used as the indicator to stain chitosan but is ineffective for chitooligosaccharides. The mutant population was generated by irradiating A. fumigatus CICC 2434 with Co-γ rays. Mutants were cultured on acidic plates containing colloidal chitosan and preliminary screened according to diameter of haloes formed around colonies. Then, chitosanase production of the isolates were verified by dinitrosalicylic acid assay. Lastly, molecular masses on enzymolysis products of isolated mutants were rapidly compared by aniline blue plate assay. Using this method, the mutant strain Co-8 was selected, which had chitosanase activity of 24.87 U/mL (increased by 369.2 % as compared to that of its parental strain).Taking together, the method is easy, efficient and particularly suited to rapid screen acidophilic fungal strains with high chitosanase-production. [ABSTRACT FROM AUTHOR]

A number of bacterial species produces chitosanases which has variety of applications because of its high biodegradability, non-toxicity and antimicrobial assets. In the present study chitosanase is purified from new bacterial species Bacillus licheniformis from spoiled vegetable. This novel strain of Bacillus licheniformis isolated from spoilt cucumber and pepper samples has the ability to produce the chitosanase enzyme when grown on chitosan substrate. Study also examined its antibiofilm properties against diverse bacterial species with biofilm forming ability. The purified chitosanase inhibited the biofilm formation ability for all Gram-negative and Gram-positive biofilm-forming bacteria [biofilm producers] tested in this study in congo red agar and microtiter plate's methods. Highly antibiofilm activity of chitosanase was recorded against Pseudomonas aeruginosa followed by Klebsiella pneumoniae with reduction of biofilm formation upto 22 and 29%, respectively compared with [100] % of control. Biofilm formation has multiple role including ability to enhance resistance and self-protection from external stress. This chitosanase has promising benefit as antibiofilm agent against biofilm forming pathogenic bacteria and has promising application as alternative antibiofilm agents to combat the growing number of multidrug resistant pathogen-associated infections, especially in situation where biofilms are involved. [ABSTRACT FROM AUTHOR]

Biological activities of chitosan oligosaccharides (COS) are well documented, and numerous reports of COS production using specific and non-specific enzymes are available. However, strategies for improving the overall yield by making it monomer free need to be developed. Continuous enzymatic production from chitosan derived from marine wastes is desirable and is cost-effective. Isolation of potential microbes showing chitosanase activity from various ecological niches, gene cloning, enzyme immobilization, and fractionation/purification of COS are some areas, where lot of work is in progress. This review covers recent measures to improve monomer-free COS production using chitosanase/non-specific enzymes and purification/fractionation of these molecules using ultrafiltration and column chromatographic techniques. Various bioprocess strategies, gene cloning for enhanced chitosanase enzyme production, and other measures for COS yield improvements have also been covered in this review. COS derivative preparation as well as COS-coated nanoparticles for efficient drug delivery are being focused in recent studies. [ABSTRACT FROM AUTHOR]

Chitosanase (CSN) from Aspergillus fumigatus has good thermal stability, wide pH range duration, and effective hydrolysis for chitosan. Inhere, CSN was successfully expressed in Escherichia coli followed by extracellular secretion under the guidance of an N-terminal signal peptide PelB, which effectively prompted its secretion out of E. coli cells. To facilitate its later purification, N-terminal or C-terminal 6xHis epitope tag was added to the PelB-CSN protein complex. Our results indicated that PelB-CSN without 6xHis-tag (PelB-CSN) or with N-terminal 6xHis-tag (PelB-CSN-N) can both be effectively secreted into the medium, while CSN with 6xHis-tag anchored at C-terminus was expressed as inclusion bodies. Process optimization strategies were further developed to improve the secretion efficiency of recombinant PelB-CSN-N in E. coli. Under the induction of 10 g/L lactose in shake-flask culture, the extracellular activity of CSN reached 6015 U/mL at 25 °C in TB medium containing 1 % glycine. Moreover, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 5-L fermenter, increasing the extracellular CSN activity to 14,000 U/mL in 2-day fermentation with the optimal addition of lactose and glycine. [ABSTRACT FROM AUTHOR]

Background: Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum. Results: ManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and mannooligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. Conclusion: This study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially 'prebiotic' oligosaccharides. This approach, with the enzyme of interest being displayed on the cell surface of a food-grade organism, may also be applied in production processes relevant for food industry. [ABSTRACT FROM AUTHOR]

Enzymatic depolymerization of chitosan, a β-(1,4)-linked polycationic polysaccharide composed of d -glucosamine (GlcN) and N -acetyl- d -glucosamine (GlcNAc) provides a possible route to the exploitation of chitin-rich biomass. Complete conversion of chitosan to mono-sugars requires the synergistic action of endo- and exo- chitosanases. In the present study we have developed an efficient and cost-effective chitosan-degrading enzyme cocktail containing only two enzymes, an endo-attacking bacterial chitosanase, Sc Csn46A, from Streptomyces coelicolor , and an exo-attacking glucosamine specific β-glucosaminidase, Tk -Glm, from the archaeon Thermococcus kodakarensis KOD1. Moreover, we developed a fast, reliable quantitative method for analysis of GlcN using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The sensitivity of this method is high and less than 50 pmol was easily detected, which is about 1000-fold better than the sensitivity of more commonly used detection methods based on refractive index. We also obtained qualitative insight into product development during the enzymatic degradation reaction by means of ElectroSpray Ionization-Mass Spectrometry (ESI-MS). [ABSTRACT FROM AUTHOR]

Objective: To determine the effects of artificial disulfide bridges on the thermostability and catalytic efficiency of chitosanase EAG1. Results: Five artificial disulfide bridges were designed based on the structural information derived from the three-dimensional (3-D) model of chitosanase EAG1. Two beneficial mutants (G113C/D116C, A207C-L286C) were located in the flexible surface loop region, whereas the similar substitutions introduced in α-helices regions had a negligible effect. Mut5, the most active mutant, had a longer half-life at 50 °C (from 10.5 to 69.3 min) and a 200 % higher catalytic efficiency ( K / K ) than that of the original EAG1. Conclusions: The contribution of disulfide bridges to enzyme thermostability is mainly dependent on its location within the polypeptide chain. Strategical placement of a disulfide bridge in flexible regions provides a rigid support and creation of a protected microenvironment, which is effective in improving enzyme's thermostability and catalytic efficiency. [ABSTRACT FROM AUTHOR]

A new strain Penicillium sp. IB-37-2, which actively hydrolyzes chitosan (SD ∼80-85%) but possesses low activity against colloidal chitin, was isolated. The fungus was observed to have a high level chitosanase biosynthesis (1.5-3.0 U/mL) during submerged cultivation at 28°C, with a pH of 3.5-7.0 and 220 rpm in nutrient media containing chitosan or chitin from shells of crabs. Purification of the chitosanase enzyme complex from Penicillium sp. IB-37-2 by ultrafiltration and hydrophobic chromatography, followed by denaturing electrophoresis, revealed two predominant proteins with molecular weights of 89 and 41 kDa. The purified enzyme complex demonstrated maximal activity (maximal rate of hydrolysis of dissolved chitosan) and stability at 50-55°C and a pH of 3.5-4.0. The enzyme preparation also hydrolyzed laminarin, β-(1,3)-(1,4)-glycan, and colloidal chitin. Exohydrolysis of chitosan by the preparation isolated from Penicillium sp. IB-37-2 resulted in the formation of single product, D-glucosamine. [ABSTRACT FROM AUTHOR]

The objectives of this investigation were to produce a novel chitosanase for application in industries and waste treatment. The transformation of chitinous biowaste into valuable bioactive chitooligomers (COS) is one of the most exciting applications of chitosanase. An amphiprotic novel chitosanase from Bacillus mycoides TKU038 using squid pen powder (SPP)-containing medium was retrieved from a Taiwan soil sample, which was purified by column chromatography, and characterized by biochemical protocol. Extracellular chitosanase (CS038) was purified to 130-fold with a 35% yield, and its molecular mass was roughly 48 kDa. CS038 was stable over a wide range of pH values (4-10) at 50°C and exhibited an optimal temperature of 50°C. Interestingly, the optimum pH values were estimated as 6 and 10, whereas CS038 exhibited chitosan-degrading activity (100% and 94%, respectively). CS038 had Km and Vmax values of 0.098 mg/mL and 1.336 U/min, separately, using different concentrations of water-soluble chitosan. A combination of the high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer data revealed that the chitosan oligosaccharides obtained from the hydrolysis of chitosan by CS038 comprise oligomers with multiple degrees of polymerization (DP), varying from 3-9, as well as CS038 in an endolytic fashion. The TKU038 culture supernatant and COS mixture exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities. The COS activities were dose dependent and correlated to their DP. The COS with high DP exhibited enhanced DPPH radical scavenging capability compared with COS with low DP. Furthermore, the COS exhibited inhibitory behavior on nitric oxide (NO) production in murine RAW 264.7 macrophage cells, which was induced by Escherichia coli O111 lipopolysaccharide (LPS). The COS with low DP possesses a more potent anti-inflammatory capability to decrease NO production (IC50, 76.27 ± 1.49 µg/mL) than that of COS with high DP (IC50, 82.65 ± 1.18 µg/mL). Given its effectiveness in production and purification, acidophilic and alkalophilic properties, stability over ranges of pH values, ability to generate COS, antioxidant activity, and anti-inflammatory, CS038 has potential applications in SPP waste treatment and industries for COS production as a medical prebiotic. [ABSTRACT FROM AUTHOR]

The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing. Proteins destined for secretion contain an N-terminal signal peptide that is cleaved by secretion machinery in the plasma membrane. The resulting protein is released in an active mature form. In this study, Bacillus subtilis chitosanase (Csn) was used as a model protein to compare the effect of two signal peptides on the secretion of heterologous recombinant protein. The results showed that the E. coli secretion machinery could recognize both native bacillus and E. coli signal peptides. However, only the native bacillus signal peptide could generate the same N-terminal sequence as in the wild type bacteria. When the recombinant Csn constructs contained the E. coli OmpA signal peptide, the secreted enzymes were heterogeneous, comprising a mixed population of secreted enzymes with different N-terminal sequences. Nevertheless, the E. coli OmpA signal peptide was found to be more efficient for high expression and secretion of bacillus Csn. These findings may be used to help engineer other recombinant proteins for secretory production in E. coli. [ABSTRACT FROM AUTHOR]

In the past, Lysobacter spp. were found only in temperate and sub-tropical zones. In order to assess the existence of Lysobacter spp. in tropical environments, samples were collected from grass-covered soil in Thailand. Among several bacteria obtained, isolate 521 (NCCB 100553) was identified as Lysobacter enzymogenes based on morphology and physiological tests along with 16S rRNA gene sequence analysis. Phenotypic characteristic variations of L. enzymogenes 521 with respect to the most studied strain from temperate regions, L. enzymogenes C3, were observed. The growth rate, carboxymethylcellulase (CMCase) and chitosanase activity of L. enzymogenes 521 were clearly higher than those of L. enzymogenes C3. Nucleotide and predicted amino acid sequences of the endoglucanase derived were deduced from draft genome sequencing. Phylogenetic analysis of the endoglucanase gene and multiple alignment of the deduced amino acid sequence illustrated that the endoglucanase (Cel8A) from L. enzymogenes 521 displayed homology to family 8 glycoside hydrolases (GHF-8), and was found to be similar to the bifunctional endoglucanase (Cel8A) from Lysobacter sp. IB-9374. Bifunctional CMCase and chitosanase activities of a purified endoglucanase with a mass of 41 kDa from L. enzymogenes 521 were revealed by zymogram analysis. This is the first report on the phenotypical characteristics, the endoglucanase with bifunctional CMCase and chitosanase activities, and the endoglucanase gene, from L. enzymogenes from a tropical environment. [ABSTRACT FROM AUTHOR]

Heterologous production of hydrolytic enzymes is important for green and white biotechnology since these enzymes serve as efficient biocatalysts for the conversion of a wide variety of raw materials into value-added products. Lactic acid bacteria are interesting cell factories for the expression of hydrolytic enzymes as many of them are generally recognized as safe and require only a simple cultivation process. We are studying a potentially foodgrade expression system for secretion of hydrolytic enzymes into the culture medium, since this enables easy harvesting and purification, while allowing direct use of the enzymes in food applications. Results: We studied overexpression of a chitosanase (CsnA) and a β-mannanase (ManB), from Bacillus licheniformis and Bacillus subtilis, respectively, in Lactobacillus plantarum, using the pSIP system for inducible expression. The enzymes were over-expressed in three forms: without a signal peptide, with their natural signal peptide and with the well-known OmpA signal peptide from Escherichia coli. The total production levels and secretion efficiencies of CsnA and ManB were highest when using the native signal peptides, and both were reduced considerably when using the OmpA signal. At 20 h after induction with 12.5 ng/mL of inducing peptide in MRS media containing 20 g/L glucose, the yields and secretion efficiencies of the proteins with their native signal peptides were 50 kU/L and 84% for ManB, and 79 kU/L and 56% for CsnA, respectively. In addition, to avoid using antibiotics, the erythromycin resistance gene was replaced on the expression plasmid with the alanine racemase (alr) gene, which led to comparable levels of protein production and secretion efficiency in a suitable, alr-deficient L. plantarum host. Conclusions: ManB and CsnA were efficiently produced and secreted in L. plantarum using pSIP-based expression vectors containing either an erythromycin resistance or the alr gene as selection marker. [ABSTRACT FROM AUTHOR]