Production Of Chitooligosaccharides Research Articles

Continuous production of chitooligosaccharides in a column reactor by the PUF-immobilized whole cell enzymes of Mucor circinelloides IBT-83

BackgroundChitosan oligosaccharides (COS) have great potential for applications in several fields, including agriculture, food industry or medicine. Nevertheless, the large-scale use of COS requires the development of cost-effective technologies for their production. The main objective of our investigation was to develop an effective method of enzymatic degradation of chitosan in a column reactor using Mucor circinelloides IBT-83 cells, immobilized in a polyurethane foam (PUF). These cells serve as a source of chitosanolytic enzymes.ResultsThe study revealed that the process of freeze-drying of immobilized mycelium increases the stability of the associated enzymes during chitosan hydrolysis. The use of stabilized preparations as an active reactor bed enables the production of COS at a constant level for 16 reactor cycles (384 h in total), i.e. 216 h longer compared to non-stabilized mycelium. In the hydrolysate, oligomers ranging in structure from dimer to hexamer as well as D-glucosamine were detected. The potential application of the obtained product in agriculture has been verified. The results of phytotests have demonstrated that the introduction of COS into the soil at a concentration of 0.01 or 0.05% w/w resulted in an increase in the growth of Lepidium sativum stem and root, respectively (extensions by 38 and 44% compared to the control sample).ConclusionsThe research has verified that the PUF-immobilized M. circinelloides IBT-83 mycelium, which has been stabilized through freeze-drying, is a promising biocatalyst for the environmentally friendly and efficient generation of COS. This biocatalyst has the potential to be used in fertilizers.

Structure-Assisted Design of Chitosanase Product Specificity for the Production of High-Degree Polymerization Chitooligosaccharides.

Chitosanases are valuable enzymatic tools in the food industry for converting chitosan into functional chitooligosaccharides (COSs). However, most of the chitosanases extensively characterized produced a low degree of polymerization (DP) COSs (DP = 1-3, LdpCOSs), indicating an imperative for enhancements in the product specificity for the high DP COS (DP >3, HdpCOSs) production. In this study, a chitosanase from Methanosarcina sp. 1.H.T.1A.1 (OUC-CsnA4) was cloned and expressed. Analysis of the enzyme-substrate interactions and the subsite architecture of the OUC-CsnA4 indicated that a Ser49 mutation could modify its interaction pattern with the substrate, potentially enhancing product specificity for producing HdpCOSs. Site-directed mutagenesis provided evidence that the S49I and S49P mutations in OUC-CsnA4 enabled the production of up to 24 and 26% of (GlcN)5 from chitosan, respectively─the wild-type enzyme was unable to produce detectable levels of (GlcN)5. These mutations also altered substrate binding preferences, favoring the binding of longer-chain COSs (DP >5) and enhancing (GlcN)5 production. Furthermore, molecular dynamics simulations and molecular docking studies underscored the significance of +2 subsite interactions in determining the (GlcN)4 and (GlcN)5 product specificity. These findings revealed that the positioning and interactions of the reducing end of the substrate within the catalytic cleft are crucial factors influencing the product specificity of chitosanase.

Utilizing chitooligosaccharides from shrimp waste biodegradation via recombinant chitinase A: a promising approach for emulsifying hydrocarbon and bioremediation

BackgroundHydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by response surface methodology (RSM) which delvs into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers.ResultsIn this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number "OR643436". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts.ConclusionsThe recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.

Response surface optimization, purification, characterization and short-chain chitooligosaccharides production from an acidic, thermostable chitinase from Thermomyces dupontii

Chitin, recovered in huge amounts from coastal waste, may biocatalytically valorized for utilization in food and biotech sectors. Conventional chemical-based conversion makes use of significant volumes of hazardous acid and alkali. Alternatively, enzymes offer better process control and generation of homogeneous products. Process variables were derived to achieve augmented levels of chitinase (3.8809 Ul−1 h−1) productivity from a novel thermophilic fungal strain Thermomyces dupontii, ITCC 9104 following incubation (96 h, 45 °C). An acidic thermostable chitinase TdChiT having molecular mass of 60 kDa has been purified. Optimal TdChiT activity has been demonstrated at 70 °C and pH 5. Notably decreased activity over a broad range of temperature and pH was observed following deglycosylation. Half-life, activation energy, Gibbs free energy, enthalpy and entropy for denaturation of TdChiT at its optimum temperature were 197.40 min, 105.48 kJ mol−1, 100.59 kJ mol−1, 102.64 kJ mol−1 and 5.95 J mol−1 K−1. TdChiT has specificity towards colloidal chitin and (GlcNAc)2–4. Metal ions viz. Mn2+, Ca2+ and Co2+ and nonionic surfactants notably enhanced chitinase activity. Thin layer chromatography analysis has revealed effective hydrolysis of colloidal chitin and (GlcNAc)2–4. TdChiT may potentially be employed for design of better, eco-friendly and less resource-intensive industrial procedures for upcycling of crustacean waste into value-added organonitrogens.

Chitinase induction in Trichoderma harzianum: a solid-state fermentation approach using shrimp waste and wheat bran/commercial chitin for chitooligosaccharides synthesis

This study innovatively employed solid-state fermentation (SSF) to evaluate chitinase induction in Trichoderma harzianum. Solid-state fermentation minimizes water usage, a crucial global resource, and was applied using shrimp waste chitin and a mixture of commercial chitin with wheat bran as substrates. Shrimp waste and wheat bran were pretreated and characterized for SSF, and the fungus’s utilization of the substrates was assessed using spectrophotometric and microscopic methods. The resulting enzymes’ ability to produce chitooligosaccharides (COS) mixtures was studied. Wheat bran/commercial chitin demonstrated superior performance, with a 1.8-fold increase in chitinase activity (76.3 U/mg protein) compared to shrimp waste chitin (41.8 U/mg protein). Additionally, the COS mixture obtained from wheat bran/commercial chitin showed a higher concentration of reducing sugars, reaching 87.85 mM, compared to shrimp waste chitin (14.87 mM). The COS profile from wheat bran/commercial chitin included monomers to heptamers, while the profile from shrimp waste chitin was predominantly composed of monomers. These results highlight the advantages of SSF for chitinase induction and COS production in T. harzianum, offering potential applications as dietary fiber, antioxidants, and antimicrobial agents. The findings contribute to by-product valorization, waste reduction, and the sustainable generation of valuable products through SSF-based enzyme production.

Expression of chitosanase from Aspergillus fumigatus chitosanase in Saccharomyces cerevisiae by CRISPR-Cas9 tools.

Chitooligosaccharides (COS) find numerous applications due to their exceptional properties. Enzymatic hydrolysis of chitosan by chitosanase is considered an advantageous route for COS production. Heterologous expression of chitosanase holds significant promise, yet studies using commonly employed Escherichia coli and Pichia pastoris strains encounter challenges in subsequent handling and industrial scalability. In this investigation, we opted for using the safe yeast strain Saccharomyces cerevisiae (GRAS), obviating the need for methanol induction, resulting in successful expression. Ultimately, utilizing the GTR-CRISPR editing system, shake flask enzyme activity reached 2 U/ml. The optimal chitosanase activity was achieved at 55℃ and pH 5, with favorable stability between 30 and 50°C. Following a 2-h catalytic reaction, the product primarily consisted of chitobiose to chitotetraose, predominantly at the chitotriose position, with a slight increase in chitobiose content observed during the later stages of enzymatic hydrolysis. The results affirm the feasibility of heterologous chitosanase expression through Saccharomyces cerevisiae, underscoring its significant industrial potential.

Secretory production and characterization of a highly effective chitosanase from Streptomyces coelicolor A3(2) M145 in Pichia pastoris.

In this study, a glycoside hydrolase family 46 chitosanase from Streptomyces coelicolor A3(2) M145 was firstly cloned and expressed in Pichia pastoris GS115 (P. pastoris GS115). The recombinant enzyme (CsnA) showed maximal activity at pH 6.0 and 65°C. Both thermal stability and pH stability of CsnA expressed in P. pastoris GS115 were significantly increased compared with homologous expression in Streptomyces coelicolor A3(2). A stable chitosanase activity of 725.7±9.58 U mL-1 was obtained in fed-batch fermentation. It's the highest level of CsnA from Streptomyces coelicolor expressed in P. pastoris so far. The hydrolytic process of CsnA showed a time-dependent manner. Chitosan oligosaccharides (COSs) generated by CsnA showed antifungal activity against Fusarium oxysporum sp. cucumerinum (F. oxysporum sp. cucumerinum). The secreted expression and hydrolytic performance make the enzyme a desirable biocatalyst for industrial controllable production of chitooligosaccharides with specific degree of polymerization, which have potential to control fungi that cause important crop diseases.

Response surface bioprocess provenance for enhanced chitinase production by Thermomyces dupontii for translation of chitinous waste to short-chain chitooligosaccharides

Chitinase, a chitin degrading glycosyl hydrolase, has received a great deal of interest because of the wide range of potential uses in the bio-based industries. A large output of the enzyme at a reasonable manufacturing cost is essential for its sustainable product oriented applications. Thermomyces dupontii ITCC 9104 has been identified as a thermophilic fungal chitinase producer with maximum chitinase production (168.30 ± 0.86 Ul−1). Nutritional components have been considered to exert profound effect on enzyme production. Derivation of optimum levels of five nutritional components as independent variables under submerged fermentation was carried out by employing central composite design resulted into 1.70 fold enhancement in the extracellular chitinase titres. The level of factors and design has been further validated by linear regression as well. Thin layer chromatographic analysis has revealed that chitinase derived from T. dupontii efficiently hydrolyzes colloidal chitin into a mixture of short-chain chitooligosaccharides and glucosamine. The findings presented here provided evidence that the Desert Ecosystem of Rajasthan could be a rich source for the exploration and prospecting of thermophilic chitinase producing microbial strains. The optimization strategies have opened up perspectives for better production of chitooligosaccharides aimed at the clean-up of coastal chitin pollution with generation of ‘products of value’ in an environmental friendly, economical and sustainable manner.

Enhancing the thermostability and catalytic activity of Bacillus subtilis chitosanase by saturation mutagenesis of Lys242.

Catalysis activity and thermostability are some of the fundamental characteristic of enzymes, which are of great significance to their industrial applications. Bacillus subtilis chitosanase BsCsn46A is a kind of enzyme with good catalytic activity and stability, which can hydrolyze chitosan to produce chitobiose and chitotriose. In order to further improve the catalytic activity and stability of BsCsn46A, saturation mutagenesis of the C-terminal K242 of BsCsn46A was performed. The results showed that the six mutants (K242A, K242D, K242E, K242F, K242P, and K242T) showed increased catalytic activity on chitosan. The catalytic activity of K242P increased from 12971±597 Umg-1 of wild type to 17820±344 Umg-1 , and the thermostability of K242P increased by 2.27%. In order to elucidate the reason for the change of enzymatic properties, hydrogen network, molecular docking, and molecular dynamics simulation were carried out. The hydrogen network results showed that all the mutants lose their interaction with Asp6 at 242 site, thereby increasing the flexibility of Glu19 at the junction sites of α1 and loop1. Molecular dynamics results showed that the RMSD of K242P was lower at both 313 and 323 K than that of other mutants, which supported that K242P had better thermostability. The catalytic activity of mutant K242P reached 17820.27 Umg-1 , the highest level reported so far, which could be a robust candidate for the industrial application of chitooligosaccharide (COS) production.

Green-Chemical Strategies for Production of Tailor-Made Chitooligosaccharides with Enhanced Biological Activities.

Chitooligosaccharides (COSs) are b-1,4-linked homo-oligosaccharides of N-acetylglucosamine (GlcNAc) or glucosamine (GlcN), and also include hetero-oligosaccharides composed of GlcNAc and GlcN. These sugars are of practical importance because of their various biological activities, such as antimicrobial, anti-inflammatory, antioxidant and antitumor activities, as well as triggering the innate immunity in plants. The reported data on bioactivities of COSs used to contain some uncertainties or contradictions, because the experiments were conducted with poorly characterized COS mixtures. Recently, COSs have been satisfactorily characterized with respect to their structures, especially the degree of polymerization (DP) and degree of N-acetylation (DA); thus, the structure-bioactivity relationship of COSs has become more unambiguous. To date, various green-chemical strategies involving enzymatic synthesis of COSs with designed sequences and desired biological activities have been developed. The enzymatic strategies could involve transglycosylation or glycosynthase reactions using reducing end-activated sugars as the donor substrates and chitinase/chitosanase and their mutants as the biocatalysts. Site-specific chitin deacetylases were also proposed to be applicable for this purpose. Furthermore, to improve the yields of the COS products, metabolic engineering techniques could be applied. The above-mentioned approaches will provide the opportunity to produce tailor-made COSs, leading to the enhanced utilization of chitin biomass.

Chitosanase Production from the Liquid Fermentation of Squid Pens Waste by Paenibacillus elgii.

Chitosanases play a significant part in the hydrolysis of chitosan to form chitooligosaccharides (COS) that possess diverse biological activities. This study aimed to enhance the productivity of Paenibacillus elgii TKU051 chitosanase by fermentation from chitinous fishery wastes. The ideal parameters for achieving maximum chitosanase activity were determined: a squid pens powder amount of 5.278% (w/v), an initial pH value of 8.93, an incubation temperature of 38 °C, and an incubation duration of 5.73 days. The resulting chitosanase activity of the culture medium was 2.023 U/mL. A chitosanase with a molecular weight of 25 kDa was isolated from the culture medium of P. elgii TKU051 and was biochemically characterized. Liquid chromatography with tandem mass spectrometry analysis revealed that P. elgii TKU051 chitosanase exhibited a maximum amino acid identity of 43% with a chitosanase of Bacillus circulans belonging to the glycoside hydrolase (GH) family 46. P. elgii TKU051 chitosanase demonstrated optimal activity at pH 5.5 while displaying remarkable stability within the pH range of 5.0 to 9.0. The enzyme displayed maximum efficiency at 60 °C and demonstrated considerable stability at temperatures ≤40 °C. The presence of Mn2+ positively affected the activity of the enzyme, while the presence of Cu2+ had a negative effect. Thin-layer chromatography analysis demonstrated that P. elgii TKU051 chitosanase exhibited an endo-type cleavage pattern and hydrolyzed chitosan with 98% degree of deacetylation to yield (GlcN)2 and (GlcN)3. The enzymatic properties of P. elgii TKU051 chitosanase render it a promising candidate for application in the production of COS.

A green approach for depolymerization of chitosan: applications in hydrogels

Chitooligosaccharides (COS) are depolymerized chains produced from the natural polymer chitosan and has been determined to exhibit improved biological activities, high solubility in neutral to slightly alkaline pH, because of the lower molecular weight (MW). This makes COS more attractive in biomedical applications. However, earlier studies focused on depolymerization techniques that were either cumbersome or expensive. Here, a convenient two-stage, green synthesis approach was developed and optimized, where gamma irradiation and oxidative degradation with H2O2 were used to depolymerize chitosan to produce COS for biomedical applications. The gamma radiation dose level, H2O2 degradation reaction’s temperature, time and H2O2 concentration were varied to obtain the mildest combination of reaction conditions. The most optimum set of conditions (15 kGy, 25oC, overnight reaction with 2% H2O2) yielded COS that was soluble in physiological pH range (7–8.5). The COS had a MW of 12.8 ± 1.6 kDa (which was a 95% reduction in MW), a 62.3% degree of deacetylation, and a crystallinity index of 33%. A photopolymerized hydrogel using this COS cross-linked with polyethylene glycol diacrylate (PEGDA) and carboxymethyl cellulose (CMC) was also developed. The hydrogel exhibited high swelling ratio (6.44–10.24), a porous morphology, a compression modulus of 4.5 ± 2.7 kPa (similar to soft tissues), and more than 95% biocompatibility with mammalian cells. This newly developed COS hydrogel involves a simple and green approach for the production of COS and shows promise as a scaffold for artificial soft tissue.

Efficient production of chito-oligosaccharides through microwave-assisted hydrothermal and low-acid-mediated degradation and deacetylation of chitin

The conventional method for producing chitin oligosaccharides involves high-temperature hydrolysis in high-concentration acid. In this study, we present an alternative method using microwave-assisted hydrothermal and diluted acid (0.12 mol/L) hydrolysis. After batched reactions, the liquefaction rate of chitin can reach almost 50%, and the sugar product yield is about 27%. Furthermore, this method leads to the reduction of the average particle size of chitin by approximately 70%, a decrease in the degree of acetylation by about 35%, partial destruction of the intermolecular hydrogen bond network, and the breaking of glycosidic bonds. These changes result in the exposure of more hydroxyl groups and enzyme binding sites, which may facilitate a subsequent enzymatic treatment. The method is a feasible alternative to obtain chitin oligosaccharides with a low environmental impact and operating cost.

Genome mining reveals chitin degradation potential of \(\textit{Streptomyces parvulus}\) VCCM 22513

The genus Streptomyces is not only known as a natural producer of antibiotics but also a prolific source of chitinolytic enzymes that digest recalcitrant chitin to chitooligosaccharides. However, only a few reports have used whole-genome sequencing to study chitin degradation of Streptomyces to date. In the present study, out of 22 Streptomyces strains, Streptomyces parvulus VCCM 22513 produced the highest chitinase activity. Time courses of incubation revealed that the maximum chitinase (0.91 ± 0.04 U/mL) of this strain was observed after 96 hours in the yeast extract salts medium supplemented with 10.0 g/L colloidal chitin. Additional genomic analysis of VCCM 22513 was also conducted to discover the genomic information related to chitin degradation. The VCCM 22513 genome consists of 341 CAZy genes divided into 6 families including glycoside hydrolase (134 genes), carbohydrate-binding module (88 genes), glycosyl transferase (87 genes), carbohydrate esterase (18 genes), polysaccharide lyase (7 genes), and auxiliary activity (7 genes). Further genome mining revealed the presence of 10 chitinases, 4 lytic polysaccharide monooxygenases, and 14 β-N-acetylhexosaminidases, which mainly contribute to the degradation of chitin polymers. This is the first report revealing the mechanism underlying the chitin degradation of S. parvulus. Further investigations are required to characterize chitinolytic enzymes found in this study for the bioeconomic production of high-quality chitooligosaccharides from chitin food wastes.

Structural and binding studies of a new chitin-active AA10 lytic polysaccharide monooxygenase from the marine bacterium Vibrio campbellii.

Vibrio spp. play a crucial role in the global recycling of the highly abundant recalcitrant biopolymer chitin in marine ecosystems through their ability to secrete chitin-degrading enzymes to efficiently hydrolyse chitinous materials and use them as their major carbon source. In this study, the first crystal structures of a complete four-domain chitin-active AA10 lytic polysaccharide monooxygenase from the chitinolytic bacterium Vibrio campbellii type strain ATCC BAA-1116 are reported. The crystal structures of apo and copper-bound VhLPMO10A were resolved as homodimers with four distinct domains: an N-terminal AA10 catalytic (CatD) domain connected to a GlcNAc-binding (GbpA_2) domain, followed by a module X domain and a C-terminal carbohydrate-binding module (CBM73). Size-exclusion chromatography and small-angle X-ray scattering analysis confirmed that VhLPMO10A exists as a monomer in solution. The active site of VhLPMO10A is located on the surface of the CatD domain, with three conserved residues (His1, His98 and Phe170) forming the copper(II)-binding site. Metal-binding studies using synchrotron X-ray absorption spectroscopy and X-ray fluorescence, together with electron paramagnetic resonance spectroscopy, gave consistently strong copper(II) signals in the protein samples, confirming that VhLPMO10A is a copper-dependent enzyme. ITC binding data showed that VhLPMO10A could bind various divalent cations but bound most strongly to copper(II) ions, with a Kd of 0.1 ± 0.01 µM. In contrast, a Kd of 1.9 nM was estimated for copper(I) ions from redox-potential measurements. The presence of ascorbic acid is essential for H2O2 production in the reaction catalysed by VhLPMO10A. MALDI-TOF MS identified VhLPMO10A as a C1-specific LPMO, generating oxidized chitooligosaccharide products with different degrees of polymerization (DP2ox-DP8ox). This new member of the chitin-active AA10 LPMOs could serve as a powerful biocatalyst in biofuel production from chitin biomass.

Utilization of Parapeneopsis stylifera shrimp shell waste for the production of chitooligosaccharides using different non-specific enzymes and their characterization

Utilization of Parapeneopsis stylifera shrimp shell waste for the production of chitooligosaccharides using different non-specific enzymes and their characterization

Enhancing Enzyme Activity and Thermostability of Bacillus amyloliquefaciens Chitosanase BaCsn46A Through Saturation Mutagenesis at Ser196

Chitosanase plays an important role in chitooligosaccharides (COS) production. We found that the chitosanase (BaCsn46A) of Bacillus amyloliquefacien was a good candidate for chitosan hydrolysis of COS. In order to further improve the enzyme properties of BaCsn46A, the S196 located near the active center was found to be a critical site impacts on enzyme properties by sequence alignment analysis. Herein, saturation mutation was carried out to study role of 196 site on BaCsn46A catalytic function. Compared with WT, the specific enzyme activity of S196A increased by 118.79%, and the thermostability of S196A was much higher than WT. In addition, we found that the enzyme activity of S196P was 2.41% of that of WT, indicating that the type of amino acid in 196 site could significant affect the catalytic activity and thermostability of BaCsn46A. After molecular docking analysis we found that the increase in hydrogen bonds and decrease in unfavorable bonds interacting with the substrate were the main reason for the change of enzyme properties which is valuable for future studies on Bacillus species chitosanase.

Purification and biochemical characterization of a novel chitosanase cloned from the gene of Kitasatospora setae KM-6054 and its application in the production of chitooligosaccharides.

Chitosanase could degrade chitosan efficiently under mild conditions to prepare chitosan oligosaccharides (COSs). COS possesses versatile physiological activities and has wide application prospects in food, pharmaceutical and cosmetic fields. Herein, a new glycoside hydrolase (GH) family 46 chitosanase (CscB) was cloned from Kitasatospora setae KM-6054 and heterologously expressed in Escherichia coli. The recombinant chitosanase CscB was purified by Ni-charged magnetic beads and showed a relative molecular weight of 29.19kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). CscB showed the maximal activity (1094.21 U/mg) at pH 6.0 and 30°C. It was revealed that CscB is a cold-adapted enzyme. CscB was determined to be an endo-type chitosanase with a polymerization degree of the final product mainly in the range of 2-4. This new cold-adapted chitosanase provides an efficient enzyme tool for clean production of COSs.

Customized chitooligosaccharide production-controlling their length via engineering of rhizobial chitin synthases and the choice of expression system.

Chitooligosaccharides (COS) have attracted attention from industry and academia in various fields due to their diverse bioactivities. However, their conventional chemical production is environmentally unfriendly and in addition, defined and pure molecules are both scarce and expensive. A promising alternative is the in vivo synthesis of desired COS in microbial platforms with specific chitin synthases enabling a more sustainable production. Hence, we examined the whole cell factory approach with two well-established microorganisms-Escherichia coli and Corynebacterium glutamicum-to produce defined COS with the chitin synthase NodC from Rhizobium sp. GRH2. Moreover, based on an in silico model of the synthase, two amino acids potentially relevant for COS length were identified and mutated to direct the production. Experimental validation showed the influence of the expression system, the mutations, and their combination on COS length, steering the production from originally pentamers towards tetramers or hexamers, the latter virtually pure. Possible explanations are given by molecular dynamics simulations. These findings pave the way for a better understanding of chitin synthases, thus allowing a more targeted production of defined COS. This will, in turn, at first allow better research of COS' bioactivities, and subsequently enable sustainable large-scale production of oligomers.

OPTIMASI KITOOLIGOSAKARIDA CANGKANG RAJUNGAN (Portunus pelagicus) MENGGUNAKAN ENZIM KITOSANASE DARI Bacillus sp.

Chitooligosaccharides (COS) are glycoprotein complex compounds derived from chitosan linked by 1,4 glucosidic bonds with chain lengths of 20 or less. Crab shell is a crustacean waste that contains 20-30% chitin as the basic material for making chitooligosaccharides. Chitooligosaccharide hydrolysis can be carried out enzymatically. Enzymatic hydrolysis is specific and controlled. Enzymes that can be used to hydrolyze chitosan into chitooligosaccharides are chitosanase enzymes. Chitosanase enzyme is a glycosyl hydrolase enzyme that catalyzes the hydrolysis of -1,4 glycosidic bonds of chitosan to produce low molecular weight chitooligosaccharides. One of the bacteria that produces chitosanase enzymes, namely Bacillus sp. The purpose of this study was to optimize the production of chitooligosaccharides by treating the enzyme concentration, hydrolysis time, and hydrolysis temperature. This research was conducted using a completely randomized design (CRD) with the following factors: enzyme concentration (0.5%, 1%, 1.5%, 2%, 2.5%) and hydrolysis temperature (30oC, 40oC, 50oC, 60oC, 70oC, 80oC) and time of hydrolysis (1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours and 8 hours). This enzyme works optimally at pH 4.5. The results showed that this chitosanase enzyme had optimum activity at a concentration of 2% (w/w) chitosan, a temperature of 50oC, and a hydrolysis time of 5 hours. The chitooligosaccharide has a reducing sugar content of 8.49 ± 0.38 mg/mL, a viscosity of 19.08 ± 0.01 cPs, and a molecular weight of 4.51 kDa.
 Keywords: chitooligosaccharides, crab shells, chitosanase enzyme, hydrolysis