Crab-Eating Monkey Acidic Chitinase (CHIA) Efficiently Degrades Chitin and Chitosan under Acidic and High-Temperature Conditions

Chitooligosaccharides, the degradation products of chitin and chitosan, possess anti-bacterial, anti-tumor, and anti-inflammatory activities. The enzymatic production of chitooligosaccharides may increase the interest in their potential biomedical or agricultural usability in terms of the safety and simplicity of the manufacturing process. Crab-eating monkey acidic chitinase (CHIA) is an enzyme with robust activity in various environments. Here, we report the efficient degradation of chitin and chitosan by monkey CHIA under acidic and high-temperature conditions. Monkey CHIA hydrolyzed α-chitin at 50 °C, producing N-acetyl-D-glucosamine (GlcNAc) dimers more efficiently than at 37 °C. Moreover, the degradation rate increased with a longer incubation time (up to 72 h) without the inactivation of the enzyme. Five substrates (α-chitin, colloidal chitin, P-chitin, block-type, and random-type chitosan substrates) were exposed to monkey CHIS at pH 2.0 or pH 5.0 at 50 °C. P-chitin and random-type chitosan appeared to be the best sources of GlcNAc dimers and broad-scale chitooligosaccharides, respectively. In addition, the pattern of the products from the block-type chitosan was different between pH conditions (pH 2.0 and pH 5.0). Thus, monkey CHIA can degrade chitin and chitosan efficiently without inactivation under high-temperature or low pH conditions. Our results show that certain chitooligosaccharides are enriched by using different substrates under different conditions. Therefore, the reaction conditions can be adjusted to obtain desired oligomers. Crab-eating monkey CHIA can potentially become an efficient tool in producing chitooligosaccharide sets for agricultural and biomedical purposes.

Enzymatic Production of Biologically Active 3-Methoxycinnamoylated Lysophosphatidylcholine via Regioselctive Lipase-Catalyzed Acidolysis

Enzymatic acidolysis of egg-yolk phosphatidylcholine (PC) with 3-methoxycinnamic acid (3-OMe-CA) was investigated to produce biologically active 3-methoxycinnamoylated phospholipids. Four commercially available lipases were screened for their ability to incorporate 3-OMe-CA into PC. The results showed that Novozym 435 is the most effective biocatalyst for this process, while during the examination of organic solvents, heptane was found propriate reaction medium. The other reaction parameters including the substrate molar ratio, enzyme load and reaction time were designed using an experimental factorial design method. According to three-level-3-factor Box-Behnken model it was shown that all of studied parameters are crucial variables for the maximization of the synthesis of structured PLs. The optimum conditions derived via response surface methodology (RSM) were: 30% of lipase of the total weight of substrates, 1:15 molar ration of PC/3-OMe-CA and reaction time 4 days. The process of acidolysis performed on the increased scale at optimized parameters afforded two products. The major product, 3-methoxycinnamoylated lysophosphatidylcholine (3-OMe-CA-LPC) was isolated in high 48% yield, while 3-methoxycinnamoylated phosphatidylcholine (3-OMe-CA-PC) was produced in trace amount only in 1.2% yield. Obtained results indicate that presented biotechnological method of synthesis of 3-methoxycinnamoylated lysophosphatidylcholine is competitive to the previously reported chemical one.

Heterologous Expression and Characterization of a High-Efficiency Chitosanase From Bacillus mojavensis SY1 Suitable for Production of Chitosan Oligosaccharides

Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase from Bacillus mojavensis SY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed into Pichia pastoris X33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.

Heterologous Expression of a Thermostable Chitinase from Myxococcus xanthus and Its Application for High Yield Production of Glucosamine from Shrimp Shell

Glucosamine (GlcN) is a widely used food supplement. Hence, enormous attention has been concerned with enzymatic production of GlcN owing to its advantage over a chemical approach. In this study, a previously unstudied chitinase gene (MxChi) in the genome of Myxococcus xanthus was cloned, expressed in recombinant soluble form and purified to homogeneity. TLC-, UPLC-, and microplate-reader- based activity tests confirmed MxChi hydrolyzes colloidal chitin to chitobiose as sole product. The optimal catalytic pH and temperature of MxChi was identified as 7.0 and 55 °C, respectively. MxChi exhibited 80% activity after 72 h incubation at 37 °C. The site-directed mutagenesis revealed that the amino acids D323A, D325A, and E327A of MxChi were in the DXDXE catalytic motif of GH18. When coupled with β-N-acetylhexosaminidase (SnHex) and deacetylase (CmCBDA), the enzyme allowed one-pot extraction of GlcN from colloidal chitin and shrimp shell. The optimal condition was 37 °C, pH 8.0, and 1/3/16.5 (MxChi/SnHex/CmCBDA), conducted by orthogonal design for the enzymatic cascades. Under this condition, the yield of GlcN was 26.33 mg from 400 mg shrimp shell. Facile recombinant in E. coli, robust thermostability and pure product herein makes newly discovered chitinase a valuable candidate for the green recycling of chitin rich waste.

Enzymatic Production of Ecodiesel by Using a Commercial Lipase CALB, Immobilized by Physical Adsorption on Mesoporous Organosilica Materials

The synthesis of two biocatalysts based on a commercial Candida antarctica lipase B, CALB enzyme (E), physically immobilized on two silica supports, was carried out. The first support was a periodic mesoporous organosilica (PMO) and the second one was a commercial silica modified with octyl groups (octyl-MS3030). The maximum enzyme load was 122 mg enzyme/g support on PMO and 288 mg enzyme/g support on octyl-MS3030. In addition, the biocatalytic efficiency was corroborated by two reaction tests based on the hydrolysis of p-nitrophenylacetate (p-NPA) and tributyrin (TB). The transesterification of sunflower oil with ethanol was carried out over the biocatalysts synthesized at the following reaction conditions: 6 mL sunflower oil, 1.75 mL EtOH, 30 °C, 25 μL NaOH 10 N and 300 rpm, attaining conversion values over 80% after 3 h of reaction time. According to the results obtained, we can confirm that these biocatalytic systems are viable candidates to develop, optimize and improve a new methodology to achieve the integration of glycerol in different monoacylglycerol molecules together with fatty acid ethyl esters (FAEE) molecules to obtain Ecodiesel.

Oligosaccharides from Coprophilous Fungi: An Emerging Functional Food with Potential Health-Promoting Properties: a Recent Appraisal

Functional foods are essential food products that possess health-promoting properties for the treatment of infectious diseases. In addition, they provide energy and nutrients, which are required for growth and survival. They occur as prebiotics or dietary supplements, including oligosaccharides, processed foods, and herbal products. However, oligosaccharides are more efficiently recognized and utilized, as they play a fundamental role as functional ingredients with great potential to improve health in comparison to other dietary supplements. They are low molecular weight carbohydrates with a low degree of polymerization. They occur as fructooligosaccharide (FOS), inulooligosaccharadie (IOS), and xylooligosaccahride (XOS), depending on their monosaccharide units. Oligosaccharides are produced by acid or chemical hydrolysis. However, this technique is liable to several drawbacks, including inulin precipitation, high processing temperature, low yields, high production costs, etc. As a consequence, the application of microbial enzymes for oligosaccharide production is recognized as a promising strategy. Microbial enzymatic production of FOS and IOS occurs by submerged or solid-state fermentation in the presence of suitable substrates (sucrose, inulin) and catalyzed by fructosyltransferases and inulinases. Incorporation of FOS and IOS enriches the rheological and physiological characteristics of foods. They are used as low cariogenic sugar substitutes, suitable for diabetics, and as prebiotics, probiotics & nutraceutical compounds. In addition, these oligosaccharides are employed as anticancer & antioxidant agents and aid in mineral absorption, lipid metabolism, immune regulation etc. This review, therefore, focuses on the occurrence, physico-chemical characteristics, and microbial enzymatic synthesis of FOS and IOS from coprophilous fungi. In addition, the potential health benefits of these oligosaccharides were discussed in detail.