Biodegradation of Crystalline Chitin:A Review of Recent Advancement,Challenges,and Future Study Directions

Chitin is the second most abundant renewable polysaccharide on Earth.The degradation of chitin into soluble and bioactive N-acetyl chitooligosaccharides(NCOSs)and N-acetyl-D-glucosamine(GlcNAc)has emerged as a pivotal step in the efficient and sustainable utilization of chitin resources.However,because of its dense structure,high crystallinity,and poor solubility,chitin typically needs pretreatment via chemical,physical,and other methods before enzymatic conversion to enhance the accessibility between substrates and enzyme molecules.Consequently,there has been considerable interest in exploring the direct biological degradation of crystalline chitin as a cost-effective and environment-friendly technology.This review endeavors to present several biological methods for the direct degradation of chitin.We primarily focused on the importance of chitinase containing chitin-binding domain(CBD).Additionally,various modification strategies for increasing the degradation efficiency of crystalline chitin were introduced.Subsequently,the review systematically elucidated critical components of multi-enzyme catalytic systems,highlighting their potential for chitin degradation.Furthermore,the application of microorganisms in the degradation of crystalline chitin was also discussed.The insights in this review contribute to the explorations and investigations of enzymatic and microbial approaches for the direct degradation of crystalline chitin,thereby fostering advancements in biomass conversion.

Separation of chitin from shrimp shells enabled by transition metal salt aqueous solution and ionic liquid

Chitin is a widely used important industrial polymer mainly from shrimp shells, but its commercial preparation is under the great challenge of serious pollution due to the requirement of HCl and Na OH.Herein, we demonstrated that high purity chitin can be obtained from waste shrimp shells(WSSs) by cascade separation with transition metal salt aqueous solution and ionic liquid(IL). Firstly, calcium carbonate of WSSs was effectively removed in the metal salt aqueous solution driven by the ion exchange interaction. Subsequently, 1-butyl-3-methylimidazolium chloride([Bmim]Cl) had bifunctional abilities to remove residual protein and introduced metal salts simultaneously by hydrogen bonding and coordination interactions. The key experimental factors affecting the separation process were systematically studied, including the type of metal salts, temperature, and [Bmim]Cl loading. After sequential treatment with a 20%(mass) Ni SO4aqueous solution at 130 ℃ and [Bmim]Cl at 150 ℃, the purity of a-chitin can be up to 96.5%(mass) that meets commercial requirements. The use of metal salts with higher coordination ability makes the preparation of chitin no longer depend on the commonly acid-base reaction, which is conducive to the preservation of chitin structure.

新型催化剂chitin/BiOBr的构建及用于抗生素污染物去除

利用溶剂热法制备了一种新型光催化剂chitin/BiOBr。在可见光照射下,chitin/BiOBr对环丙沙星表现出了优异的光催化降解性能,180 min内可对环丙沙星(250 mL,20mg·L^(-1))的降解率为78%,其降解速率是纯溴氧化铋的5.2倍。通过一系列表征技术对样品的形貌结构、光电性能和化学元素组成等进行了研究。活性成分测定实验表明,羟基自由基(·OH)和空穴(h+)共同作用于环丙沙星的降解。循环实验表明chitin/BiOBr有较稳定的可见光催化性能。同时根据实验结果对环丙沙星的降解机理进行了探讨。

Isolation and Screening of Strains for Silkworm Puparium Chitin Degradation and the Optimization of Chitinase Production by Response Surface Methodology

[Objective] The aim of this study was to optimize the conditions of chitinase-produce strains.[Method] A kind of screened chitinase-produce strain G-254 was habituated cultured,and then the single factor experiment was carried out to explore the effects of different carbon source,nitrogen source and inorganic salt on the activity of produced chitinase;the response surface test was used to determine the optimal conditions for chitinase production.[Result] The optimal conditions for chitinase production were：8% of glucose,5% of beef extract and 0.07% of MgSO4,and the activity of chitinase reached the maximal value（6.86 U）under these conditions.[Conclusion] The study improved the activity of chitinase produced by strain G-254 and provided good foundation for industrial production.

Chitinolytic Assay and Identification of Bacteria Isolated from Shrimp Waste Based on 16S rDNA Sequences

Shrimp waste contains 20% - 60% chitin and possible to be source of chitinolytic bacteria. Chitinolytic bacteria are capable of hydrolyzing of chitin progressively to produce N-acetylglucosamine monomer which can be used to overcome the shrimp waste. The objectives of this research were to identify species of bacteria with high activity of chitin degradation in shrimp waste and to analyze their potency as chitin degradation agent. The research consists of screening of chitinolytic bacteria based on chitinolytic index, activity assay of chitinase using colorimetric method, and molecular identification of bacteria based on 16S rDNA sequences. Two of eighteen isolates of chitinolytic bacteria (PBK 2 and SA 1.2 isolates) showed the highest chitinolytic index, which were 2.069 and 2.084, whereas chitinase activity was 0.213 and 0.219 U/ml respectively. Based on 16S rDNA sequences, isolate of PBK 2 was identified as Acinetobacter johnsonii 3-1, whereas SA 1.2 was identified as Bacillus amyloliquefaciens GR53 with 99.78% similarity.

IMPROVING NONTHROMBOGENICITY OF CHITIN WITH ZWITTERIONIC STRUCTURE OF SULFOBETAINE

In order to improve the nonthrombogenicity of chitin,a new monomer,N,N-dimethyl(β-hydroxyethyloxyethyl) ammonium propanesulfonate(DHAPS)was designed,synthesized and grafted onto the chitin membrane by using hexamethylene diisocyanate(HDI)as a coupling agent.Surface analysis of the grafted membranes by ATR-FTIR and XPS confirms that DHAPS has been successfully grafted onto the membrane surface.The platelet resistant property of the grafted membranes was evaluated by a platelet-rich plasma adhesion method.The results showed that platelet-adhesive resistance of the modified membrane has been greatly improved.

Degradation of Chitin and Chitosan by a Recombinant Chitinase Derived from a Virulent <i>Aeromonas hydrophila</i>Isolated from Diseased Channel Catfish

A chitinase was identified in extracellular products of a virulent?Aeromonas hydrophila?isolated from diseased channel catfish (Ictalurus punctatus). Recombinant chitinase (rChi-Ah) was produced in?Escherichia coli. Purified rChi-Ah had optimal activity at temperature of 42℃?and pH 6.5. The affinity (Km) for chitosan was 4.18 mg·ml-1?with?Vmax?of 202.5 mg·min-1·mg-1. With colloidal chitin as substrate, rChi-Ah generated N,N’-diacetyl-glucosamine predominantly. Conversion of chitosan (≥75% deacetylated) by rChi-Ah revealed five major products: 2 to 4 units of glucosamine, all of which had at least one acetyl group. It was determined that N-acetylated glucosamine was the recognition and cleavage site of rChi-Ah;the minimal and maximal cleavages were two and four glucosamine units, respectively. Functional analysis of rChi-Ah suggests that?A. hydrophilachitinase is a bioactive chitinolytic enzyme, which may benefit the pathogen for survival and/or infection.

Bioluminescence and Chitinase Production during Chitin Fermentation by Vibrio harveyi

Vibrio harveyi, like other luminescent bacteria, is capable of producing extracellular chitinases. Microbial chitinases are utilized to depolymerize chitin into chitooligosaccharides and N-acetylglucosamine for the acquisition of carbon and possibly nitrogen, needed for survival. For many luminous marine bacteria （Vibrio spp.）, quorum-sensing is highly speculated to be responsible for bioluminescence; however, in terrestrial species （Photorhabdus spp.） luminosity seems to be controlled through unknown mechanism of phase variation. In the present work, the correlation between bacterial luminosity and chitinase production of F. harveyi was studied. The utilization of bioluminescence could prove to be an easier and more convenient method to monitor chitin fermentations that employ luminous bacteria. Results from the fermentation study indicate that luminosity of F. harveyi inversely correlates with chitinase production. In other words, during chitin fermentation, chitinase production was seen to increase while luminosity decreased with respect to growth and growth conditions. Furthermore, the results also suggest that V. harveyi may utilize an alternate mechanism that can counter quorum-sensing mechanisms to ensure bacterial survival under deteriorating growth conditions. The inverse relationship observed in this study may lead to a basic understanding of monitoring and studying chitin fermentations and anti-quorum-sensing/phase variation mechanisms exhibited by luminous bacteria.

Bridging peripheral nerves using a deacetyl chitin conduit combined with short-term electrical stimulation

Previous studies have demonstrated that deacetyl chitin conduit nerve bridging or electrical stimulation can effectively promote the regeneration of the injured peripheral nerve. We hypoth-esized that the combination of these two approaches could result in enhanced regeneration. Rats with right sciatic nerve injury were subjected to deacetyl chitin conduit bridging combined with electrical stimulation （0.1 ms, 3 V, 20 Hz, for 1 hour）. At 6 and 12 weeks after treatment, nerve conduction velocity, myelinated axon number, ifber diameter, axon diameter and the thickness of the myelin sheath in the stimulation group were better than in the non-stimulation group. The results indicate that deacetyl chitin conduit bridging combined with temporary electrical stimu-lation can promote peripheral nerve repair.

Activities of the Deriviatives of Chitin on the Osbeoblast Proliferation and the Effect on Bone Strength in Ovariectomized Rats

This study intends to examine the effects of different concentrations of four kinds of degradations of chitin： glucosamine （GLC）, N-acetyl-D-glucosamine （NAG）, chitooligosaccharide（COS）, CM-chitooligosaccharide （CM-COS）—on the proliferation of MC3T3-E1 cell line cultured in vitro. Results suggest that all of the glucoses mentioned above promoted the proliferation of osteoblast, and various concentrations have different effects： the proliferation was remarkable when the concentration of GLC, NAG, COS, CM-COS was 100 , 100, 500, 500 μg/mL ,respectively. Furthermore we choose the glucosamine as the material and study the effect on the bone strength in ovariectomized rats. The results showed that the middle does of glucosamine can significantly increase the hone strength of femur in ovariectomized rats.

Recovery of Chitin and Protein from Shrimp Head Waste by Endogenous Enzyme Autolysis and Fermentation

The industrial processing of shrimp produces massive quantities of solid waste that is a notable source of animal protein, chitin, carotenoids, and other bioactive compounds that are not appropriately utilized. In the present study, chitin and protein extraction from shrimp head with autolysis and fermentation using Bacillus licheniformis were investigated. The results showed that when shrimp heads were autolyzed with a natural pH at 50℃ for 4 h, the total amino acid nitrogen in the supernatant was 5.01 mg mL^-1. Then, when a 50%(v/m) inoculum of the hydrolysate was incubated at 60℃ for 10 h, a deproteinization rate of 88.3% could be obtained. The fermented supernatant was processed into a dry protein powder, while the residues were demineralized by 10% citric acid for chitin. The recovered protein powder contained 5.5% moisture, 11.5% ash, and 66.7% protein, while the chitin contained 3.5% moisture, 2.1% ash, and 3.1% protein. In addition, amino acids, minerals, heavy metals, the degree of acetylation, microstructure, and Fourier-transform infrared(FT-IR) spectroscopy results were analyzed. Furthermore, the statistics of the large scale trial after treatment with 20 kg of shrimp heads were analyzed. Thus, this work made the shrimp waste utilization environmentally sound and valuable.

Synthesis of Novel Benzoylphenylurea Chitin Inhibitors from Chlorothalonil

Twenty-six novel benzoylphenylurea chitin inhibitor derivatives have been synthesized in over 30~50% yield from chlorothalonil 1 via sequential fluorine exchange, aminolysis, hydrolysis, decarboxylation and acylation reactions.

Adsorption of Heavy Metal Ions by Adsorbent from Waste Mycelium Chitin

The adsorption properties of chitin adsorbent from mycelium of fermentation industries for the removal of heavy metal ions were studied. The result shows that the chitin adsorbent has high adsorption capacity for many heavy metal ions and Ni2+ in citric acid. The influence of pH was significant:When pH is higher than 4.0, the high adsorption capacity is obtained,otherwise H+ ion inhibits the adsorption of heavy metal ions. The comparison of the chitin adsorbent with some other commercial adsorbents was made, in which that the adsorption behaviorchitin adsorbent is close to that of commercial cation exchange adsorbents, and its cost is much lower than those commercial adsorbents.

Comparison of the defluoridation efficiency of calcium phosphate and chitin in the exoskeleton of Antarctic krill

Calcium （Ca）, phosphorus （P）, and chitin are the main components of the exoskeleton of krill. Defluoridation of a solution of sodium fluoride （NaF） using calcium phosphate （Ca3（PO4）2） and chitin as defluoridation agents was studied. Orthogonal experiments were designed to find the optimum reaction conditions for defluoridation, to obtain the maximum defluoridation efficiency and fluoride removal capacity of calcium phosphate and chitin. At the same time, a comparison of the capacity of the two defluoridation agents was made. The results suggest that calcium phosphate has a far greater capability than chitin for the removal of fluoride （F） from water under similar reaction conditions. It is also suggested that Antarctic krill is likely to adsorb fluoride via compounds such as calcium phosphate, hydroxyapatite, and other compounds of Ca and P with the general form （Ca, X）x（PO4, HPO4, Y）y（OH, Z）z, in addition to chitin.

SDS-Chitin-PAGE电泳法分析大葱叶几丁质酶特性

使用Sodium dodecyl sulfate-几丁质-聚丙烯胺酰胺凝胶电泳(SDS-Chitin-PAGE)方法分析了大葱葱叶几丁质酶的特性。在大葱叶组织中共检测到三种几丁质酶同工酶,分别命名为CH-A、CH-B、CH-C。分析结果表明,这三种几丁质酶具有SDS抗性,β-巯基乙醇对其活性也无显著影响。在SDS-Chitin-PAGE前,样品缓冲溶液与样品蛋白粗提液孵化处理的最适温度为24℃、时间为10～20min。凝胶电泳酶活分析表明,大葱葱叶中CH-A、CH-B的耐热温度可达60℃,CH-C的耐热温度达到70℃。葱叶中CH-A的最适pH为5.2～5.6,CH-B的最适pH为4.0～4.4,CH-C的最适pH为6.6～8.0。

真菌作为生物医学材料的应用——以灵芝子实体SACCHACHITIN为例的研发过程

到目前为止,SACCHACHITIN为唯一将真菌应用为生物医学材料的案例。灵芝残渣是一种坚韧的纤维。过去,学者对真菌细胞壁的研究也发现真菌细胞壁主要的组成是多糖（Polysaccharide）和几丁质（Chitin）,以及一些类似黑色素（Melanin）的成分。几丁质最早发现在甲壳类之外骨骼,蟹壳几丁质在1992时已发展成为商品化的伤口敷料,对伤口愈合具有优良促进功能。灵芝残渣同样含有几丁质,其以热碱（IN NaOH 90oC）去除残余脂蛋白、核酸、脂肪再以过氧化氢漂白去除黑色素,再做成模膜,是一种滤纸状多孔性白色薄膜。此一薄膜几丁质的含量约在40%~50%,其余为以葡萄糖为主的多糖（50%~60%）的复合体,称之为SACCHACHITIN。经过一连串的细胞试验、动物试验、人体试验证实具有生物兼容性、生物降解性、并无过敏性及抗体反应;同时对急性及慢性皮肤创伤均具有极优良的愈合促进作用。其作用机理在于吸附伤口中由人体免疫细胞所产生过量的金属型基质蛋白水解脢（MMP）,以及细菌所产生的蛋白水解脢,避免细菌感染,结束发炎反应,提早进入肉芽组织生长期。此作用也会保护生长因子（Growth factors,TGF,PDGF和VEGF）不受破坏而加速愈合。SACCHACHITIN的专利已进行技术转移;目前产品以镭射（激光）除斑手术的术后修复面膜,以及化妆品为商品诉求。

Chitin scaffold combined with autologous small nerve repairs sciatic nerve defects

Although autologous nerve transplantation is the gold standard for treating peripheral nerve defects,it has many clinical limitations.As an alternative,various tissue-engineered nerve grafts have been developed to substitute for autologous nerves.In this study,a novel nerve graft composed of chitin scaffolds and a small autologous nerve was used to repair sciatic nerve defects in rats.The novel nerve graft greatly facilitated regeneration of the sciatic nerve and myelin sheath,reduced atrophy of the target muscle,and effectively restored neurological function.When the epineurium of the small autogenous nerve was removed,the degree of nerve regeneration was similar to that which occurs after autogenous nerve transplantation.These findings suggest that our novel nerve graft might eventually be a new option for the construction of tissue-engineered nerve scaffolds.The study was approved by the Research Ethics Committee of Peking University People's Hospital(approval No.2019 PHE27)on October 18,2019.

In vitro toxicologic study of chitin short fiber reinforced polycaprolactone composite

Objective: To investigate the cytotoxicity and cytocompatibility of chitin fiber reinforced polycaprolactone composite in vitro in order to provide useful scientific basis for clinical application. Methods: Cell morphology observation, MTT and DNA assay were used to evaluate the influence of the composite on the morphology, growth and proliferation of cultured L-929 cells. Results: The composite did not impair the morphology of cultured cells in vitro. MTT and DNA assay demonstrated that the growth and proliferation of the cultured cells were not significantly inhibited by the composite. Conclusion : The composites have fine cytocompatibility and are safe for clinical use of reconstruction of chest wall defects.

Myelin-associated glycoprotein combined with chitin conduit inhibits painful neuroma formation after sciatic nerve transection

Studies have shown that myelin-associated glycoprotein(MAG)can inhibit axon regeneration after nerve injury.However,the effects of MAG on neuroma formation after peripheral nerve injury remain poorly understood.In this study,local injection of MAG combined with nerve cap made of chitin conduit was used to intervene with the formation of painful neuroma after sciatic nerve transfection in rats.After 8 weeks of combined treatment,the autotomy behaviors were reduced in rats subjected to sciatic nerve transfection,the mRNA expression of nerve growth factor,a pain marker,in the proximal nerve stump was decreased,the density of regenerated axons was decreased,the thickness of the myelin sheath was increased,and the ratio of unmyelinated to myelinated axons was reduced.Moereover,the percentage of collagen fiber area and the percentage of fibrosis marker alpha-smooth muscle actin positive staining area in the proximal nerve stump were decreased.The combined treatment exhibited superior effects in these measures to chitin conduit treatment alone.These findings suggest that MAG combined with chitin conduit synergistically inhibits the formation of painful neuroma after sciatic nerve transection and alleviates neuropathic pain.This study was approved by the Animal Ethics Committee of Peking University People’s Hospital(approval No.2019PHE027)on December 5,2019.

Ionic Liquid as Useful Media for Dissolution, Derivatization, and Nanomaterial Processing of Chitin

This paper reviews studies on dissolution, derivatization, and nanomaterial processing of chitin using an ionic liquid as useful media. Because chitin is the second most abundant polysaccharide on the earth after cellulose, there is major interest in conversion of native chitin resources into various useful materials after proper dissolution in suitable solvents. For the derivatization and nanomaterial processing of chitin, the author has been focusing on ionic liquids because which have been found to be used as good solvents for cellulose in a past decade. The author found that an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), dissolved chitin in concentrations up to 4.8 wt% and mixtures of the higher amounts of chitin with AMIMBr gave ion gels. Acetylation, the simplest derivatization, of chitin using acetic anhydride was achieved in the AMIMBr solvent under mild conditions. Furthermore, the chitin nanofibers were fabricated by regeneration technique from the chitin ion gel with AMIMBr using methanol. Moreover, filtration of the chitin nanofiber dispersion with methanol was carried out to give a chitin nanofiber film. The chitin nanofiber-poly(vinyl alcohol) composite film was also prepared from the ion gel by co-regeneration method.