Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

High-level production of chitinase by multi-strategy combination optimization in Bacillus licheniformis.

In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2△abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2△abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA.

Bacterial chitinases: genetics, engineering and applications.

Chitinases are a group of enzymes that catalyze chitin hydrolysis and are present in all domains of life. Chitinases belong to different glycosyl hydrolase families with great diversity in their sequences. Microorganisms such as bacteria and fungi produce chitinases for nutrition, and energy, and to parasitize the chitinous hosts. But chitinases from bacteria are of special interest due to their ubiquitous nature and ability to perform under extreme conditions. Chitinases produced by bacteria have been explored for their use in agriculture and industry. In agriculture, their main role is to control chitin-containing insect pests, fungal pathogens, and nematodes. In the seafood industry, they found their role in the management of processing wastes which are mainly chitinous substances. Chitinases are also used to synthesize low molecular weight chitooligomers which are proven bioactive compounds with activities such as anti-tumour, antimicrobial, and immunity modulation. Considering their importance in ecology and biotechnological applications, several bacterial chitinases have been studied in the last two decades. Despite their potential, bacterial chitinases have a few limitations such as low production and lack of secretion systems which make the wild-type enzymes unfit for their applications in industries and other allied sectors. This review is an attempt to collate significant works in bacterial chitinases and their application in various industries and the employment of various tools and techniques for improvement to meet industrial requirements.

Recombinant production and characterisation of two chitinases from Rasamsonia emersonii, and assessment of their potential industrial applicability.

Rasamsonia emersonii (previously Talaromyces emersonii) is a thermophilic filamentous fungus displaying optimum growth at 45 °C. It has a history of use in commercial food enzyme production. Its unfractionated chitinolytic secretome was partially characterised in the early 1990s; however, no individual chitinase from this source has been described in literature previously. This study describes two GH18 chitinases originating from the R. emersonii genome, expressed in the methylotrophic yeast P. pastoris. Chit1 comprises of a GH18 catalytic domain and Chit2 comprises of a GH18 catalytic domain and a chitin-binding motif at the C-terminal. The chitinases were expressed as glycoproteins. The apparent molecular weight of Chit1 was 35.8-42.1 kDa with a smearing tail associated with glyco-sidechains visible up to 72.2 kDa. This became two bands of 30.8 and 29.0 kDa upon de-glycosylation. The apparent molecular weight of Chit2 was 50.4 kDa, reducing to 48.2 kDa upon de-glycosylation. Both chitinases displayed endo-chitinase and chitobiosidase activity, temperature optima of 50-55 °C and low pH optima (pH 4.5 or lower); Chit1 displayed a pH optimum of 3.5, retaining > 60% maximum activity at pH 2.2, a pH range lower than most enzymes of fungal origin. Chit2 displayed the highest chitin-degrading ability at 3456 µmol/mg on 4-NP-triacetylchitotriose, but lost activity faster than Chit1, which displayed 403 µmol/mg on the same substrate. The predicted D values (time required to reduce the enzyme activity to 10% of its original value at 50 °C) were 19.2 and 2.3 days for Chit1 and Chit2, respectively. Thus, Chit1 can be considered one of few hyperthermostable chitinase enzymes described in literature to date. Their physicochemical properties render these chitinases likely suitable for shrimp chitin processing including one-step chitin hydrolysis and alternative sustainable protein processing and the attractive emerging application of mushroom food waste valorisation.Key points• Two GH18 chitinases originating from the industrially relevant thermophilic fungus R. emersonii were cloned and expressed in P. pastoris.• The purified recombinant chitinases showed low pH and high temperature optima and appreciable thermostability at industrially relevant temperatures.• The chitinases displayed characteristics that indicate their likely suitability to several industrial applications including sustainable alternative protein processing, food waste valorisation of commercial mushroom production and one-step shrimp chitin processing.

Salicylate-Induced Chitinases in Pea Roots.

Three proteins induced by salicylic acid were revealed in pea roots. These proteins were identified as chitinase isozymes belonging to the glycoside hydrolases family 18. The PsCam050724 transcript encoding at least one of these isoforms was found, allowing us to determine its primary structure, which lacks the signal peptide.

Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc.

Chitinase and chitin-oligosaccaride can be used in multiple field, so it is important to develop a high-yield chitinase producing strain. Here, a recombinant Pichia pastoris with 4 copies of ChiA gene from Bacillus licheniformis and co-expression of molecular chaperon HAC1 was constructed. The amount of recombinant ChiA in the supernatant of high-cell-density fermentation reaches a maximum of 12.7 mg/mL, which is 24-fold higher than that reported in the previous study. The recombinant ChiA can hydrolyze 30% collodidal chitin with 74% conversion ratio, and GlcNAc is the most abundant hydrolysis product, followed by N, N'-diacetylchitobiose. Combined with BsNagZ, the hydrolysate of ChiA can be further transformed into GlcNAc with 88% conversion ratio. Additionally, the hydrolysate of ChiA can obviously accelerate the germination growth of rice and wheat, increasing the seedling height and root length by at least 1.6 folds within 10 days.

Chitinase family genes in grape differentially expressed in a manner specific to fruit species in response to Botrytis cinerea.

Chitinases (Chi), an important resistance-related protein, act against fungal pathogens by catalyzing the fungal cell wall, whereas are involved in different biological pathways in grape. In this study, we found 42 Chi family genes in Vitis vinifera L. (VvChis) and evaluated their expression levels after Botrytis infection, stress hormones like ethylene (ETH) and methyl-jasmonate (MeJA), and abiotic stresses like salinity and temperature changes in ripened fruits. VvChis were categorized into five groups including A, B, C, D, and E belonged to glycoside hydrolase family 18 and 19 (GH18 and GH19) according to genes structure, which expression analysis showed distinct temporal and spatial expression patterns changed in different tissues and various development stages. Different responsive elements to biotic and abiotic stresses were determined in the promoter regions of VvChis, specially elicitor-responsive element that was conserved among all VvChis genes. The expression levels of VvChis in groups A, B, and E increased after Botrytis cinerea infection in leaves and berries. Meanwhile, VvChis in glycoside hydrolase family 18 (GH18) were up-regulated under MeJA and ETH treatment, although the induction of VvChis by low temperature was more significant than high temperature. The expression of VvChis was also positively correlated with the concentration of NaCl treatment. Furthermore, differential gene-overexpression of VvChi5, VvChi17, VvChi22, VvChi26, and VvChi31 in strawberry and tomato fruits demonstrated the involvement of various isoforms in resistance to Botrytis infection through antioxidant system and lignin accumulation, which led to a reduction of damage. Among different isoforms of VvChis, we confirmed the interaction of Chi17 with Metallothionein (MTL) as oxidative stress protection, which suggests VvChis can modulate oxidative stress during postharvest storage in ripened fruits.

Chitinase production by Trichoderma koningiopsis UFSMQ40 using solid state fermentation.

The chitinases have extensive biotechnological potential but have been little exploited commercially due to the low number of good chitinolytic microorganisms. The purpose of this study was to identify a chitinolytic fungal and optimize its production using solid state fermentation (SSF) and agroindustry substrate, to evaluate different chitin sources for chitinase production, to evaluate different solvents for the extraction of enzymes produced during fermentation process, and to determine the nematicide effect of enzymatic extract and biological control of Meloidogyne javanica and Meloidogyne incognita nematodes. The fungus was previously isolated from bedbugs of Tibraca limbativentris Stal (Hemiptera: Pentatomidae) and selected among 51 isolated fungal as the largest producer of chitinolytic enzymes in SSF. The isolate UFSMQ40 has been identified as Trichoderma koningiopsis by the amplification of tef1 gene fragments. The greatest chitinase production (10.76 U gds-1) occurred with wheat bran substrate at 55% moisture, 15% colloidal chitin, 100% of corn steep liquor, and two discs of inoculum at 30 °C for 72 h. Considering the enzymatic inducers, the best chitinase production by the isolated fungus was achieved using chitin in colloidal, powder, and flakes. The usage of 1:15 g/mL of sodium citrate-phosphate buffer was the best ratio for chitinase extraction of SSF. The Trichoderma koningiopsis UFSMQ40 showed high mortality of M. javanica and M. incognita when applied to treatments with enzymatic filtrated and the suspension of conidia.

Bioconversion of chitin and concomitant production of chitinase and N-acetylglucosamine by novel Achromobacter xylosoxidans isolated from shrimp waste disposal area.

Marine pollution is a significant issue in recent decades, with the increase in industries and their waste harming the environment and ecosystems. Notably, the rise in shellfish industries contributes to tons of shellfish waste composed of up to 58% chitin. Chitin, the second most ample polymer next to cellulose, is insoluble and resistant to degradation. It requires chemical-based treatment or enzymatic hydrolysis to cleave the chitin polymers. The chemical-based treatment can lead to environmental pollution, so to solve this problem, enzymatic hydrolysis is the best option. Moreover, the resulting biopolymer by-products can be used to boost the fish immune system and also as drug delivery agents. Many marine microbial strains have chitinase producing ability. Nevertheless, we still lack an economical and highly stable chitinase enzyme for use in the industrial sector. So we isolate a novel marine bacterial strain Achromobacter xylosoxidans from the shrimp waste disposal site using chitin minimal medium. Placket-Burman and central composite design statistical models for culture condition optimisation predicted a 464.2 U/ml of chitinase production. The culture conditions were optimised for maximum chitinase production recording up to 467 U/ml. This chitinase from the A. xylosoxidans was 100% active at an optimum temperature of 45 °C (withstand up to 55 °C) and pH 8 with 80% stability. The HPLC analysis of chitinase degraded shellfish waste reveals a major amino acid profile composition-arginine, lysine, aspartic acid, alanine, threonine and low levels of isoleucine and methionine. These chitinase degraded products and by-products can be used as supplements in the aquaculture industry.

Cloning, characterization, and functional analysis of chitinase-like protein 1 in the shell of Pinctada fucata.

Biomineralization, especially shell formation, is a sophisticated process regulated by various matrix proteins. Pinctada fucata chitinase-like protein 1 (Pf-Clp1), which belongs to the GH18 family, was discovered by our group using in-depth proteomic analysis. However, its function is still unclear. In this study, we first obtained the full-length cDNA sequence of Pf-Clp1 by RACE. Real-time polymerase chain reaction results revealed that Pf-Clp1 was highly expressed in the important biomineralization tissues, the mantle edge and the mantle pallial. We expressed and purified recombinant protein rPf-Clp1 in vitro to investigate the function of Pf-Clp1 on CaCO3 crystallization. Scanning electron microscopy imaging and Raman spectroscopy revealed that rPf-Clp1 was able to affect the morphologies of calcite crystal in vitro. Shell notching experiments suggested that Pf-Clp1 might function as a negative regulator during shell formation in vivo. Knockdown of Pf-Clp1 by RNAi led to the overgrowth of aragonite tablets, further confirming its potential negative regulation on biomineralization, especially in the nacreous layer. Our work revealed the potential function of molluscan Clp in shell biomineralization for the first time and unveiled some new understandings toward the molecular mechanism of shell formation.

Functional expression of recombinant hybrid enzymes composed of bacterial and insect's chitinase domains in E. coli.

To elucidate the functional alteration of the recombinant hybrid chitinases composed of bacterial and insect's domains, we cloned the constitutional domains from chitinase-encoding cDNAs of a bacterial species, Bacillus thuringiensis (BtChi) and a lepidopteran insect species, Mamestra brassicae (MbChi), respectively, swapped one's leading signal peptide (LSP) - catalytic domain (CD) - linker region (LR) (LCL) with the other's chitin binding domain (ChBD) between the two species, and confirmed and analyzed the functional expression of the recombinant hybrid chitinases and their chitinolytic activities in the transformed E. coli strains. Each of the two recombinant cDNAs, MbChi's LCL connected with BtChi's ChBD (MbLCL-BtChBD) and BtChi's LCL connected with MbChi's ChBD (BtLCL-MbChBD), was successfully introduced and expressed in E. coli BL21 strain. Although both of the two hybrid enzymes were found to be expressed by SDS-PAGE and Western blotting, the effects of the introduced genes on the chitin metabolism appear to be dramatically different between the two transformed E. coli strains. BtLCL-MbChBD remarkably increased not only the cell proliferation rate, extracellular and cellular chitinolytic activity, but also cellular glucosamine and N-acetylglucosamine levels, while MbLCL-BtChBD showed about the same profiles in the three tested subjects as those of the strains transformed with each of the two native chitinases, indicating that a combination of the bacterial CD of TIM barrel structure with characteristic six cysteine residues and insect ChBD2 including a conserved six cysteine-rich region (6C) enhances the attachment of the enzyme molecule to chitin compound by MbChBD, and so increases the catalytic efficiency of bacterial CD.

Expression and characterization of a chitinase from Serratia marcescens.

A chitinase gene from Serratia marcescens was cloned and expressed in Escherichia coli BL21(DE3) and the properties of recombinant chitinase rCHI-2 were characterized. The optimum catalytic pH of rCHI-2 was 6.0. It was stable in the pH range of 6.0-9.0 and could maintain more than 90% of its relative enzyme activity after incubation at 37 °C for 1 h. The optimum catalytic temperature of the enzyme was 55 °C and 85% of enzyme activity was remained after incubation at 45 °C for 1 h. The activation energy of the thermal inactivation of the enzyme was 10.9 kJ/mol and the Michaelis-Menten constant was 3.2 g/L. The purified rCHI-2 was found to be highly stable at 45 °C with half-life (t1/2) of 289 min and thermodynamic parameters ΔH*, ΔG* and ΔS* revealed high affinity of rCHI-2 for chitin. Hg2+ was found to be able to inhibit the enzyme activity reversibly, while IC50 and inhibition constant of Hg2+ on the enzyme were 34.8 µmol/L and 44.6 µmol/L, respectively. Moreover, rCHI-2 could specifically hydrolyze colloidal chitin into GlcNAc2 as the major product.

Statistical Optimization for Coproduction of Chitinase and Beta 1, 4-Endoglucanase by Chitinolytic Paenibacillus elgii PB1 Having Antifungal Activity.

A bacterial strain PB1 with antagonistic activity against pathogenic fungi was isolated from marine soil and was identified as Paenibacillus elgii based on phenotypic and genotypic characterization. The isolate showed good antifungal activity against "Aspergillus niger (MTCC 282), Trichophyton rubrum (MTCC 791), Microsporum gypseum (MTCC 2819), Candida albicans (MTCC 227), and Saccharomyces cerevisiae (MTCC 170)". Chitinase and beta 1, 4-endoglucanase are known for their capability to degrade fungal cell wall, thus we analyzed its productivity in PB1 strain using Plackett-Burman and Central Composite Design. The factors that affect the productivity of chitinase and beta 1, 4-endoglucanase were identified and optimized. A 7.77-fold increase (3.157 to 24.53 ± 1.33 U/mL) in chitinase and 7.422-fold increase (6.476 to 48.066 ± 0.676 U/mL) in beta 1, 4-endoglucanase versus basal medium was achieved. Chitinase and beta 1, 4-endoglucanase produced by Paenibacillus elgii strain PB1 represents the new source for biotechnological, medical, and agricultural applications.

Anticancer and antifungal efficiencies of purified chitinase produced from Trichoderma viride under submerged fermentation.

Trichoderma viride AUMC 13021 isolated from Mangrove soil of Ras Mohammed protected area at Sharm El-Sheikh, Egypt, was optimized to promote chitinase activity under submerged fermentation. The maximum enzyme yield (38.33 U/mg protein) was obtained at 1.4% of colloidal chitin, 96 h of incubation, 35°C, pH 6.5 and 125, rpm and using maltose (1%) and yeast extract (1%) as supplementation of salt basal medium. The enzyme has been purified with an overall yield of 73.1% and 5.48 purification fold, and a specific activity of 210.16 U/mg protein. The molecular mass of the purified chitinase was 62 kDa. Maximal activity of chitinase was recorded at pH 6.5 and 40°C. The highest activity was recorded in the case of colloidal chitin, with an apparent Km value of 6.66 mg/ml and Vmax of 90.8 U/ml. The purified chitinase was activated by Ca2+ and Mn2+ while the activity was inhibited by Hg2+, Zn2+, Cu2+, Co2+, dodecyl sulphate and EDTA. In vivo, the median lethal dose (LD50) was approximately 18.43 mg/kg body weight of Sprague Dawley rats. MTT assay showed that the purified chitinase has a toxic effect to MCF7 with an IC50 value 20 µg/ml, and HCT-116 cell lines with an IC50 value 44 µg/ml. Moreover, the purified enzyme showed significant antifungal activity against Fusarium oxysporum f. sp. lycopersici race 3 the causal agent of tomato wilt.

Comprehensive Analysis of the Chitinase Gene Family in Cucumber (Cucumis sativus L.): From Gene Identification and Evolution to Expression in Response to Fusarium oxysporum.

Chitinases, a subgroup of pathogenesis-related proteins, are responsible for catalyzing the hydrolysis of chitin. Accumulating reports indicate that chitinases play a key role in plant defense against chitin-containing pathogens and are therefore good targets for defense response studies. Here, we undertook an integrated bioinformatic and expression analysis of the cucumber chitinases gene family to identify its role in defense against Fusarium oxysporum f. sp. cucumerinum. A total of 28 putative chitinase genes were identified in the cucumber genome and classified into five classes based on their conserved catalytic and binding domains. The expansion of the chitinase gene family was due mainly to tandem duplication events. The expression pattern of chitinase genes was organ-specific and 14 genes were differentially expressed in response to F. oxysporum challenge of fusarium wilt-susceptible and resistant lines. Furthermore, a class I chitinase, CsChi23, was constitutively expressed at high levels in the resistant line and may play a crucial role in building a basal defense and activating a rapid immune response against F. oxysporum. Whole-genome re-sequencing of both lines provided clues for the diverse expression patterns observed. Collectively, these results provide useful genetic resource and offer insights into the role of chitinases in cucumber-F. oxysporum interaction.

Microbial chitinases: properties, current state and biotechnological applications.

Chitinases are a group of hydrolytic enzymes that catalyze chitin, nd are synthesized by a wide variety of organisms. In nature, microbial chitinases are primarily responsible for chitin decomposition. Several chitinases have been reported and characterized, and they are garnering increasing attention for their uses in a wide range of applications. In the food industry, the direct fermentation of seafood, such as crab and shrimp shells, using chitinolytic microorganisms has contributed to increased nutritional benefits through the enhancement of chitin degradation into chitooligosaccharides. These compounds have been demonstrated to improve human health through their antitumor, antimicrobial, immunomodulatory, antioxidant, and anti-inflammatory properties. Moreover, chitinase and chitinous materials are used in the food industry for other purposes, such as the production of single-cell proteins, chitooligosaccharides, N-acetyl D-glucosamines, biocontrol, functional foods, and various medicines. The functional properties and hydrolyzed products of chitinase, however, depend upon its source and physicochemical characteristics. The present review strives to clarify these perspectives and critically discusses the advances and limitations of microbial chitinase in the further production of functional foods.

Identification and Expression Analysis of Chitinase Genes in Pitchers of Nepenthes sp. during Development.

Fifteen chitinases of classes I-V were identified in the transcriptomes of pitchers and adult leaves of the carnivorous plant Nepenthes sp. Ten of these chitinases were identified for the first time, including the chitinases of classes II and V. The expression levels of all found chitinase genes in leaves and at three stages of pitcher development were determined. The maximum level of transcriptional activity in an open pitcher was observed for the genes encoding chitinase NChi4 (class II) and its isoforms. The expression levels of these genes significantly increased as the pitcher developed. In addition, for the first time, transcription of the genes encoding chitinases of all five classes was detected in the leaves of this plant.

The deletion of the AcMNPV ac124 gene resulted in a decrease in chitinase transcription.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac124 gene has been previously characterized as a viral pathogenicity factor. In this study, an ac124-knockout virus (vAc124KO) was generated to examine the role of the ac124 gene in the context of the AcMNPV genome during infection. Our results showed that the absence of ac124 does not affect the production of budded virus (BV) and occlusion bodies (OBs) in infected Sf9 cells. Western blotting analysis showed that the deletion of ac124 does not affect the temporal expression and the relative levels of GP64, VP39, P6.9, and polyhedrin. qRT-PCR analysis showed that the transcription level of chitinase but not the adjacent cathepsin in vAc124KO infected cells was significantly lower than that of the vAcWT infected cells from 24 to 96 h p.i. Luciferase assays showed that the overexpression of Ac124 could significantly improve the ability of chitinase promoter to initiate reporter genes. Based on the above data, we hypothesize that Ac124 binds to the promoter of chitinase to regulate the expression of chitinase gene.

Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H.

Chitin is an abundant biopolymer found mainly in the exoskeleton of crustaceans and insects. The degradation of chitin using chitinases is one way to address the accumulation of chitin waste streams in the environment, and research has therefore focused on the identification, improvement and expression of suitable enzymes. Here we describe the production, purification and characterization of Bacillus licheniformis chitinase A in the Pichia pastoris expression system. Optimal enzyme activity occurred at pH 4.0-5.0 and within the temperature range 50-60 °C. With colloidal chitin as the substrate, the Km (2.307 mM) and Vmax (0.024 mM min-1) of the enzyme were determined using a 3,5-dinitrosalicylic acid assay. The degradation products of colloidal chitin and hexa-N-acetylchitohexaose were compared by thin-layer chromatography. The activity of the glycosylated enzyme produced in P. pastoris was compared with the in vitro deglycosylated and aglycosylated version produced in Escherichia coli. We showed that the glycosylated chitinase was more active than the deglycosylated and aglycosylated variants.

Expression of M2 macrophage markers YKL-39 and CCL18 in breast cancer is associated with the effect of neoadjuvant chemotherapy.

PURPOSE: High activity of enzyme TOP2a in tumor cells is known to be associated with sensitivity to anthracycline chemotherapy, but 20% of such patients do not show clinical response. Tumor microenvironment, including tumor-associated macrophages (TAM), is an essential factor defining the efficiency of chemotherapy. In the present study, we analyzed the expression of M2 macrophage markers, YKL-39 and CCL18, in tumors of breast cancer patients received anthracycline-based NAC. METHODS: Patients were divided into two groups according to the level of doxorubicin sensitivity marker TOP2a: DOX-Sense and DOX-Res groups. Expression levels of TOR2a, CD68, YKL-39 and CCL18 genes were analyzed by qPCR, the amplification of TOR2a gene locus was assessed by the microarray assay. Clinical and pathological responses to neoadjuvant chemotherapy were assessed. RESULTS: We found that the average level of TOP2a expression in patients of DOX-Sense group was almost 10 times higher than in patients of DOX-Res group, and the expression of CD68 was 3 times higher in the DOX-Sense group compared to DOX-Res group. We demonstrated that expression levels of M2-derived cytokines but not the amount of TAM is indicative for clinical and pathological chemotherapy efficacy in breast cancer patients. Out of 8 patients from DOX-Sense group who did not respond to neoadjuvant chemotherapy (NAC), 7 patients had M2+ macrophage phenotype (YKL-39+CCL18- or YKL-39-CCL18+) and only one patient had M2- macrophage phenotype (YKL-39-CCL18-). In DOX-Res group, out of 14 patients who clinically responded to NAC 9 patients had M2- phenotype and only 5 patients had M2+ macrophage phenotype. Among pathological non-responders in DOX-Sense group, 19 (82%) patients had M2+ tumor phenotype and only 4 (18%) patients had M2- phenotype. In DOX-Res group, all 5 patients who pathologically responded to NAC had M2 phenotype (YKL-39-CCL18-). Unlike the clinical response to NAC, the differences in the frequency of M2+ and M2- phenotypes between pathologically responding and non-responding patients within DOX-Sense and DOX-Res groups were statistically significant. CONCLUSIONS: Thus, we showed that in patients with breast cancer who received anthracycline-containing NAC the absence of clinical response is associated with the presence of M2+ macrophage phenotype (YKL-39-CCL18 + or YKL-39 + CCL18-) based on TOP2a overexpression data.