Saccharification of chitin using solid-state culture of Aspergillus sp. S1-13 with shellfish waste as a substrate

Saccharification of chitin was performed in a suspension (mash) of a solid-state culture of chitinase-producing Aspergillus sp. Sl-13 with acid-treated shellfish waste as a substrate. The conditions for the saccharifying reaction and the solid-state cultivation were examined from the viewpoint of saccharification in the mash. Optimum cultivation conditions were defined: a solid-state medium consisting of 5 g of 10% lactic acid-treated crab shells (0.50-2.36 mm in size) and 3 ml of a basal medium (0.028% KH2PO4 0.007% CaCl2.2H2O, and 0.025% MgSO4.7H2O) supplemented with 0.3% peptone was inoculated with 4 ml of spore suspension (1 x 10(7) spores/ml), and the water content of the medium was adjusted to 75%; static cultivation at 37 degrees C for 7 d. When a culture obtained under the optimum conditions was suspended in 70 ml of 50 mM sodium phosphate-citrate buffer (pH 4.0) and incubated at 45 degrees C for 11-13 d, 55 mM N-acetylglucosamine (GlcNAc) was formed in the solid-state culture mash, indicating that at least 33% of the initial chitin in the solid material was hydrolyzed. Through the experiments, the amounts of G1cNAc formed in the solid-state culture mash varied in a way similar to that of the water-extractable pnitrophenyl beta-D-N-acetylglucosaminide-hydrolyzing enzyme in the culture, but not to that of the colloidal chitin-hydrolyzing enzyme. G1cNAc-assimilating lactic acid bacteria, which were inoculated into the mash after or at the start of the saccharification, formed lactic acid with decreasing GlcNAc.     

Conversion of chitinous wastes to hydrogen gas by Clostridium paraputrificum M-21

The chitinolytic bacterium Clostridium paraputrificum strain M-21 produced 2.2 and 1.5 mol hydrogen gas from 1 mol N-acetyl-D-glucosamine (GlcNAc) and ball-milled chitin equivalent to 1 mol of GlcNAc, respectively, at pH 6.0. In addition, strain M-21 efficiently degraded and fermented ball-milled raw shrimp and lobster shells to produce hydrogen gas: 11.4 mmol H2 from 2.6 g of the former and 7.8 mmol H2 from 1.5 g of the latter. Hydrogen evolution from these shell wastes were enhanced two fold by employing acid and alkali pretreatment. Waste from the starch industry was also converted to hydrogen. When C. paraputrificum M-21 was cultivated on ball-milled chitin and ball-milled shrimp shells for 14 and 12 h, respectively, chitinases ChiA and/or ChiB were detected as the major chitinase species in the supernatant of the cultures, suggesting that the play a critical role in the degradation of chitinous materials.     

Purification and characterization of a 49-kDa chitinase from Streptomyces griseus HUT 6037

A 49-kDa chitinase (pI7.3) was purified to homogeneity from the culture supernatant of Streptomyces griseus HUT 6037 by ultrafiltration, DEAE-Sephadex A-50 and Sephadex G-100 column chromatographies, and chromatofocusing. The purified enzyme was stable up to 40 degrees C. The N-terminal amino acid sequence of the enzyme was highly homologous to the N-terminal region of the fibronectin type III-like domain of S. olivaceoviridis chitinase 01 belonging to family 18 glycosyl hydrolases. The 49-kDa chitinase hydrolyzed partially N-acetylated chitosan more easily than colloidal chitin. The hydrolyzate of 54% deacetylated chitosan by the enzyme was separated by CM-Sephadex C-25 column chromatography. The structures of the oligosaccharides obtained were determined by MALDI-TOF MS analysis combined with exo-glycosidase digestion. In addition to GlcNAc, (GlcNAc)2, and (GlcNAc)3, hetero-chitooligosaccharides with GlcNAc at the reducing end were detected. Thus, the specificity of the enzyme for the hydrolysis of the beta-1,4-glycosidic linkages in partially N-acetylated chitosan was similar to that of the family 18 chitinases.     

Identification and characterization of Clostridium paraputrificum M-21, a chitinolytic, mesophilic and hydrogen-producing bacterium

A strictly anaerobic, mesophilic and chitinolytic bacterial strain, M-21, was isolated from a soil sample collected from Mie University campus and identified as Clostridium paraputrificum based on morphological and physiological characteristics, and 16S rRNA sequence analysis. C. paraputrificum M-21 utilized chitin and N-acetyl- -glucosamine (GlcNAc), a constituent monosaccharide of chitin, to produce a large amount of gas along with acetic acid and propionic acid as major fermentation products. Hydrogen and carbon dioxide accounted for 65% and 35% of the gas evolved, respectively. The conditions for 1 l batch culture of C. paraputrificum, including pH of the medium, incubation temperature and agitation speed, were optimized for hydrogen production with GlcNAc as the carbon source. The bacterium grew rapidly on GlcNAc with a doubling time of around 30 min, and produced hydrogen gas with a yield of 1.9 mol H₂/mol GlcNAc under the following cultivation conditions: initial medium pH of 6.5, incubation temperature of 45°C, agitation speed of 250 rpm, and working volume of 50% of the fermentor. The dry cell weight harvested from this culture was 2.0 g/l.     

Characterization of Clostridium paraputrificum chitinase A from a recombinant Escherichia coli

Clostridium paraputrificum chitinase A (ChiA) was purified from a recombinant Escherichia coli. ChiA was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. ChiA showed maximum activity at pH 6.0 and its optimum temperature was 45 degrees C. ChiA was stable between pH 6.0 and 9.0 and at temperatures up to 40 degrees C. The K(m) and V(max) values of ChiA for 4-MU-(GlcNAc)2 were estimated to be 6.9 microM and 43 micromol/min/mg, respectively. Thin-layer chromatography indicated that ChiA hydrolyzes chitooligosaccharides to mainly chitobiose. ChiA was found to adsorb not only chitinous polymers but also cellulosic polymers.     

Purification and properties of extracellular chitinases from the parasitic fungus Isaria japonica

Two chitinases (P-1 and P-2) induced with colloidal chitin were purified from the culture supernatant of Isaria japonica by chromatography on DEAE Bio-Gel, chromatofocusing and gel filtration with Superdex 75 pg. The enzymes were electrophoretically homogeneous and estimated to have a molecular mass of 43,273 (±5) for P-1 and 31,134 (±6) for P-2 by MALDI-MS. The optimum pH and temperature was 3.5–4.0 and 50°C for P-1 and 4.0–4.5 and 40°C for P-2. P-1 acted against chitosan 7B (degree of deacetylation, 65–74%) = glycol chitin> colloidal CHITIN = chitosan 10B (degree of deacetylation, above 99%) and P-2 against chitosan 7B> glycol CHITIN = chitosan 10B> colloidal chitin in order of activity. The products of hydrolysis of chitin and chitosan hexamer were analyzed by MALDI-MS. The products from the chitin hexamer obtained with P-1 were almost all dimers with only a small amount of trimer whereas those obtained with P-2 were mainly trimers with some dimer and tetramer. No hydrolysis of chitosan hexamer was observed. High homology in the amino-terminal sequence for chitinase P-1 was exhibited by chitinases from Trichoderma harzianum, Candida albicans and Saccharomyces cerevisiae in the range of 48–39%. The highest homology for Chitinase P-2 was shown by an endochitinase from Metarhizium anisopliae of 66%, while 44% homology was exhibited by chitinases of Leguminosae plants.     

乳酸菌发酵蟹壳脱钙的菌株筛选及条件优化

为实现用生物发酵法替代化学法快速高效的脱除蟹壳中的碳酸钙,以15株乳酸菌作为出发菌株,采用与蟹壳共发酵的方式进行了筛选,筛选出脱钙效果最好的菌株,并进行了主要影响因素的考察.结果表明,编号为AS 1.2437的菌株脱钙效果最好.发酵时间越长、葡萄糖浓度越高、固液比越小,脱钙效果越好.在接种量10％,固液比5g/100mL,糖浓度10g/100mL,发酵温度为30℃条件下发酵5d,脱钙率高达95.5％.     

双酶协同降解胶体几丁质及其作用机制

建立一种几丁质酶(chitinase,ChiA)和β-N-乙酰基己糖胺酶HJ5N协同催化体系,可实现一步反应降解胶体几丁质制备N-乙酰氨基葡萄糖(N-acetylglucosamine,GlcNAc),探讨不同因素对双酶协同催化体系的影响和双酶协同作用机制.结果表明,双酶协同催化体系的最适反应温度为30℃,最适反应pH...     

酶在壳聚糖制备中的应用

综述了酶在壳聚糖制备中的应用;详细介绍了虾蟹壳酶法脱蛋白、甲壳素酶法脱乙酰及壳聚糖酶法降解这三方面的研究概况。     

虾蟹壳生产甲壳素废碱液的成分分析

为了探讨利用膜分离技术处理并综合利用水产品加工下脚料虾、蟹壳生产甲壳素产生的废碱液的效果,本文对海虾壳及海蟹壳碱两种原料经烧碱脱蛋白工艺产生的高碱废水进行了成分分析.分析结果表明,用现有生产工艺生产甲壳素,废碱液中残留烧碱的浓度很高,约在3%～4.5%.蟹壳废碱液中钙含量平均高迭20.5mg/g,蛋白质及其水解物含量达4.72%,虾青素含量390μg/L,化学需氧量(COD)高达11.28g/L,蟹壳废碱液中各成分指标都远高于虾壳.虾壳废碱液中蛋白水解物分子量主要分布在200-1000Da范围内,蟹壳的则分布在10000～20000Da范围内;两种原料的氨基酸分析结果表明,天冬氨酸和谷氨酸含量都明显高于其他氨基酸成分.     

Production of chitin oligosaccharides with different molecular weights and their antioxidant effect in RAW 264.7 cells

The purpose of this research is not only to produce two kinds of chitin oligosaccharides or N-acetyl chito-oligosaccharides (NA-COSs) with different molecular weights (MW) from crab chitin hydrolysis solution but also to determine their effect against oxidative stress in live cells. Two kinds of NA-COSs with MW 1–3 kDa (NA-COS 1–3 kDa) and below 1 kDa (NA-COS < 1 kDa) were obtained using an ultrafiltration membrane system. They exhibited an inhibitory effect against DNA and protein oxidation. In addition, in their presence, intracellular glutathione (GSH) level and direct intracellular radical scavenging effect were significantly increased in a time-dependent manner in mouse macrophages (RAW 264.7) and rendered inhibitory effect against cellular oxidative stress. In particular, NA-COS 1–3 kDa was more effective than NA-COS < 1 kDa in protein oxidation and production of intracellular free radicals in live cells. These results suggest that NA-COSs act as a potential scavenger against oxidative stress in cells.     

产几丁质酶菌的分离、筛选及其对烟草病原真菌的抑制作用

从福建、河南、安徽、云南、湖北采集了烟草根际土样100份,用几丁质选择培养基法共分离出产几丁质酶菌株62株,其中真菌32株、细菌22株、放线菌8株。通过透明圈法和还原糖法对分离的菌株进行了筛选,结果表明链霉菌属(Streplomycessp)产几丁质酶活性最高。并测定了几丁质酶活性较高的菌株对烟草赤星病菌和黑胫病菌的抑制效果,其中菌株9-1-1、9-2-1和19-2-1对黑胫病菌均有不同程度的抑制作用,以菌株9-1-1的抑菌效果最好,而这3种菌株对赤星病菌的抑制效果不明显。因此,菌株9-1-1有开发成为生防制剂用于防治烟草黑胫病的潜力。     

A novel thermostable chitinase (PJC) from pomegranate (Punica granatum) juice

A novel chitinase was isolated and purified to its homogeneity from pomegranate juice by a combination of ammonium sulphate precipitation and ion-exchange chromatography. The pomegranate juice chitinase (PJC) was purified to specific activity of 14.5 U/mg and a recovery of 34%. The monomeric protein migrated on SDS–PAGE at 29 kDa. The enzyme was found to be glycosylated (7.2%). It exhibited optimal activity at pH 4.5 and 70 °C. The enzyme was stable in the pH range 3.0–9.0 and up to 65 °C. The internal peptide sequence results suggest that the purified PJC shared high homology with class III chitinases of other known plant chitinases. The purified enzyme could hydrolyse colloidal chitin to its oligomers. It did not exhibit any antifungal activity.     

Purification and properties of extracellular chitinases from the parasitic fungus Isaria japonica

Two chitinases (P-1 and P-2) induced with colloidal chitin were purified from the culture supernatant of Isaria japonica by chromatography on DEAE Bio-Gel, chromatofocusing and gel filtration with Superdex 75 pg. The enzymes were electrophoretically homogeneous and estimated to have a molecular mass of 43,273 (+/-5) for P-1 and 31,134 (+/-6) for P-2 by MALDI-MS. The optimum pH and temperature was 3.5-4.0 and 50 degrees C for P-1 and 4.0-4.5 and 40 degrees C for P-2. P-1 acted against chitosan 7B (degree of deacetylation, 65-74%) = glycol chitin > colloidal chitin = chitosan 10B (degree of deacetylation, above 99%) and P-2 against chitosan 7B > glycol chitin = chitosan 10B > colloidal chitin in order of activity. The products of hydrolysis of chitin and chitosan hexamer were analyzed by MALDI-MS. The products from the chitin hexamer obtained with P-1 were almost all dimers with only a small amount of trimer whereas those obtained with P-2 were mainly trimers with some dimer and tetramer. No hydrolysis of chitosan hexamer was observed. High homology in the amino-terminal sequence for chitinase P-1 was exhibited by chitinases from Trichoderma harzianum, Candida albicans and Saccharomyces cerevisiae in the range of 48-39%. The highest homology for Chitinase P-2 was shown by an endochitinase from Metarhizium anisopliae of 66%, while 44% homology was exhibited by chitinases of Leguminosae plants.     

海洋产几丁质酶厦门加西利亚单胞菌Chi34的筛选、鉴定及酶学性质分析

目的：海洋是自然界中含几丁质最多的生态系统,孵育了大量可降解利用几丁质的微生物,因此利用海洋微生物几丁质酶降解几丁质,是获得高附加值几丁寡糖的重要方法。本试验以连云港海域海泥为样品,以期筛选获得稳定的产几丁质酶菌株。方法：利用平板筛选法和摇瓶发酵复筛法筛选获得产几丁质酶菌株Chi34,利用形态学分析和16S r DNA序列分析对菌株进行鉴定,同时还研究了温度、pH、金属离子、EDTA及SDS等因素对Chi34几丁质酶的影响,最后通过TLC实验分析了Chi34几丁质酶降解几丁质胶体的产物。结果：菌株Chi34鉴定为厦门加西利亚单胞菌(Gallaecimonas xiamenensis),其几丁质酶酶活力为0.631 U/m L,最适反应温度为35℃,最适反应p H为6.0,Na₊、Ca₂₊、Mn₂₊、K₊对Chi34几丁质酶具有显著的激活作用,Cu₂₊、Fe₃₊、Ba₂₊、Zn₂₊、Cd₂₊、...     

酶法水解蟹壳蛋白

对水解脱钙蟹壳中蛋白质的蛋白酶进行了选择,并探讨了蛋白酶水解的条件,通过扫描电镜(SEM)观察了采用不同脱除蛋白方法处理后的甲壳素表面状态,分析了蛋白水解液中的氨基酸组成及营养价值。结果表明,Alcalase2.4 L酶比较适于水解蟹壳中的蛋白质,水解度较其他蛋白酶高。Alcalase2.4 L水解蟹壳蛋白的最适条件为:温度60℃,pH8.0,料液比1∶3,酶底物比为3 000 U/g,水解5 h后水解度可达到14%左右。酶法与碱法脱蛋白对甲壳素表面微观状态影响不同,酶法脱蛋白后甲壳素表面较光洁。水解液中的氨基酸组成与FAO/WHO建议的理想模式基本一致,鲜味氨基酸含量较高。     

PRODUCTION OF N-ACETYLCHITOOLIGOSACCHARIDES BY AEROMONAS SP. DYU-TOO 7

Aeromonas sp. DYU‐Too 7 was screened to produce chitinases. The enzymes had an optimal reaction temperature of 50C and an optimal pH of 7.0. The best concentration of chitin in the fermentation process is 4%. Higher concentrations of chitin inhibited microbial growth and influenced the production of reducing sugars, the enzymatic activity and the protein content in the culture. N‐acetylglucosamine, the main product in the hydrolysate, was 0.7 g/L at 36 h of cultivation. The concentration of N‐acetylchitobiose was 0.16 g/L for the regular chitin broth and 0.28 g/L for the phosphorus‐limiting chitin broth, with the highest concentration of N‐acetylchitohexaose at about 0.062 g/L in both media. Experimental results showed that cultivation under a phosphorus‐limiting condition substantially reduced the production of biomass, chitinases as well as N‐acetylchitooligosaccharides.     

Isolation of genes coding for chitin-degrading enzymes in the novel chitinolytic bacterium, Chitiniphilus shinanonensis, and characterization of a gene coding for a family 19 chitinase

Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi.     

海洋细菌来源几丁质酶的研究进展

海洋环境中有总量巨大的几丁质资源，细菌为主的海洋微生物能用产生的几丁质酶将长链几丁质水解为可利用单体，从而海洋维持碳氮循环的平衡，具有重要的生态意义。同时，工业上利用几丁质酶制备N-乙酰-D-氨基葡萄糖和几丁寡糖具有较大应用价值。迄今为止，研究者已鉴定了30多种海洋细菌所产的几丁质酶。本文着重介绍海洋几丁质的结构和来源、几丁质酶的作用方式和海洋细菌来源几丁质酶的催化性质，并且分析了几丁质酶在农业、医药和食品领域的应用。几丁质酶有望成为一种新型的工业酶制剂。     

Role of aluminum speciation in the removal of disinfection byproduct precursors by a coagulation process

Humic acid (HA) was extracted and separated into different molecular weight (MW) fractions, then coagulated by aluminum chloride and polyaluminum chloride (PACl). The removal of disinfection byproduct (DBP) precursors and the aluminum speciation variation of the coagulants were investigated in detail. In particular, the role of aluminum speciation in the removal of DBP precursors was discussed. During the coagulation process, AlCl3 hydrolyzed into dominating in situ Al13 species at pH 5.5. The in situ Al13 species exhibited better removal ability for haloacetic acid (HAA) precursors than PACl. At pH 7.5, in situ hydrolyzed Al13 species of AlCl3 decomposed into dimeric Al species. In this case, preformed Al13 of PACl had a high removal ability of HAA precursors. Specially, the greatest reduction of HAA precursors with a low MW (<30 kDa) was through charge neutralization at pH 5.5, and that of HAA precursors in high MW (> 30 kDa) fractions was through adsorption at pH 7.5. Different from HAA precursors, the in situ Al13 species did not have a high removal ability of trihalomethane (THM) precursors. Therefore, PACl exhibited a better removal ability of THM precursors than AlCl3 at different pH values. In the different MW fractions, the greatest reduction of THM precursors was through charge neutralization at pH 5.5.