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.     

柠檬酸用于虾头、虾壳的脱钙处理

以南美白对虾虾头、虾壳为原料,利用柠檬酸对其脱钙处理制备甲壳素,通过单因素和响应面试验优化提取条件。结果表明：当柠檬酸质量浓度12.00g/L、料液比1:2.00(g/mL)、时间3.88h时,所得甲壳素灰分含量为1.0g/100g,即已经达到食品级甲壳素的要求。利用柠檬酸脱除虾头、虾壳中的钙盐制备甲壳素,不仅获得食品级的甲壳素,而且反应条件温和、污染小,生成的副产物柠檬酸钙可以作为钙强化剂,提高虾头、虾壳的资源利用率。     

A new type of beta-N-Acetylglucosaminidase from hydrogen-producing Clostridium paraputrificum M-21

A beta-N-acetylglucosaminidase gene (nag84A) was cloned from Clostridium paraputrificum M-21 in Escherichia coli. The nag84A gene consists of an open reading frame of 4647 by encoding 1549 amino acids, with a deduced molecular weight of 174,311, which have a catalytic domain belonging to family 84 of the glycoside hydrolases. Nag84A was purified from a recombinant E. coli and characterized. Although Nag84A exhibited high homology to the hyaluronidase from Clostridium perfringens, it did not degrade hyluronic acid. The enzyme hydrolyzed chitooligomers such as di-, tri-, tetra-, penta- and hexa-N-acetylchitohexaose, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta-D-glucosaminide [4-MU-(G1cNAc)], but did not hydrolyze 4-MU-beta-D-glucoside, 4-MU-alpha-D-glycoside, 4-MU-alpha-D-GlcNAc, 4-MU-alpha-D-galactoside, 4-MU-beta-D-xyloside, PNP-beta-D-galactoside, and PNP-alpha-D-xyloside. The enzyme was optimally active at 50 degrees C and pH 6.5, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 8.5 microM and 1.39 micromol/min/mg of protein, respectively. SDS-PAGE, zymogram, and immunological analyses suggested that Nag84A was inducible by ball-milled chitin. Since Nag84A has a high molecular weight with a family 84 catalytic domain with high homology to hyaluronidases but no hyaluronidase activity, the enzyme is a novel beta-N-acetylglucosaminidase different from others reported having low molecular weights and belonging to family 3 and family 18.     

离子液体在虾壳甲壳素提取中的应用研究进展

文章综述了虾壳在离子液体中的溶解与再生、甲壳素提取物的组成及结构表征,分析了离子液体种类、溶解条件对甲壳素提取效果的影响规律,并展望了今后的研究方向。     

产蛋白酶菌株的鉴定及其对虾壳的脱蛋白研究

从虾塘沉积物中分离到一株产蛋白酶的菌株C1,采用菌落形态、生理生化特征和16S rDNA基因序列分析相结合的方法进行鉴定,进一步探究其在提取虾壳甲壳素工艺中脱蛋白的应用,并与枯草芽孢杆菌（Bacillus subtilis）对虾壳蛋白脱除效果进行比较分析。结果表明,菌株C1被鉴定为一株蜡样芽孢杆菌（Bacillus cereus）,其对虾壳的脱蛋白能力高于枯草芽孢杆菌。当发酵培养基中葡萄糖添加量为50 g/L,虾壳粉添加量为20 g/L,酵母膏添加量为1 g/L时,枯草芽孢杆菌和蜡样芽孢杆菌发酵5 d的蛋白酶活力分别为145.7 U/mL、220.8 U/mL,脱蛋白率分别达到80.4%、90.8%。     

EXTRACTION OF CAROTENOPROTEIN FROM BLACK TIGER SHRIMP SHELLS WITH THE AID OF BLUEFISH TRYPSIN

The effect of bluefish (Pomatomus saltatrix) trypsin on the recovery and characteristics of carotenoprotein from black tiger shrimp (Penaeus monodon) shells was investigated. Trypsin concentration and reaction time both affected the hydrolysis and the recovery of carotenoproteins ( P <  0.05). The recovery of carotenoproteins from shrimp shells was maximized by the hydrolysis of shrimp shells using 1.2 trypsin units/g shrimp shells for 1 h at 25C. Freeze-dried carotenoprotein recovered contained 70.20% protein, 19.76% lipid, 6.57% ash, 1.50% chitin, and 87.91 µg total astaxanthin/g sample, indicating a substantial reduction in the levels of antinutrients associated with shrimp waste, while enriching the product in carotenoid pigments and valuable essential nutrients (proteins and lipids). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the recovered carotenoprotein revealed that protein with molecular weight of 45 kDa was the major constituent. When hydrolytic activities of bluefish and bovine trypsins toward carotenoproteins in black tiger shrimp shells were compared, the recovery efficacy of protein and pigment by bluefish trypsin was similar to that achieved by trypsin from bovine pancreas. Therefore, bluefish trypsin could be used as an alternative cheap proteinase for extraction of carotenoproteins from black tiger shrimp shells.

PRACTICAL APPLICATIONS

Carotenoproteins from black tiger shells, the byproduct of shrimp processing, can be recovered with the aid of fish trypsin. This product can be used for both food and feed applications. Additionally, the fish trypsin can be used instead of bovine trypsin. As a whole, the utilization of fish and shellfish processing wastes can be maximized.     

Electrotransformation of Clostridium paraputrificum M-21 with some plasmids

Clostridium paraputrificum M-21 was transformed with several shuttle plasmids constructed for Clostridium acetobutylicum-Escherichia coli and Clostridium perfringens-E. coli by electroporation. The Clostridium stercorarium xylanase gene xyn10B was successfully expressed in C. paraputrificum M-21 and the expressed protein did not suffer from proteolysis by host protease(s). This system will provide us with a genetic tool for genetic and metabolic engineering of this bacterium.     

外源硫化氢对氧化应激下铜绿假单胞菌生长和生物膜的调节作用

探究外源硫化氢(H2S)对铜绿假单胞菌(Pseudomonas aeruginosa,PAO1)在氧化应激(H₂O₂)胁迫条件下的作用。选取硫氢化钠(NaHS)作为外源H;S的供体。将铜绿假单胞菌分为两组;对照组的培养基中不添加Na HS;实验组的培养基中添加0.2mmol/L NaHS;观察两组菌株在0、1、2、2.5 mmol/L H₂O₂的压力下存活率、细胞形态、DNA外渗量、生物膜生长的变化情况。研究结果表明,随着时间的推移(从0.5 h到1 h再到1.5 h),与对照组菌株相比,实验组菌株的存活率显著降低(p<0.05),其中在1 mmol/L H₂O₂的处理下,实验组比对照组显著降低24.83%、15.69%、11.36%;在2 mmol/L H₂O₂的处理下,实验组比对照组显著降低4.92%、11.16%、5.75%;在2.5mmol/LH₂O₂的处理下,实验组比对照组降低6.83%、2.98%、0.39%。实验组较对照组相比,细胞形态受损和DNA外渗更加严重。利用结晶紫染色、扫描电子显微镜和激光共聚焦显微镜检测在不同浓度H₂O₂胁迫下生物膜形成的变化,结果表明,与对照组菌株的生物膜相比,在含有1 mmol/L和2 mmol/L H₂O₂的Luria Bertani(LB)培养基中实验组菌株的生物膜形成在培养48 h和72 h后显著减少。结果表明,H2S能增强H₂O₂对铜绿假单胞菌细胞的损伤。     

虾、蟹壳利用的研究进展

虾、蟹壳是虾、蟹加工过程中产生的主要废弃物,含有较大量的蛋白质、灰分和甲壳素,以及少量的脂肪、游离氨基酸和虾青素等。近年来,随着我国养殖、捕捞技术的进步以及伏季休渔制度的实施,虾、蟹产量逐年上升。因此,有效利用虾、蟹壳副产物,开发基于虾、蟹壳废弃物的利用途径和产品类型,以提高产品附加值,减少环境污染,对于虾、蟹产业的健康发展具有重要意义。目前,采用酸碱法制备甲壳素是虾、蟹壳利用的主要方法,该方法易于操作,但能耗高且污染严重,近年来研究人员对传统的酸碱法制备甲壳素的工艺进行了优化,并积极探索酶法和发酵法等新型提取工艺。此外,虾、蟹壳中其他可利用成分(蛋白质、脂肪、钙质和虾青素)的提取和利用也获得了许多研究成果。本文主要综述了虾、蟹壳的组成成分,虾、蟹壳整体利用途径以及虾、蟹壳中甲壳素、蛋白质、脂肪、钙质、虾青素等成分的提取和利用途径的研究进展,以期为虾、蟹壳的高效、低成本、无污染和高附加值利用提供借鉴。     

虾壳在1-乙基-3-甲基咪唑醋酸盐离子液体中的溶解特性

为探索从对虾加工废弃物中回收甲壳素的绿色工艺,本文以离子液体1-乙基-3-甲基咪唑醋酸盐([C₂mim]Ac)为溶剂,系统考察了溶解时间、溶解温度、固-液比3个因素对虾壳的溶解度、回收率以及再生后的组分、表观形貌和晶体结构的影响规律。结果表明,固-液比为1:20 w/w、100℃下加热1 h后,虾壳溶解度最高,为15.30%。与未处理的虾壳相比,经过[C₂mim]Ac溶解再生后的样品表面多孔、粗糙或扭曲变形,而且结晶结构被破坏,蛋白质和矿物质脱除率可分别达到67.13%和42.84%。当温度超过120℃,溶解时间超过2 h时,不利于[C₂mim]Ac对虾壳的溶解。     

三株产几丁质酶菌株的筛选、产酶条件优化及水解虾壳的研究

目的:筛选鉴定产几丁质酶的菌株并优化其发酵条件,最终应用于虾壳降解研究。方法:以盐城市滩涂海泥为样品,利用平板筛选水解几丁质的菌株,运用生物信息学方法鉴定菌株,通过单因素优化其发酵条件,并将筛选得到的菌株和优化后的发酵条件用于虾壳发酵。结果:鉴定得到三株显著降解胶体几丁质的菌,分别是发光杆菌（Photobacterium sp. LYM-1）、需钠弧菌（Vibrio sp. WM-1）和希瓦氏菌（Shewanella sp. ZXY-1）;优化发酵条件:发光杆菌的碳源为几丁质10 g/L,氮源为NH₄Cl 2.0 g/L,接种量为3%,发酵液pH为6.5,温度为32 ℃,发酵1 d酶活最高为15.37±0.55 U/mL,是优化前的4.37倍;需钠弧菌的碳源为几丁质10 g/L,氮源为NH₄Cl 2.0 g/L,接种量为3%,发酵液pH为7.5,温度为22 ℃,发酵2 d酶活最高为40.82±6.03 U/mL,是优化前的1.60倍;希瓦氏菌的碳源为几丁质10 g/L,氮源为(NH₄)₂SO₄ 2.0 g/L,接种量为3%,发酵液pH为6.5,温度为22 ℃,发酵1 d酶活最高为25.64±3.29 U/mL,是优化前的2.47倍;三株菌均能利用虾壳产几丁质酶,但利用效率均低于几丁质,酶活力分别为10.25±0.95、32.16±2.25和21.81±4.27 U/mL。结论:本研究从盐碱地筛选得到三株产几丁质酶的菌株,优化后酶活力均得到提高,且均能利用虾壳产几丁质酶,为发酵虾壳制备几丁质酶提供新的菌株来源。     

New route of chitosan extraction from blue crabs and shrimp shells as flocculants on soybean solutes

The chitosan extracted from blue crabs and shrimp shells using calcium oxide (deproteinization) followed by deacetylation which eliminated the demineralization step to reduce the chemical usage and environmental protection. The extracted chitosan examined the flocculation to soybean solutes. The optical density (OD), solid%, and purity% (carbohydrates/soluble solids) after flocculation were measured. The OD was significantly decreased from 0.76 to 0.16 with blue crabs and 0.06 with shrimp shells chitosan-acetate dosing (0.5 g/L). The removal of about 68 and 66% solids was achieved by the addition of 0.5 g/L chitosan-acetate. The purity% was reached about 80% with blue crabs, and 78% with shrimp shells chitosan-acetate. The results of this study verified that the calcium oxide treatment should remove protein and increase the chitin extraction yield on blue crab and shrimp shells. This new route of chitosan extraction should be a useful method for making flocculants in the soybean solutes.     

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₊、...     

One-step bio-extraction of chitin from shrimp shells by successive co-fermentation using Bacillus subtilis and Lactobacillus plantarum

Chitin was bio-extracted in one-step from shrimp shells by successive co-fermentation using Bacillus subtilis and Lactobacillus plantarum in this study. To construct this co-fermentation system, B. subtilis was first cultured for 3 days for deproteinization (DP), and then L. plantarum was inoculated for demineralization (DM). After 6 days of co-fermentation, the final DP and DM efficiency reached 94.1% and 96.3%, respectively. The molecular weight, degree of acetylation, and crystalline index of chitin were reduced by 9.8, 5.6 and 1.4% with the DP time of 2 days extending to 3 days. Meanwhile, L. plantarum instead of B. subtilis became the dominant bacterium on day 5 of co-fermentation, with L. plantarum count of 6.19 log CFU/mL and lactic acid concentration of 28.22 g/L, respectively. The chitin prepared in this study exhibited similar structural characterization as commercial chitin. One step successive co-fermentation was a simple and feasible approach for high-quality chitin preparation.Industrial relevance: This study establishes one-step bio-extraction of chitin from shrimp shells by successive co-fermentation using B. subtilis followed by L. plantarum. One-step successive co-fermentation simplifies the intermediate processes of conventional two-step fermentation for extracting chitin. DP and DM happen during the entire co-fermentation period, which helps to achieve satisfactory DP and DM efficiency. This innovative yet simple method provides more possibilities for the biotechnological production of chitin on an industrial scale.     

桔橙小单孢菌产几丁质脱乙酰酶的酶学性质研究

从海边红树林虾场土壤中筛选的一株产几丁质脱乙酰酶（CDA）的放线菌桔橙小单孢菌（Micromonospora aurantiaca）,研究其产CDA的酶学性质。结果表明,CDA的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)电泳结果显示为单一条带,分子质量为81.8 ku。最适pH值为7.0;最适温度为40 ℃;Ca2+对此CDA酶活有促进作用,而Cu2+、Zn2+、Mg2+表现出了抑制作用。CDA对虾壳来源的几丁质有明显的脱乙酰效果,红外光谱测得样品脱乙酰度由39.03%提高至78.40%。扫描电镜发现CDA酶解过的几丁质样品表面疏松多孔、出现凹槽、晶体消失,进一步印证了该酶的良好脱乙酰效果,也力证了该酶拥有较好的特性。     

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.     

Microbial hydrogen production from sweet potato starch residue

Clostridium butyricum could produce hydrogen from a sweet potato starch residue upon supplementation of nitrogen sources. A repeated batch culture using a mixed culture of C. butyricum and Enterobacter aerogenes produced hydrogen with a yield of 2.4 mol H2/mol glucose under a controlled culture pH of 5.25 in a medium consisting of the sweet potato starch residue and 0.1% Polypepton without addition of any reducing agents. Rhodobacter sp. M-19 produced hydrogen from the supernatant of the culture broth obtained in the repeated batch culture containing C. butyricum and E. aerogenes when 50 microg/l Na2MoO4.2H2O and 20 mg/l EDTA were added to the supernatant and it was cultured under a controlled culture pH of 7.5. A high yield of hydrogen of 7.0 mol H2/mol glucose from the starch remaining in the starch residue was attained in two-step repeated batch cultures containing C. butyricum and E. aerogenes, and by Rhodobacter sp. M-19.     

克雷伯杆菌利用生物柴油副产物甘油生产氢气和1,3-丙二醇的研究

以Klebsiella pneumoniaeDSM2026为出发菌株,通过紫外线诱变,选育得到能耐较高浓度生物柴油副产物甘油生产H2和1,3-丙二醇(1,3-PD)的菌株21株,命名为Kp1~Kp21。通过比较,Kp8菌株产量最高,1,3-PD和H2产量分别达到0.36 g/50 mL和0.99 mmol/50 mL,比出发菌株分别提高了3.5倍和4.2倍。对Kp8菌株发酵条件进行优化,得到最佳培养条件为pH 7.0,培养温度37℃,接种量10%(v/v),废甘油浓度为30 g/L。在该条件下H2产量为1.0 mmoL/50 mL,1,3-PD产量为7.5 g/L,甘油转化率为83.3%。     

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...