Purification and Properties of β-1,4-Xylanases 2 and 3 from Aeromonas caviae W-61

A system of multiple xylaiiase enzymes was detected in the culture supernatant of Aeromonas caviae W-61. Among the detected xylanases, two β-l,4-xylanases (l,4-β-D-xylan xylanohydrolases, EC 3.2.1.8), designated xylanases 2 and 3, have been purified to homogeneity, by using ultrafiltration, ammonium sulfate precipitation, DEAE-Toyopearl 650M, CM-Sephadex C-50, and high-pressure liquid chromatographies. Endoxylanase 2 was a basic protein of 41 kDa, and endoxylanase 3 was an acidic protein of 58 kDa. The two xylanases had different pH and temperature optima, as well as thermal stabilities. The two purified enzymes had no activity on β-1,3-xylan, cellulose, carboxymethyl cellulose, or water-soluble starch. Various xylo-oligosaccharides such as xylotriose, xylotetraose, xylopentaose, xylohexaose, and higher oligosaccharides were formed, and only a small amount of xylobiose was detected as the hydrolysis products of oat spelt xylan by endoxylanase 2. Endoxylanase 3 released higher xylo-oligosaccharides as main products with very small amounts of xylotetraose and xylopentaose.     

A Novel Culture Method for High Level Production of Heterologous Protein in Saccharomyces cerevisiae

A high level production system for heterologous protein by cold culture of yeast transformants at 15°C was developed. The yeast transformants, carrying a plasmid containing cDNA for Aspergillus oryzae α-amylase (Taka-amylase A) or human lysozyme synthetic DNA, were cultivated in a selective medium for 1 or 2 days until full growth at 30°C. The yeast cells were harvested by centrifugation from the culture fluid and then were transferred to YPD medium. These inoculated broths were incubated for 2 days at 15°C and then for another 2 days at 30°C. By the cold culture method described above, higher amounts of Taka-amylase A (28.6 mg/liter) and human lysozyme (6.1 mg/liter) were produced by the yeast transformants compared to those by conventional methods.

Heterologous protein productions using YEp, YCp, and YIp types of yeast expression vectors with ADH1 or GAPDH promoter by the cold culture method showed effective productivity of about 2-fold compared to those by the conventional method of culture at 30°C. The high level production of heterologous protein by this method was not specific to the S. cerevisiae strains examined.     

Functional Analysis of the Carbohydrate-Binding Module of an Esterase from Neisseria sicca SB Involved in the Degradation of Cellulose Acetate

An esterase gene from Neisseria sicca SB encoding CaeA, which catalyzes the deacetylation of cellulose acetate, was cloned. CaeA contained a putative catalytic domain of carbohydrate esterase family 1 and a carbohydrate-binding module (CBM) family 2. We constructed two derivatives, with and without the CBM of CaeA. Binding assay indicated that the CBM of CaeA had an affinity for cellulose.     

Studies on Pectolytic Enzymes of Molds

The clarification of apple juice has been studied using six pectolytic enzymes produced by Coniothyrium diplodiella, endo-PG (polygalacturonase) I, II and III, exo-PG and PE (pectinesterase) I and II. Each of these six enzymes had no effect on the clarification of apple juice when acted alone, whereas mixtures of any one of endo-PGs and PEs were all able to clarify the juice. Mixtures of exo-PG and either of PEs has no effect on the clarification. Clarifying activities of PG-PE mixtures were varied with the kind of endo-PG used in each mixture and not with the kind of PE. Clarifying activity of PG-PE mixture depended on either endo-PG or PE activities when the other was kept constant.

Crude enzyme from the mold and a mixture of the four PGs and PE in the ratio of the crude enzyme had essentially identical effect on apple juice as well as on artificial pectin and pectic acid.     

Studies on Pectolytic Enzymes of Molds

Exopolygalacturonase from Coniothyrium diplodiella has been purified by ammonium sulfate fractionation, chromatography on DEAE-cellulose and column zone electrophoresis. The enzyme was concentrated about 5-fold with a yield of 0.24% on the basis of polygalacturonase activity per weight of total nitrogen. The purified enzyme was homogenous On free-boundary electrophoresis. The enzyme was most active in the pH range 4.0~4.5. The enzyme was stable at 50°C and pH range of 3.5~6.0, but inactivated at higher than 55°C. Hydrolysis of pectic acid by the enzyme went to completion via galacturonic acid liberation from the end of the chain, but pectin was little affected by the enzyme.     

Studies on Pectolytic Enzymes of Molds

Experiments have been made on fractionation of the pectolytic enzymes produced by Coniothyrium diplodiella. It has been observed that 30 to 35% of the polygalacturonase (PG) activity of the pectolytic enzymes of the said microorganism is salted out with ammonium sulfate, and this portion contains cndo-PG I, endo-PG II and pectin esterase (PE) (with a trace of exo-PG). The endo-PG I accounts for 60 to 65% of the total PG activity, and the endo-PG II, 25 to 30%. Both types of endo-PG scarcely act on pectin, and hydrolyze pectic acid to the extent of 65 to 70%.     

Studies on Pectolytic Enzymes of Molds

Two hundred and fifty strains of molds including plant pathogenic microorganisms were cultured on solid media, and the production of pectolytic enzymes was followed by the clarification of fruit juice. Forty-four of them were found to have the action of clarifying fruit juice.

Out of the said 44 strains, the following 7 strains, Coniothyrium diplodiella, Agaricus capentris, Botrytis cinerea, Penicillium citrinum, Sclerotinia libertiana, Carpenteles javanicus and Aspergillus niger, were choosen as producers of effective pectolytic enzymes, and C. diplodiella proved the most active of all in clarifying fruit juice and hydrolyzing pectin or pectic acid.     

Studies on Pectolytic Enzymes of Molds

The process of apple juice clarification by pectolytic enzymes has been successfully observed turbidimetrically and macroscopically by heating of reaction mixtures. It has been shown that the process of apple juice clarification varies with the varieties and conditions of apple juices as well as with the sources of enzyme preparations. From a study of the turbidimetry of apple juice clarification, α method for determination of clarification values been described.     

Pectolytic Enzymes in Three Populations of Ditylenchus dipsaci

Extracts of nematodes of the Raleigh, North Carolina (RNC), Waynesville, N. C. (WNC), and onion populations of Ditylenchus dipsaci were examined for pectolytic activity. RNC nematodes contained a NaCl-stimulated endo-polymethylgalacturonase with optimal pH for activity of 6.0, whereas nematodes of the WNC and onion populations possessed a NaCl-stimulated endo-polygalacturonase with pH optimum of 4.0. Nematodes of each population also contained a CaCl₂-activated endo-pectin methyl-trans-eliminase with optimal pH of 9.0. Nematode extracts containing 0.5 M NaCl macerated potato discs. RNC and onion nematodes induced gall formation in Wando pea seedlings, but WNC nematodes induced a resistant, hypersensitive response. Thus pectolytic activity was not correlated with pathogenicity of D. dipsaci on Wando pea.     

Structure of Lily Glucomannan Deduced from Enzymic Hydrolyzate

A thermostable acetylxylan esterase gene, TTE0866, which catalyzes the deacetylation of cellulose acetate, was cloned from the genome of Caldanaerobacter subterraneus subsp. tengcongensis. The pH and temperature optima were 8.0 and 60 °C. The esterase was inhibited by phenylmethylsulfonyl fluoride. A mixture of the esterase and cellulolytic enzymes efficiently degraded insoluble cellulose acetate with a higher degree of substitution.     

邻单胞菌(Plesiomonas sp.90-1)降解直链烷基苯磺酸钠酶的诱导

在Plesiomonas sp.90-1中,降解直链烷基苯环酸钠(LAS)相关的酶为诱导酶.使用正交多因子法研究了细胞降解LAS酶活诱导的最佳条件为:细胞培养温度30℃,LAS诱导浓度10ppm,酵母膏0.008%,pH8.0,通气.在此条件下,LAS酶活比未经诱导者提高1.4倍.碳源的加入会阻遏LAS降解酶的活力形成.在所试氮源中,以(NH_4)H_2PO_4对LAS降解酶的形成最有利.低浓度的磷酸盐对LAS降解酶活形成无影响,但高浓度的磷酸盐对LAS降解酶活形成不利.利用微量检压技术发现经此条件诱导的细胞耗氧量上升2—3倍.     

裂褶菌纤维二糖脱氢酶吸附纤维素性质

纤维二糖脱氢酶（ＣＤＨ）可以吸附棉花、微晶纤维素和酸处理纤维素,４ｍｉｎ便都达到平衡。与纤维素酶明显不同,该酶的Ｓｃａｔｃｈａｒｄ吸附曲线都是一条直线,为典型的单结合位点模型（ｏｎｅ－ｂｉｎｄｉｎｇ－ｓｉｔｅ ｍｏｄｅｌ）。观察到ｐＨ值、温度、乙二醇和ＮａＣｌ对ＣＤＨ吸附微昌纤维素有影响,并进行了讨论。     

寄主植物对松墨天牛幼虫体内酶系活性的影响

室内采用在马尾松、黑松、火炬松、湿地松和雪松皮下接入松墨天牛Monochamus alternatus Hope幼虫的方法,探明5种寄主植物对松墨天牛幼虫的存活、钻蛀取食及纤维素酶、酯酶、羧酸酯酶活性的影响。结果表明,5种寄主植物对松墨天牛幼虫存活和钻蛀取食有显著影响,接种于马尾松的天牛幼虫存活率100%;接种于雪松的松墨天牛幼虫存活率仅55.56%。松墨天牛取食不同寄主后,其体内纤维素酶、酯酶和羧酸酯酶活性存在显著差异。其中以取食火炬松的Cx酶活性最高,为(4.5754±0.1975)μmol·g-1(FW)·min-1,C1酶和β-l,4-葡萄糖苷酶活性以取食马尾松的松墨天牛幼虫消化道中最高,分别为(2.8199±0.0510)μmol·g-1(FW)·min-1与(3.0793±0.1733)μmol·g-1(FW)·min-1;而以取食黑松和马尾松的酯酶、羧酸酯酶活性最高,分别为1372.74mmoD/min·μg和744.82mmoD/min·μg。     

Effect of surfactants on cellulose hydrolysis

The effect of surfactants on the heterogeneous enzymatic hydrolysis of Sigmacell 100 cellulose and of steam-exploded wood was studied. Certain biosurfactants (sophorolipid, rhamnolipid, bacitracin) and Tween 80 increased the rate of hydrolysis of Sigmacell 100, as measured by the amount of reducing sugar produced, by as much as seven times. The hydrolysis of steam-exploded wood was increased by 67% in the presence of sophorolipid. At the same time, sophorolipid was found to decrease the amount of enzyme adsorbed onto the cellulose at equilibrium. Sophorolipid had the greatest effect on cellulose hydrolysis when it was present from the beginning of the experiment and when the enzyme/cellulose ratio was low. (c) 1993 John Wiley & Sons, Inc.     

Production of Pectolytic Enzymes by Rhizopus stolonifer Sporangiospores After "Lethal" Gamma Irradiation

Irradiated sporangiospores of Rhizopus stolonifer excreted pectolytic enzymes, which hydrolyzed pectin and macerated potato tuber discs, into the suspending medium. Pectin glycosidase, but not pectin methylesterase, activity developed regardless of the amount of radiation the spores had received, unless the dose exceeded about 1 megarad. The ability to produce pectolytic enzymes was found to be more radiation-resistant than the potential for colony formation or the ability to germinate. Spores made incapable, through irradiation, of forming colonies continued to produce pectolytic enzymes after a 6-day period following irradiation treatment.     

Analysis of the Components Released from Potato Tuber Tissues during Maceration by Pectolytic Enzymes

Endo-pectin lyase and endo-polygalacturonase of Aspergillus japonicus attack the middle lamella of plant tissue and cause tissue maceration. Galacturonides, neutral sugars, and proteins were released from potato tuber tissues during maceration by both purified enzymes. These three components accounted for 92% of the soluble products. The neutral sugars released were rhamnose, arabinose, and galactose with a molar ratio of 1:3:15. They were covalently linked to galacturonides. Over 85% of the galacturonides released by the enzymes were short chain products, which indicated that a large portion of the main chain of pectic substances is a homogalacturonan. The results of chromatography on columns of Sephadex G-100 and DEAE-cellulose suggested that a protein component may be attached to pectic substances. This protein did not contain hydroxyproline and, therefore, was different from the cell wall structural glycoprotein.     

木质素降解酶及相关基因研究进展

生物质的高效综合利用已成为全球关注的热点问题。生物质的主要成分是木质素、纤维素和半纤维素,其利用的关键是如何去除木质素,从而提高纤维素和半纤维素的得率。其中利用真菌的生物预处理方法因条件温和、无二次污染等优点符合全球经济可持续发展需要,受到研究者的普遍关注。综述了近年国内外真菌分泌的主要木质素降解酶,包括木质素过氧化物酶(Li P)、锰过氧化物酶(Mn P)、漆酶(laccase)和多功能过氧化物酶(VP)的主要特点,总结了木质素降解相关酶的基因工程、基因组学的研究成果,并对其发展前景进行了展望。