Production of cellulose- and xylan-degrading enzymes by a koji mold, aspergillus oryzae, and their contribution to the maceration of rice endosperm cell wall

The production of cellulose- (CEL), xylan- (XYL), and pectin-degrading enzymes (PEC) by a koji mold, Aspergillus oryzae, was studied, and their contributions to the maceration of the rice endosperm cell wall were investigated with regard to the utilization of available rice in the sake mash. The sake koji mold showed higher CEL and XYL productivities, whereas the miso and soy sauce koji molds showed higher PEC productivity. Statistical analyses indicated that CEL and XYL contribute predominantly and synergistically to the maceration of the rice endosperm cell wall. A. oryzae produced at least three kinds of CEL (Cel-1, 2, 3) and two kinds of XYL (Xyl-1, 2) when cultured in a wheat bran medium. In the solid-state culture, the production of Cel-3 and Xyl-2 was markedly stimulated by decreasing the moisture content of the solid substrate, although the production levels of Cel-1 and Xyl-1 were almost the same. These data suggest that the production of Cel-3 and Xyl-2 is strongly influenced by culture conditions, and that water activity is one of the dominant factors in the regulation of their production.     

Genetic Engineering of white Shochu-Koji to achieve Higher Levels of Acid-Stable α-Amylase and Glucoamylase and other Properties when used for Shochu Making on a Laboratory Scale

The genes for acid-stable α-amylase and glucoamylase were cloned from white shochu-koji, Aspergillus kawachii. Both genes were used to transform the parent strain of white shochu-koji which carried a dominant selective marker gene, amdS, that originated from A. oryzae. Three lines of transformants were identified that secreted about 6-fold more acid-stable α-amylase activity and about 7-fold more glucoamylase activity than the parent strain in liquid culture. In solid culture, all three transformants had 2-fold higher acid-stable α-amylase activity and 2.4-fold higher glucoamylase activity than the parent strain. When koji was prepared on a laboratory scale, acid-stable α-amylase activity was 5.7-fold higher and glucoamylase activity was 3.8-fold higher than when the parent strain was used. Shochu was produced with a koji ratio of 33% or 10% using one line of transformants on a laboratory scale. Even with a koji ratio of 10%, the weight of the mash obtained with the transformant decreased to almost the same extent as with a koji ratio of 33% and the parent strain. Levels of flavour compounds in shochu produced with koji of the transformant were higher than in the shochu prepared with koji of the parent strain. In particular, levels of isoamyl acetate and β-phenethyl acetate were as high as 12.9 mg/litre and 3.8 mg/litre, respectively.     

短小芽孢杆菌固态发酵生产木聚糖酶的培养条件优化

采用单因素试验和正交试验的方法研究了短小芽孢杆菌固态发酵生产木聚糖酶的培养条件,确定了利用麸皮作为在主要基质进行固态发酵生产木聚糖酶的适宜条件：pH9、温度35℃、料液比1：2、接种量10％。在500mL三角瓶中固态培养72h左右,木聚糖酶的活力可达4915U／g干曲。     

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.     

响应面优化药食同源基质制曲工艺及酒体成分分析

以12 种药食同源物质为制曲基质,研究根霉接种量、基质含水量、培曲时间和培曲温度对实验曲糖化酶和液化酶活力的影响。在单因素试验基础上,采用Box-Behnken试验设计原理,以糖化酶活力为响应值,对制曲工艺条件进行优化。结果表明,最佳工艺条件为根霉接种量0.3%、培曲时间40 h、基质含水量65%和培曲温度28 ℃,在此条件下糖化酶活力预测值为778.65 mg/（g·h）。最佳培曲条件经过3 次验证实验,所得糖化酶活力实测平均值为（779.233±0.577）mg/（g·h）,实测值与预测值较为接近,说明此方案可行。且实验曲糖化酶和液化酶活力分别约为传统曲的1.3 倍和2.8 倍。对传统曲酿造米酒和实验曲酿造米酒的33 种酒体成分进行检测,其两种曲酿酒差异最大的物质分别为异戊醇（10.065、109.394 μg/100 mL）、丁酸乙酯（0.867、187.552 μg/100 mL）、乙酸（135.240、560.939 μg/100 mL）、苯酚（0.156、0.858 μg/100 mL）。综合以上结果,实验曲具有良好的发酵性能,所酿造米酒具有更加丰富的滋味,有较好的开发前景。     

高糖化力菌种的筛选及诱变育种

固体曲中精化酶能将淀粉水解为葡萄精,进而被微生物发酵生成酒精.以分离筛选出的1株产糖化酶的黑曲霉(酶活265.21U/g干曲)为出发菌,经紫外线及原生质体紫外线复合诱变育种,获得1株糖化酶活力较高、生产性能稳定的菌株,其酶活达到3367.79U/g干曲,比原始菌株提高1169.86%.     

Utilization of shrimp shellfish waste as a substrate for solid-state cultivation of Aspergillus sp. S1-13: evaluation of a culture based on chitinase formation which is necessary for chitin-assimilation

The utilization of shrimp shellfish waste as a substrate for solid-state cultivation of a filamentous fungus, Aspergillus sp. S1-13, was investigated. The organism was selected from among 220 isolates based on the productivity of its chitinolytic enzyme (chitinase), which might reflect microbial growth. The enzyme was produced only when the organism was grown on medium containing the shellfish waste. The addition of 58-65% water (w/w) to the medium was effective in enhancing production, and a certain amount of enzyme was observed in media of higher water content (up to about 75%). The initial pH and nitrogen source (ammonium sulfate) of the solid-state medium also affected the amount of enzyme. The amount of enzyme increased 2-fold in an optimum solid-state medium: 5 g of shrimp shellfish waste and 3 ml of basal medium (pH 5) containing 0.1% (NH4)2SO4 was inoculated with 4 ml of spore suspension; static cultivation at room temperature. The amount increased further (1.5-fold) when the cultivation was carried out at 37 degrees C, with 1.85 units of the enzyme formed from 1 g of shrimp shellfish waste. An analysis by ion-exchange column chromatography suggested the presence of at least two colloidal chitin-hydrolyzing enzymes and one p-nitrophenyl beta-D-N-acetylglucosaminide-hydrolyzing enzyme in an extract of the solid-state culture. The elution profile was similar to that obtained with a liquid culture filtrate.     

衡水老白干酒大曲分离具有酯化力霉菌及其特性研究

以衡水老白干酒大曲中分离纯化出的3株霉菌为研究对象,以酯化力为评价指标,通过二级筛选得到1株高酯化力霉菌8号,初步鉴定为毛霉。将其制作成纯种的麸曲进行培养条件优化。结果表明,大曲在目前已测定的10个碳原子以下的酸醇反应中,对己酸乙酯具有最高酯化力101.29 mg/g麸曲。8号菌在最适培养温度36 ℃,酸度0.40度,固态发酵培养基含水量50%,固态发酵培养基厚度10 mm时,具有最高酯化力263.55 mg/g麸曲（以乙酸乙酯计）。     

Glucoamylases production of Aspergillus niger in solid state fermentation using a continuous counter-current reactor

This work presents the continuous production of fungal biomass and glucoamylase by solid state fermentation (SSF) in a counter-current reactor adapted for this purpose. Pre-germinated conidia of Aspergillus niger were used as an inoculum, and sugarcane bagasse, embedded with a nutritive solution, was the solid support. The solids residence time distribution (RTD) was carried out by feeding one impule of blue-coloured humidified bagasse and its RTD was fixed at 20 h. This study demonstrated that the values of the measured parameters (pH, moisture, biomass, glucoamylases production) were similar to those reported for batch SSF using the same solid support and micro-organism. A marked increase in biomass occurred from the progressive compartment (from compartment 1 to 9) into the reactor and the enzyme production was important (40 IU g⁻¹ dry exit solids). No mycelium damage or sporulation was observed by microscopy analysis. The above results confirmed that the continuous production of enzymes by SSF under no sterile conditions was successful. Inoculation with pre-germinated conidia shortened the processing time and allowed control of the age of the mycelium in each compartment. Aeration was accomplished by natural convection and moisture content had to be controlled. This process can be applied to the continuous production of fungal biomass and metabolites in SSF with industrial applications using environmental-friendly biotechnology.     

固态发酵生产高活力纤维二糖酶

在固态发酵条件下,对７个纤维二糖酶生产菌株进行了筛选,发现ＡｓｐｅｒｇｉｌｕｓｎｉｇｅｒＬＯＲＲＥ０１２为纤维二糖酶高产菌株。研究了培养时间、温度、培养基含水量及初始ｐＨ等因子对该菌形成纤维二糖酶的影响。当培养基采用自然ｐＨ（约６．０）、含水量７０％,接种后在３０℃下先培养２ｄ,再在２５℃下培养２ｄ,每克干酶曲所含的纤维二糖酶活力可达到４３０．５６ＩＵ。由此获得的纤维二糖酶曲与Ｔｒｉｃｈｏｄｅｒｍａｒｅｓｅｉ产生的纤维素酶曲在糖化纸浆过程中有明显的协同作用,当两者以１∶１０混合使用时（纤维二糖酶与滤纸酶活力之比约为０．４４）,可有效地消除水解产物中纤维二糖的累积,使纤维纸浆的酶水解得率高达９０．７％。     

Electricity production from cellulose in a microbial fuel cell using a defined binary culture

Microbial fuel cells (MFCs) convert biodegradable materials into electricity, potentially contributing to an array of renewable energy production strategies tailored for specific applications. Since there are no known microorganisms that can both metabolize cellulose and transfer electrons to solid extracellular substrates, the conversion of cellulosic biomass to electricity requires a syntrophic microbial community that uses an insoluble electron donor (cellulose) and electron acceptor (anode). Electricity was generated from cellulose in an MFC using a defined coculture of the cellulolytic fermenter Clostridium cellulolyticum and the electrochemically active Geobacter sulfurreducens. In fed-batch tests using two-chamber MFCs with ferricyanide as the catholyte, the coculture achieved maximum power densities of 143 mW/ m2 (anode area) and 59.2 mW/m2 from 1 g/L carboxymethyl cellulose (CMC) and MN301 cellulose, respectively. Neither pure culture alone produced electricity from these substrates. The coculture increased CMC degradation from 42% to 64% compared to a pure C. cellulolyticum culture. COD removal using CMC and MN301 was 38 and 27%, respectively, with corresponding Coulombic efficiencies of 47 and 39%. Hydrogen, acetate, and ethanol were the main residual metabolites of the binary culture. Cellulose conversion to electricity was also demonstrated using an uncharacterized mixed culture from activated sludge with an aerobic aqueous cathode.     

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27°C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.     

浓香型大曲中优势蛋白酶产生细菌的分离及产酶条件研究

对浓香型大曲中蛋白酶产生细菌进行分离,并对其最适产酶条件进行研究。采用酪素培养基,从大曲中分离出5株产蛋白酶菌株,筛选出酶活力最高的菌株,其酶活为53.40 U/mL。以小麦为固体培养基,从pH值、培养基水分含量、碳源添加量、氮源添加量、接种量、培养温度和培养时间等方面研究此菌株的最适产酶条件。结果表明,在水分含量65%、初始pH6.0、蔗糖添加量6%、硫酸铵添加量1%、接种量12%、温度40℃,培养时间5 d的条件下,其酶活可达262.18 U/mL,为菌种复筛时的4.91倍。     

Improvement of bacterial cellulose production by addition of agar in a jar fermentor

Bacterial cellulose (BC) was produced by Acetobacter xylinum BPR 2001 and its acetan nonproducing mutant EP1 in corn steep liquor-fructose medium in a 10-l jar fermentor supplemented with different agar concentrations ranging from 0% to 1.0% (w/v). The BC productivity of the two strains was increased by adding agar. The maximum BC production of BPR 2001 at an agar concentration of 0.4% was 12.8 g/l compared with 8 g/l without agar. The mutant EP1 produced 11.6 g/l of BC at an agar concentration of 0.6%, while only 5.5 g/l was produced in the control. Enhanced productivity is associated with an increase in viscosity of the culture, dispersion of BC pellets, and number of free cells due to agar addition, suggesting that acetan produced by BPR 2001 has a critical role in enhanced BC production.     

Changes in selected physical property and enzyme activity of rice and barley koji during fermentation and storage

Koji are solid-state fermentation products made by inoculating steamed grains with the spores of fungi, particularly Aspergillus spp. This research was undertaken to identify the fermentation and storage conditions optimal for the production and maintenance of selected hydrolytic enzymes, such as α-amlyase and protease, in koji. Steamed rice and barley were inoculated with 2 × 10 (11) Aspergillus oryzae spores per kilogram of grains and fermented for 118 h in a growth chamber at 28 to 32 °C with controlled relative humidities. Samples were drawn periodically during fermentation and storage at -20, 4, or 32 °C, and α-amylase and protease activity, mold counts, a(w), moisture contents, and pH of collected samples were determined. It was observed that the a(w), moisture contents, and pH of the koji were influenced by the duration of fermentation and temperature of storage. The α-amylase activity of both koji increased as the populations of A. oryzae increased during the exponential growth phase. The enzyme activity of barley koji was significantly higher than that of rice koji, reaching a peak activity of 211.87 or 116.57 U at 46 and 58 h, respectively, into the fermentation process. The enzyme activity in both products started to decrease once the mold culture entered the stationary growth phase. The protease activities of both koji were low and remained relatively stable during fermentation and storage. These results suggest that rice and barley koji can be used as sources of α-amylase and desired enzyme activity can be achieved by controlling the fermentation and storage conditions. PRACTICAL APPLICATION: Amylases and proteases are 2 important hydrolytic enzymes. In the food industry, these enzymes are used to break down starches and proteins while reducing the viscosity of foods. Although amylases and proteases are found in plants and animals, commercial enzymes are often produced using bacteria or molds through solid state fermentation, which is designed to use natural microbial process to produce enzymes in a controlled environment. A properly produced and maintained koji with a high hydrolytic enzyme activity can serve as an important source of the enzymes for the food industry.     

米曲霉A100-8种曲的培养工艺的研究

米曲霉A100-8菌种,是经离子注入诱变育种后,是以沪酿3．042为对照筛选得到的其特点为蛋白酶、糖化酶以及纤维素酶活力均有明显提高,且以偏酸性蛋白酶活力为主。因此,本文着重研究了A100-8种曲的培养条件。通过对原料的加水量、培养时间及有关无机盐的添加等的有效试验,最终由正交试验确立米曲霉A100-8的最佳培养工艺为,原料的加水量为120％;硫酸锰的添加量为0．04％、氯化钙为0．4％、磷酸盐为O．2％;培养时间为72h。成曲孢子数可达192亿／g左右;出芽率为95％以上（均以干基计）,较优化前均提高30％以上。     

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27 degrees C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.     

黑曲霉产粥化酶的发酵条件优化和浓缩酶液的制备

对作者所在实验室保藏的高产果胶酶的粥化酶产生菌株A．niger 537进行发酵营养和培养条件的优化，确定优化的工艺条件，果胶酶、纤维素酶、木聚糖酶、蛋白酶和糖化酶的干曲酶活分别达到4973，199．8，4993．3，26636．7，3170μ／g．同时对浸提条件及浓缩酶液的制备进行了考察．     

黑根霉Q3Y在旱胁迫效应下的菌丝生长特性及糖化产物

选取产糖化酶较强和糖化能力较高的黑根霉（Rhizopus nigricans）Q3Y进行菌丝体的抗旱逆境液态发酵培养,分析了菌株Q3Y菌丝生长状况及菌丝对糯米的利用。结果表明,在土豆汁培养基中添加0.50%的琼脂,使其黏度达到500 mPa·s,黑根霉菌Q3Y在该培养基中菌丝生长状态良好,细胞分散均匀;在料水比为6∶10（g∶mL）,含水量为62.5%糯米培养液中,黑根霉菌株Q3Y菌丝体能得到更好的生长,培养72 h还原糖、可溶性总糖及糖化酶酶活分别达到26 g/100mL、32 g/100mL、23.0 U/mL。     

Production and some properties of salt-tolerant beta-xylosidases from a shoyu koji mold, Aspergillus oryzae in solid and liquid cultures

Beta-xylosidase production from a shoyu (soy sauce) koji mold, Aspergillus oryzae HL15, cultured in solid and liquid media was examined and some properties of the enzymes were studied. Three beta-xylosidases (Xy11, Xy12 and Xy13) were easily extracted with 0.5% NaCl from a solid medium and purified homogeneously on SDS-PAGE by chromatography. On the other hand, in a liquid medium, A. oryzae HL15 produced mainly cell-wall-bound beta-xylosidases which could not be extracted with 0.5% NaCl or any detergent. Cell-wall-bound beta-xylosidases, Xy11-CB and Xy12-CB, were liberated by digestion of mycelia with Yatalase and purified to a homogeneous state on SDS-PAGE by HPLC column chromatography. Four beta-xylosidases (Xy11, Xy12, Xy11-CB and Xy12-CB) exhibited not only high activity at high NaCl concentrations, but also similar properties; on the other hand, Xy13 differed in terms of thermostability and halophilic properties. The salt tolerance of beta-xylosidases in A. oryzae suggests that these enzymes are highly active and involved in releasing xylose in shoyu moromi mash.