Lignocellulosic biomass, is a great resource for the production of bio-energy and bio-based material since it is largely abundant, inexpensive and renewable. The requirement of new energy sources has led to a wide search for novel effective enzymes to improve the exploitation of lignocellulose, among which the importance of thermostable and halotolerant cellulase enzymes with high pH performance is significant. The primary aim of this study was to discover a novel alkali-thermostable endo-β-1,4-glucanase from the sheep rumen metagenome. At first, the multi-step in-silico screening approach was utilized to find primary candidate enzymes with superior properties. Among the computationally selected candidates, PersiCel4 was found and subjected to cloning, expression, and purification followed by functional and structural characterization. The enzymes’ kinetic parameters, including Vmax, Km, and specific activity, were calculated. The PersiCel4 demonstrated its optimum activity at pH 8.5 and a temperature of 85 °C and was able to retain more than 70% of its activity after 150 h of storage at 85 °C. Furthermore, this enzyme was able to maintain its catalytic activity in the presence of different concentrations of NaCl and several metal ions contains Mg2+, Mn2+, Cu2+, Fe2+ and Ca2+. Our results showed that treatment with MnCl2 could enhance the enzyme’s activity by 78%. PersiCel4 was ultimately used for enzymatic hydrolysis of autoclave pretreated rice straw, the most abundant agricultural waste with rich cellulose content. In autoclave treated rice straw, enzymatic hydrolysis with the PersiCel4 increased the release of reducing sugar up to 260% after 72 h in the harsh condition (T = 85 °C, pH = 8.5). Considering the urgent demand for stable cellulases that are operational on extreme temperature and pH conditions and due to several proposed distinctive characteristics of PersiCel4, it can be used in the harsh condition for bioconversion of lignocellulosic biomass.

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A novel high performance in-silico screened metagenome-derived alkali-thermostable endo-β-1,4-glucanase for lignocellulosic biomass hydrolysis in the harsh conditions.

Deletion of the middle region of the transcription factor ClrB in Penicillium oxalicum enables cellulase production in the presence of glucose.

Degradation of various types of lignocellulosic biomass that require harsh pretreatment and abnormal operating conditions often occur in presence of a large amount of salt. Therefore, identification and use of stable and halotolerant cellulases is essential for industrial lignocellulose applications under extreme pH and temperature. This study focuses on discovering a novel thermostable and halotolerant cellulase from rumen microbiota by employing a multi-stage in-silico screening pipeline. According to this cost-effective strategy, the new PersiCel3 was cloned, expressed, purified, and characterized. The enzyme demonstrated suitable thermal and storage stability. To improve the thermal stability and halotolerance of the enzyme, it was immobilized on the CMC-based hydrogel. The maximum activity of the PersiCel3 could be seen in the concentration of 3 M NaCl for both free (132.46%) and immobilized (197.47%) enzyme. Applying both the free and immobilized PersiCel3 on degrading the rice straw in saline condition leads to an increment in generating the reducing sugars. The immobilized enzyme presented a significant enhancement in the hydrolysis of rice straw in saline conditions compared to its free form. Our results demonstrated the potential of the robust PersiCel3 in the harsh condition and the superb performance of the immobilized enzyme for the lignocellulosic biomass industries to increase the yield of value-added products in high temperatures and saline conditions.

Upgrading the enzymatic hydrolysis of lignocellulosic biomass by immobilization of metagenome-derived novel halotolerant cellulase on the carboxymethyl cellulose-based hydrogel

The unique GH5 cellulase, AgCMCase, from Aspergillus glaucus CCHA was identified and characterized as having high cellulose and straw hydrolysis activities that were thermostable, pH stable and salt-tolerant. Therefore, it is a potential straw-degradation enzyme that can release reducing sugars in industrial applications. To increase the efficiency of the AgCMCase’ hydrolysis of straw to release simple sugars, response surface methodology (RSM) was introduced to optimize hydrolysis parameters such as pH, temperature, reaction time and enzyme dose. The enzyme showed only one major protein band from the fermentation broth by the Pichia pastoris GS115 expression. The crude enzyme (without purification) showed a satisfactory capability to hydrolyze CMC-Na after 4 days of production. Here, the crude AgCMCase also showed cellulose and straw hydrolysis capabilities as assessed by scanning electron microscopic and Fourier-transform infrared spectroscopic analyses. A high-performance liquid chromatographic analysis demonstrated that the degradation of corn and rice straw by crude AgCMCase mainly produced glucose and cellobiose. Temperature, reaction time and enzyme dose were the significant variables affecting corn and rice straw degradation. After the optimization of RSM, a model was proposed to predict 1.48% reducing sugar yield with the optimum temperature (51.45 °C) and reaction time (3.84 h) from the straw degradation. The reaction of crude AgCMCase and rice straw in the optimized condition resulted in reducing sugar production of 1.61% that agrees the prediction. Our findings suggest that the crude AgCMCase is suitable to be used in straw conversion.

Statistical optimization of a cellulase from Aspergillus glaucus CCHA for hydrolyzing corn and rice straw by RSM to enhance yield of reducing sugar.

Numerous thermostable enzymes have been reported from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which made it an attractive resource for gene cloning. This research reported a glycosyl hydrolase (Tk-ChiA) form T. Kodakarensis with dual hydrolytic activity due to the presence of three binding domains with affinity toward chitin and cellulose. The Tk-ChiA gene was cloned and expressed on Pichia pastoris GS115. The molecular weight of the purified Tk-ChiA is about 130.0 kDa. By using chitosan, CMC-Na and other polysaccharides as substrates, we confirmed that Tk-ChiA with dual hydrolysis activity preferably hydrolyzes both chitosan and CMC-Na. Purified Tk-ChiA showed maximal activity for hydrolyzing CMC-Na at temperature 65°C and pH 7.0. It showed thermal stability on incubation for 4 h at temperatures ranging from 70 to 80°C and remained more than 40% of its maximum activity after pre-incubation at 100°C for 4 h. Particularly, Tk-ChiA is capable of degrading shrimp shell and rice straw through scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR) analysis. The main factors affecting shell and straw degradation were determined to be reaction time and temperature; and both factors were optimized by central composite design (CCD) of response surface methodology (RSM) to enhance the efficiency of degradation. Our findings suggest that Tk-ChiA with dual thermostable hydrolytic activities maybe a promising hydrolase for shell and straw waste treatment, conversion, and utilization.

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An Archaeal Chitinase With a Secondary Capacity for Catalyzing Cellulose and Its Biotechnological Applications in Shell and Straw Degradation.

In a halotolerant fungus Aspergillus glaucus CCHA, several functional proteins with stress-tolerant activity have been studied, but no secretory enzymes have been identified yet. The unique GH5 cellulase candidate from A. glaucus, an endoglucanase termed as AgCMCase, was cloned, expressed in the Pichia pastoris system and the purified enzyme was characterized. A large amount of recombinant enzyme secreted by the P. pastoris GS115 strain was purified to homogeneity. The molecular weight of the purified endoglucanase is about 55.0 kDa. The AgCMCase exhibited optimum catalytic activity at pH 5.0 and 55 °C. However, it remained relatively stable at temperatures ranging from 45 to 80 °C and pH ranging from 4.0 to 9.0. In addition, it showed higher activity at extreme NaCl concentrations from 1.0 to 4.0 M, suggesting it is an enzyme highly stable under heat, acid, alkaline and saline conditions. To evaluate the catalytic activity of AgCMCase, the hydrolysis products of rice and corn straws were successfully studied. In conclusion, the AgCMCase is a thermostable and salt-tolerant cellulase with potential for industrial application.

The unique GH5 cellulase member in the extreme halotolerant fungus Aspergillus glaucus CCHA is an endoglucanase with multiple tolerance to salt, alkali and heat: prospects for straw degradation applications.

The invention belongs to the technical field of bioengineering and enzyme engineering, and discloses a polysaccharide hydrolase gene from pyrococcus horikoshii and application. A nucleotide sequence of the polysaccharide hydrolase gene Tk1765 is shown as SEQ ID NO:1. A coding sequence of the polysaccharide hydrolase gene Tk1765 is shown as SEQ ID NO:2. Polysaccharide hydrolase has the efficient cellulose gum and chitose double digest character. According to cellulose gum of the polysaccharide hydrolase, the optimum pH value is 10.0, and the optimum enzyme reaction temperature is 65 DEG C. According to hydrolysis chitose, the optimum pH value is 11.0, and the optimum enzyme reaction temperature is 80 DEG C. The hydrolase is heated for 4 h at the temperature being 100 DEG C, and the activity of the hydrolase can be kept to be 40% or above. A recombinant vector of the hydrolase is constructed and excessively expressed in pichia pastoris, a product of the hydrolase has the function of hydrolyzing maize straw, and the polysaccharide hydrolase and the coding gene thereof can be widely applied to cellulose and chitin degradation.

Polysaccharide hydrolase gene from pyrococcus horikoshii and application

The invention relates to a cellulase gene from extreme saline-alkali resistant aspergilli and application, and belongs to the technical field of bioengineering and enzyme engineering. A nucleotide sequence of the cellulase gene CCHA333 is shown in SEQ ID NO.1, a coding sequence of the cellulase gene CCHA333 is shown in SEQ ID NO.2, and the cellulase gene CCHA333 can be applied to dynamic characteristic identification of an enzyme capable of hydrolyzing sodium carboxymethyl cellulose; the optimal pH value of the cellulase is 6, the optimal enzymatic reaction temperature is 55 DEG C, the pH ranges from 4 to 12, heating is conducted for 2 h at 80 DEG C, and the enzyme activity can be maintained 50 percent or above. Under the environment that many metal ions such as Cu , Co and Na exist, the enzyme activity is obviously improved. Due to the fact that a recombinant vector is built, and a product over expressed in pichia pastoris has the function of efficiently hydrolyzing the sodium carboxymethyl cellulose and maize straw, the cellulase and the coding gene thereof can be widely applied to cellulose degradation.

Li Zhengqun

Papers

The unique GH5 cellulase member in the extreme halotolerant fungus Aspergillus glaucus CCHA is an endoglucanase with multiple tolerance to salt, alkali and heat: prospects for straw degradation applications.

Polysaccharide hydrolase gene from pyrococcus horikoshii and application

Cellulase gene from extreme saline-alkali resistant aspergilli and application