Alkaline cellulases from alkaliphilic Bacillus: Enzymatic properties, genetics, and application to detergents

We have isolated a number of alkaliphilic Bacillus that produce alkaline exoenzymes and found a possible use for alkaline cellulase (carboxymethylcellulase) as an additive for improving the cleaning effect of detergents. Enzymatic properties of some candidate cellulases fulfilled the essential requirements for enzymes to be used practically in laundry detergents. Here I describe the properties and possible catalytic mechanism of the hydrolytic reaction and the gene for the industrial alkaline cellulase produced by one of the isolates, Bacillus sp. KSM-635. Received: October 4, 1996 / Accepted: December 2, 1996     

An overview of Bacillus proteases: from production to application

Proteases have a broad range of applications in industrial processes and products and are representative of most worldwide enzyme sales. The genus Bacillus is probably the most important bacterial source of proteases and is capable of producing high yields of neutral and alkaline proteolytic enzymes with remarkable properties, such as high stability towards extreme temperatures, pH, organic solvents, detergents and oxidizing compounds. Therefore, several strategies have been developed for the cost-effective production of Bacillus proteases, including optimization of the fermentation parameters. Moreover, there are many studies on the use of low-cost substrates for submerged and solid state fermentation. Other alternatives include genetic tools such as protein engineering in order to obtain more active and stable proteases and strain engineering to better secrete recombinant proteases from Bacillus through homologous and heterologous protein expression. There has been extensive research on proteases because of the broad number of applications for these enzymes, such as in detergent formulations for the removal of blood stains from fabrics, production of bioactive peptides, food processing, enantioselective reactions, and dehairing of skins. Moreover, many commercial proteases have been characterized and purified from different Bacillus species. Therefore, this review highlights the production, purification, characterization, and application of proteases from a number of Bacillus species.     

Restricting detergent protease action to surface of protein fibres by chemical modification

Due to their excellent properties, such as thermostability, activity over a broad range of pH and efficient stain removal, proteases from Bacillus sp. are commonly used in the textile industry including industrial processes and laundry and represent one of the most important groups of enzymes. However, due to the action of proteases, severe damage on natural protein fibres such as silk and wool result after washing with detergents containing proteases. To include the benefits of proteases in a wool fibre friendly detergent formulation, the soluble polymer polyethylene glycol (PEG) was covalently attached to a protease from Bacillus licheniformis. In contrast to activation of PEG with cyanuric chloride (50%) activation with 1,1′-carbonyldiimidazole (CDI) lead to activity recovery above 90%. With these modified enzymes, hydrolytic attack on wool fibres could be successfully prevented up to 95% compared to the native enzymes. Colour difference (ΔE) measured in the three dimensional colour space showed good stain removal properties for the modified enzymes. Furthermore, half-life of the modified enzymes in buffers and commercial detergents solutions was nearly twice as high as those of the non-modified enzymes with values of up to 63 min. Out of the different modified proteases especially the B. licheniformis protease with the 2.0-kDa polymer attached both retained stain removal properties and did not hydrolyse/damage wool fibres.     

Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated <Emphasis Type="Italic">Bacillus</Emphasis> species Y

Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about 1.5 kb) of its 16S rDNA it was identified as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purification of the enzyme by ammonium sulfate (50–70% saturation) yielded 8-fold purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C up to 2 h. The protease showed good hydrolysis specificity with different substrates tested. The presence of Mn²⁺, Co²⁺ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up to 3 h retaining almost 70–80% activity and 10–20% activity at room temperature (30°C) and 50°C, respectively, indicating the potential role of this enzyme for detergent application.     

Utility of enzymes from <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> and <Emphasis Type="Italic">Prevotella ruminicola</Emphasis> as detergent additives

In this study, we investigated the application of cellulase and protease purified from rumen bacteria as detergent additives. Cellulase and protease were purified from the rumen cellulytic bacteria Fibrobacter succinogenes S85, and Prevotella ruminicola 23, respectively. An inhibitor test indicated that the purified protease belongs to the category of serine proteases and metalloproteases. Both the enzymes were effective at a high temperature (50 degrees C) and neutral pH (pH 7-8), but the protease activity increased with the increase in temperature and pH. The purified protease was treated with ten types of surfactants/detergents; it was found to retain over 60% of its activity in the presence of anionic and nonionic detergents. The cellulose plus protease combination was still effective after treatment with Triton X-100 and Tween 80, but the residual activity was low after treatment with Tween 20 than that after treatment with other nonionic detergents. Washing tests indicated that enzyme addition produced no significant improvement in the removal of grass stains, but individual enzyme addition in surfactants/detergents, especially in nonionic detergents, could improve the washing performance of the detergents by improving its ability to remove blood stains. This suggested that the surfactant/detergent class, enzyme properties, and the mixing ratio of ingredients should be considered simultaneously to enhance the washing performance.     

Immunochemical characterization of commercial protease products

Total protease activity at pH 7 and 10.3 of 23 commercial grade enzymes was determined. The type and amount of enzymatic activity varied widely among the products. The wide variation in pH 7.0/pH 10.3 proteolytic activity ratios among products indicated that the products studied contained differing levels of alkaline and neutral proteases. Antisera were prepared against the purified enzyme in detergent grade Enzyme AP, neutral protease from B. megaterium, detergent grade ALK Enzyme, and Thermolysin. The commercial (unpurified) products were classified as neutral subtilopeptidase A and subtilopeptidase B from three Bacillus species using these antisera. It was concluded that standard immunochemical techniques provide rapid and sensitive methods for the preliminary identification of sources and types of proteases present in commercial enzyme products.     

Genomic and Functional Diversity of Bacteria Isolated from Hot Springs in Odisha,India

This study focused on culture-dependent survey of important bacterial community diversity of hot springs of Odisha. India. Molecular and cultural techniques were employed for assessing and exploiting the genetic and functional variability among the isolates obtained from three alkaline and mesophilic hot springs. A total of 48 isolates belonging to family Bacillaceae, Paenibacillaceae, Planococcaceae, Pseudomonadaceae and Enterobacteriaceae were identified. Majority of the bacterial isolates were affiliated with the genus Bacillus. Morphologically all the isolated bacteria were either Gram-positive spore-forming rods, or Gram-negative rods. The optimum temperature for growth of the isolates varied between 37°C to 50°C. The functional diversity revealed that many of the predominant and scarce isolates produced a variety of extracellular enzymes such as amylase, cellulase, lipase, phosphatase and protease, and genus Bacillus dominated for extracellular enzymatic activity. We employed two molecular markers to characterize the isolates. The hsp60 universal target sequence was found to be more discriminatory than 16s rRNA gene sequences. The cultivable bacterial community structure that colonized in the investigated thermal springs did not reveal much overlapping. Our results indicate that bacteria in the geothermal environment are metabolically active and cultivable populations may have great potential in biotechnology.     

Enzyme treatment to reduce solids and improve settling of sewage sludge

The effect of microbial enzymes in reducing the disposable solid content of sludge was investigated. A mixture of industrial cellulase, protease, and lipase, in equal proportion by weight, reduced total suspended solids (TSS) by 30–50% and improved settling of solids. An increase in solid reduction was observed with increasing enzyme concentration. The effect of combinations of enzyme treatments indicated that two-enzyme combinations of protease and cellulase produced better solid reduction than individual enzymes and that lipase further augmented this effect. Among the individual enzymes, protease produced a more settleable sludge as compared to cellulase and lipase. Adjustment of the pH of the enzymatically treated sludge to the acidic range (pH 2–4) further improved solid reduction, and adjustment to the alkaline range (pH 10–12) improved settleability. Journal of Industrial Microbiology & Biotechnology (2001) 26, 383–386. Received 01 November 2000/ Accepted in revised form 29 April 2001     

杜比亚蟑螂肠道菌的分离鉴定及产消化酶功能菌株的筛选

【背景】杜比亚蟑螂(Blaptica dubia)可用于活体饲料、化妆品和医药保健品的生产,其肠道菌的研究对杜比亚蟑螂的饲养和肠道菌资源的开发与利用都十分重要。【目的】揭示杜比亚蟑螂肠道可培养菌的种类,筛选具有产消化酶功能的菌株,为理解肠道菌对宿主的影响机理及功能菌株的利用提供科学依据和研究材料。【方法】采用体外培养法获得杜比亚蟑螂肠道菌,结合形态学和分子生物学方法进行鉴定;用水解圈法分别筛选产纤维素酶、蛋白酶、脂肪酶和淀粉酶菌株。【结果】在杜比亚蟑螂肠道中共分离出4属7种细菌,其中芽孢杆菌属(Bacillus)2种,沙雷氏菌属(Serratia)和柠檬酸杆菌属(Citrobacter)各2种,肠球菌属(Enterococcus)1种。从获得的20个菌株中筛选出10个具有产消化酶功能的菌株。其中,芽孢杆菌属的菌株D6、D12和D20具有产纤维素酶、蛋白酶、淀粉酶及脂肪酶4种消化酶的功能;沙雷氏菌属的菌株D3、D7、D9、D11和D15具有产纤维素酶、蛋白酶和脂肪酶3种消化酶的能力;柠檬酸杆菌属的菌株D5具有产纤维素酶的功能;肠球菌属的菌株D17具有产蛋白酶的能力。【结论】杜比亚蟑螂肠道多种细菌具有产消化酶帮助降解大分子营养物质的功能,可通过协助食物消化影响宿主健康。菌株D12、D7和D11分别具有最强产纤维素酶、蛋白酶和脂肪酶的能力,是可进一步开发利用的肠道功能菌株资源。     

Alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain KSM-LD1 contains a record number of subtilisin-like serine proteases genes

The presence of 11 genes encoding subtilisin-like serine proteases was demonstrated by cloning from the genome of alkaliphilic Bacillus sp. strain KSM-LD1. This strain exoproduces the oxidatively stable alkaline protease LD-1 (Saeki et al. Curr Microbiol, 47:337–340, 2003). Among the 11 genes, six genes encoding alkaline proteases (SA, SB, SC, SD, SE, and LD-1) were expressed in Bacillus hosts. However, the other five genes for subtilisin-like proteases (SF, SG, SH, SI, and SJ) were expressed in neither Bacillus hosts nor Escherichia coli. The deduced amino acid sequences of SA, SB, SC, SF, SG, SH, SI, and SJ showed similarity to those of other subtilisin-like proteases from Bacillus strains with only 38 to 86% identity. The deduced amino acid sequence of SD was completely identical to that of an oxidatively stable alkaline protease from Bacillus sp. strain SD521, and that of SE was almost identical to that of a high-molecular mass subtilisin from Bacillus sp. strain D-6 with 99.7% identity. There are four to nine subtilisin-like serine protease genes in the reported genomes of Bacillus strains. At least 11 genes for the enzymes present in the genome of Bacillus sp. strain KSM-LD1, and this is the greatest number identified to date.     

An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations

An investigation on the properties of an alkaline protease secreted by Bacillus circulans BM15 strain isolated from a mangrove sediment sample was carried out in order to characterize the enzyme and to test its potency as a detergent additive. The protease was purified to apparent homogeneity by ammonium sulphate precipitation and was a 30-kDa protease as shown by SDS-PAGE and its proteolytic activity was detected by casein zymography. It had optimum activity at pH 7, was stable at alkaline pH range (7 to 11), had optimum temperature of activity 40°C and was stable up to a temperature of 55°C after incubation for one hour. Hg²⁺, Zn²⁺, Co²⁺, and Cu²⁺completely inhibited the enzyme activity, while Ca²⁺, Mg²⁺, K⁺ and Fe³⁺ were enhancing the same. The serine protease inhibitor PMSF and metal chelator EDTA inhibited the activity of this protease while the classic metalloprotease inhibitor 1, 10 phenanthroline did not show inhibition. The enzyme was stable in SDS, Triton-X-100 and H₂ O₂ as well as in various commercial detergents after incubation for one hour. The extracellular production of the enzyme, the pH and temperature stability and stability in presence of oxidants, surfactants and commercial detergents suggest its possible use as a detergent additive.     

The State of Amino Acid Residues in Alkaline Protease Produced by Bacillus No. 221

The state of amino acid residues in alkaline protease of Bacillus No. 221 and that of subtiiisin BPN’ were compared by spectrophotometric tiiration of tyrosine residues and by several reagents: β-naphtoqumone-4,6-disulfonic acid and monochlorofluoroquinone for amino groups, H₂O₂-dioxane for tryptophan, glyoxal for arginine, and tetranitromethane for tyrosine.

The reactivity of both proteases was fairly similar to those reagents.

The helix content of alkaline protease of Bacillus No. 221 (37%) was higher than that of subtilisin BPN’ (20%).

The Km and V_max of alkaline protease of Bacillus No. 221 toward ATEE and BTEE were obtained from Lineweaver-Burk plot and compared with those of α-chymotrypsin and subtiiisin BPN’.     

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H₂O₂, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.     

Molecular Cloning,Nucleotide Sequence,and Expression of the Structural Gene for Alkaline Serine Protease from Alkaliphilic Bacillus sp. 221

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus suhtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6—61.70/0). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.     

Effects of adsorption properties and mechanical agitation of two detergent cellulases towards cotton cellulose

Abstract

The impacts of two hybrid cloned commercial cellulases designed for detergency on cotton fibres were compared. HiCel45 has a family 45 catalytic domain and a fungal cellulose binding module (CBM) from the fungus Humicola insolens. BaCel5 has a family 5 catalytic domain and a fungal CBM from Bacillus spp. BaCel5 bound irreversibly to cellulose under the buffer conditions tested while HiCel45 was found to bind reversibly to cellulose because it showed low adsorption. BaCel5 seems to yield more activity towards cotton than HiCel45 under mild stirring conditions, but under strong mechanical agitation both enzymes produce similar amount of sugars. HiCel45 had a more progressive production of residual reducing ends on the fabric than BaCel5. These studies seem to indicate that HiCel45 is a more cooperative enzyme with detergent processes where high mechanical agitation is needed.     

Multiple-layer substrate zymography for detection of several enzymes in a single sodium dodecyl sulfate gel

We have developed a system to detect three hydrolytic enzymes (cellulase, lipase, and protease) using a single sodium dodecyl sulfate (SDS) gel and an electrotransfer system. After electrophoresis, proteins in the gel were transferred to three sandwiched substrate gels containing glycerol tributyrate, azo-carboxymethyl cellulose (Azo-CMC), and fibrin for detection of cellulase, lipase, and protease, respectively. We show that three cellulases (from a Paenibacillus sp. and two Bacillus sp. strains), one lipase (from a Staphylococcus sp.), and two proteases (from two Bacillus sp. strains) can be detected simultaneously with our zymogram system.     

Phylogenetic Diversity and Characterization of Novel and Efficient Cellulase Producing Bacterial Isolates from Various Extreme Environments

A set of 300 bacterial strains isolated from various extreme environments were screened for the presence of cellulase activity on CMC agar plates. Phylogenetic analysis of the positive strain, based on 16S rRNA gene sequences indicated that the isolates were clustered within Firmicutes and Actinobacteria. A majority (17) of the isolates were identified as Bacillus, Paenibacillus, and Lysinibacillus sp., and the remaining three were identified as Arthobacter, Rhodococcus, and Bhargavaea cecembensis. Among the 20 positive isolates, 6 were evaluated for the production of cellulases on five different cellulosic substrates. Two isolates, B. cecembensis and Bacillus sp., based on maximum enzyme production on all cellulosic substrates, especially CMC and rice straw, were evaluated in terms of enzyme properties and kinetics. The enzymes of these two isolates are found to be active over broad range of pH and temperature. Such thermostable enzymes facilitate the development of efficient and cost-effective forms of the simultaneous saccharification and fermentation process converting lignocellulosic biomass into biofuels and value-added products.     

Compatibility of alkaline xylanases from an alkaliphilic<Emphasis Type="Italic"> Bacillus</Emphasis> NCL (87-6-10) with commercial detergents and proteases

Alkaline xylanases from alkaliphilic Bacillus strains NCL (87-6-10) and Sam III were compared with the commercial xylanases Pulpzyme HC and Biopulp for their compatibility with detergents and proteases for laundry applications. Among the four xylanases evaluated, the enzyme from the alkaliphilic Bacillus strain NCL (87-6-10) was the most compatible. The enzyme retained its full activity (40 °C for 1 h) in the presence of detergents, whereas Pulpzyme HC and Sam III showed only 30% and 50% of their initial activity, respectively. Biopulp, though stable to detergents, had only marginal activity (5%)at pH 10. However, all four enzymes retained significant activity (80%) for 60 min in the presence of the proteases Alcalase and Conidiobolus protease. Supplementation of the enzyme enhanced the cleaning ability of the detergents.     

Sequence of the gene for a high-alkaline mannanase from an alkaliphilic Bacillus sp. strain JAMB-750, its expression in Bacillus subtilis and characterization of the recombinant enzyme

A novel alkaline mannanase Man26A has been found in the culture of an alkaliphilic Bacillus sp. strain JAMB-750 and the optimal pH for the mannanase activity of the enzyme was around pH 10 (J Biol Macromol 4: 67–74, 2004). This optimal pH is the highest among those of the mannanases reported to date. The gene man26A coding the enzyme was cloned from the genomic DNA of strain JAMB-750 and sequenced. It encodes a protein of 997 amino acids including a signal peptide. The N-terminal half (Glu27–Val486) of the enzyme exhibited moderate similarities to other mannanases belonging to glycoside hydrolase family 26, such as the enzymes from Cellvibrio japonicus (37% identity), Cellulomonas fimi (33% identity), and Bacillus sp. strain AM-001 (28% identity). The C-terminal half was found to contain four domains. The first, second, third, and fourth domains exhibited similarities to the carbohydrate-binding module, the mannan-binding module, the Homo sapiens collagen type IX alpha I chain, and the membrane anchor region of Gram-positive surface proteins, respectively. Its recombinant mannanase was produced extracellularly using Bacillus subtilis as the host. The optimal pH for the mannanase activity of the recombinant enzyme was around pH 10. The enzyme was very resistant to surfactants, for example, SDS up to 2.0% (w/v).     

Extracellular hydrolase profiles of fungi isolated from koala faeces invite biotechnological interest

The extracellular enzymes of seven fungal strains isolated from koala faeces have been comprehensively characterised for the first time, revealing potential for biotechnological applications. The fungal isolates were grown in a hydrolase-inducing liquid medium and the supernatants were analysed using enzyme assays and zymogram gels. Temperature and pH profiles were established for xylanase (EC 3.2.1.8 endo-1,4-β-xylanase), mannanase (EC 3.2.1.78 mannan endo-1,4-β-mannosidase), endoglucanase (EC 3.2.1.4 cellulase), β-glucosidase (EC 3.2.1.21 β-glucosidase), amylase (EC 3.2.1.1 α-amylase), lipase (EC 3.1.1.3 triacylglycerol lipase) and protease (EC 3.4 peptidase) activities. Comparisons were made to the high-secreting hypercellulolytic mutant strain Trichoderma reesei RUT-C30 and the wild-type T. reesei QM6a. The isolates from koala faeces Gelasinospora cratophora A10 and Trichoderma atroviride A2 were good secretors of total protein and heat-tolerant enzymes. Doratomyces stemonitis C8 secreted hemicellulase(s), endoglucanase(s) and β-glucosidase(s) with neutral to alkaline pH optimums. A cold-tolerant lipase was secreted by Mariannaea camptospora A11. The characteristics displayed by the enzymes are highly sought after for industrial processes such as the manufacture of paper, detergents and food products. Furthermore, the enzymes were produced at good starting levels that could be increased further by strain improvement programs.